S. cerevisiae

EMBO J, 2003 Oct 15, 22(20), 5358 - 69
Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import; Matsuura Y et al.; The yeast nucleoporin Nup2p is associated primarily with the nuclear basket of nuclear pore complexes and is required for efficient importin-alpha:beta-mediated nuclear protein import as well as efficient nuclear export of Kap60p/importin-alpha . Residues 1-51 of Nup2p bind tightly to Kap60p and are required for Nup2p function in vivo . We have determined the 2.6 A resolution crystal structure of a complex between this region of Nup2p and the armadillo repeat domain of Kap60p . Nup2p binds along the inner concave groove of Kap60p, but its interaction interface is different from that employed for nuclear localization signal (NLS) recognition although there is some overlap between them . Nup2p binds Kap60p more strongly than NLSs and accelerates release of NLSs from Kap60p . Nup2p itself is released from Kap60p by Cse1p:RanGTP only in the presence of the importin-beta binding (IBB) domain of Kap60p . These data indicate that Nup2p increases the overall rate of nuclear trafficking by coordinating nuclear import termination and importin recycling as a concerted process.

Genes Cells, 2003 Oct, 8(10), 779 - 88
Transcription elongation factor S-II maintains transcriptional fidelity and confers oxidative stress resistance; Koyama H et al.; BACKGROUND: During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts . Transcription factor S-II enhances the excision of these mis-incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro . This mechanism is considered to be transcriptional proof-reading, but its physiological relevance remains unknown . RESULTS: We report that S-II contributes to the maintenance of transcriptional fidelity in vivo . We employed a genetic reporter assay utilizing a mutated lacZ gene from which active beta-galactosidase protein is expressed when mRNA proof-reading is compromised . In S-II-disrupted mutant yeasts, beta-galactosidase activity was ninefold higher than that in wild-type . The S-II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S-II gene . The mutant S-II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity . CONCLUSION: These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation.

Mov Disord, 2003 Oct, 18 Suppl 7, S71 - 80
Dopamine transporter: basic science and human variation of a key molecule for dopaminergic function, locomotion, and parkinsonism; Uhl GR; We review the basic science of the dopamine transporter (DAT), a key neurotransmitter for locomotor control and reward systems, including those lost or deranged in Parkinson's disease (PD) . Physiology, pharmaceutical features, expression, cDNA, protein structure/function relationships, and phosphorylation and regulation are discussed . The localization of DAT provides the best marker for the integrity of just the pre-synaptic dopaminergic systems that are most affected in PD . Its function is key for the actions of several toxins that provide some of the best current models for idiopathic parkinsonism, and its variation can clearly alter movement . The wealth of information about this interesting molecule that has been developed over the last 12 years has led to increased interest in DAT among workers interested in both normal and abnormal movement .

Nucleic Acids Res, 2003 Oct 15, 31(20), 6053 - 61
Detection of regulatory circuits by integrating the cellular networks of protein-protein interactions and transcription regulation; Yeger-Lotem E et al.; The post-genomic era is marked by huge amounts of data generated by large-scale functional genomic and proteomic experiments . A major challenge is to integrate the various types of genome-scale information in order to reveal the intra- and inter- relationships between genes and proteins that constitute a living cell . Here we present a novel application of classical graph algorithms to integrate the cellular networks of protein-protein interactions and transcription regulation . We demonstrate how integration of these two networks enables the discovery of simple as well as complex regulatory circuits that involve both protein-protein and protein-DNA interactions . These circuits may serve for positive or negative feedback mechanisms . By applying our approach to data from the yeast Saccharomyces cerevisiae, we were able to identify known simple and complex regulatory circuits and to discover many putative circuits, whose biological relevance has been assessed using various types of experimental data . The newly identified relations provide new insight into the processes that take place in the cell, insight that could not be gained by analyzing each type of data independently . The computational scheme that we propose may be used to integrate additional functional genomic and proteomic data and to reveal other types of relations, in yeast as well as in higher organisms.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12510 - 5 Epub 2003 Oct 06.
Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity; Yamaguchi T et al.; We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter . Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system . The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter . It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain . Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated . Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen . Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport . The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1 . This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport . The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.

J Biol Chem, 2003 Dec 19, 278(51), 51441 - 7 Epub 2003 Oct 06.
Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase . Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade; Chavan M et al.; Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast . Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex . Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated . Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein . Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine . Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3 . It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade . It has been shown that the pkc1 null mutant exhibits lowered OT activity . Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity . We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex.

J Biol Chem, 2003 Dec 19, 278(51), 51841 - 50 Epub 2003 Oct 06.
A single protease, Apg4B, is specific for the autophagy-related ubiquitin-like proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L; Hemelaar J et al.; Apg8 is a ubiquitin-like protein involved in autophagy in yeast . Apg8 is covalently but transiently attached to membrane lipids through the actions of activating, conjugating, and processing/deconjugating enzymes . The mammalian Apg8 homologues GATE-16, GARARAP, and MAP1-LC3 have been implicated in intra-Golgi transport, receptor sorting, and autophagy, respectively . All are served by a single set of activating and conjugating enzymes . Here we identify a novel mammalian Apg8 homologue, which we name Apg8L, and describe the synthesis of electrophilic probes based on the GATE-16, GARARAP, MAP1-LC3, and Apg8L proteins . These probes not only form specific adducts in crude cell lysates, but also allow identification of the cellular proteases specific for the C termini of these Apg8 homologues . We find a single protease, Apg4B/autophagin-1, capable of acting on GATE-16, GABARAP, MAP1-LC3, and Apg8L . The Apg4B/autophagin-1 protease thus serves as a processing/deconjugating enzyme for these four highly divergent mammalian Apg8 homologues.

J Mol Biol, 2003 Oct 17, 333(2), 229 - 35
Interaction of the MAGUK family member Acvrinp1 and the cytoplasmic domain of the Notch ligand Delta1; Pfister S et al.; The evolutionarily conserved Notch signal transduction pathway regulates cell fate and cellular differentiation in various tissues and has essential functions in embryonic patterning and tumorigenesis . Cell-cell signaling by the Notch pathway is mediated by the interaction of the transmembrane receptor Notch with its ligands Delta or Jagged presented on adjacent cells . Whereas signal transduction to Notch expressing cells has been described, it is unclear whether Delta-dependent signaling may exist within the Delta-expressing cell . Here, we report on the identification of Acvrinp1, a MAGUK family member, interacting with the intracellular domain of Delta1 (Dll1) . We confirmed the interaction between Dll1 and Acvrinp1 by pull-down experiments in vitro and in a mammalian two-hybrid system in vivo . We delimited the fourth PDZ domain of Acvrinp1 and the PDZ-binding domain of Dll1 as major interacting domains . In situ expression analyses in mouse embryos revealed that Dll1 and Acvrinp1 show partly overlapping but distinct expression patterns, for example, in the central nervous system and the vibrissae buds . Further, we found that expression of Acvrinp1 is altered in Dll1 loss-of-function mouse embryos.

Mol Biol Cell, 2004 Jan, 15(1), 24 - 36 Epub 2003 Oct 03.
Vacuole size control: regulation of PtdIns(3,5)P2 levels by the vacuole-associated Vac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase; Rudge SA et al.; In the budding yeast Saccharomyces cerevisiae, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is synthesized by a single phosphatidylinositol 3-phosphate 5-kinase, Fab1 . Cells deficient in PtdIns(3,5)P2 synthesis exhibit a grossly enlarged vacuole morphology, whereas increased levels of PtdIns(3,5)P2 provokes the formation of multiple small vacuoles, suggesting a specific role for PtdIns(3,5)P2 in vacuole size control . Genetic studies have indicated that Fab1 kinase is positively regulated by Vac7 and Vac14; deletion of either gene results in ablation of PtdIns(3,5)P2 synthesis and the formation of a grossly enlarged vacuole . More recently, a suppressor of vac7Delta mutants was identified and shown to encode a putative phosphoinositide phosphatase, Fig4 . We demonstrate that Fig4 is a magnesium-activated PtdIns(3,5)P2-selective phosphoinositide phosphatase in vitro . Analysis of a Fig4-GFP fusion protein revealed that the Fig4 phosphatase is localized to the limiting membrane of the vacuole . Surprisingly, in the absence of Vac14, Fig4-GFP no longer localizes to the vacuole . However, Fig4-GFP remains localized to the grossly enlarged vacuoles of vac7 deletion mutants . Consistent with these observations, we found that Fig4 physically associates with Vac14 in a common membrane-associated complex . Our studies indicate that Vac14 both positively regulates Fab1 kinase activity and directs the localization/activation of the Fig4 PtdIns(3,5)P2 phosphatase.

J Biol Chem, 2003 Dec 26, 278(52), 52689 - 99 Epub 2003 Oct 03.
The human phosphatidylinositol phosphatase SAC1 interacts with the coatomer I complex; Rohde HM et al.; The Saccharomyces cerevisiae SAC1 gene encodes an integral membrane protein of the endoplasmic reticulum (ER) and the Golgi apparatus . Yeast SAC1 mutants display a wide array of phenotypes including inositol auxotrophy, cold sensitivity, secretory defects, disturbed ATP transport into the ER, or suppression of actin gene mutations . At present, it is not clear how these phenotypes relate to the finding that SAC1 displays polyphosphoinositide phosphatase activity . Moreover, it is still an open question whether SAC1 functions similarly in mammalian cells, since some phenotypes are yeast-specific . Potential protein interaction partners and, connected to that, possible regulatory circuits have not been described . Therefore, we have cloned human SAC1 (hSAC1), show that it behaves similar to ySac1p in terms of substrate specificity, demonstrate that the endogenous protein localizes to the ER and Golgi, and identify for the first time members of the coatomer I (COPI) complex as interaction partners of hSAC1 . Mutation of a putative COPI interaction motif (KXKXX) at its C terminus abolishes interaction with COPI and causes accumulation of hSAC1 in the Golgi . In addition, we generated a catalytically inactive mutant, demonstrate that its lipid binding capacity is unaltered, and show that it accumulates in the Golgi, incapable of interacting with the COPI complex despite the presence of the KXKXX motif . These results open the possibility that the enzymatic function of hSAC1 provides a switch for accessibility of the COPI interaction motif.

Gene, 2003 Sep 18, 314, 1 - 13
A POLYCOMB group gene of rice (Oryza sativa L . subspecies indica), OsiEZ1, codes for a nuclear-localized protein expressed preferentially in young seedlings and during reproductive development; Thakur JK et al.; The SET domains are conserved amino acid sequences present in chromosomal proteins that contribute to the epigenetic control of gene expression by altering regional organization of the chromatin structure . The SET domain proteins are divided into four subgroups as categorized by their Drosophila members; enhancer of zeste (E(Z)), trithorax (TRX), absent small or homeotic 1 (ASH1) and supressor of variegation (SU(VAR)3-9) . Homologs of all four classes have been characterized in yeast, mammals and plants . We report here the isolation and characterization of rice (Oryza sativa L . subspecies indica) cDNA, OsiEZ1, as a monocot member of this family . The OsiEZ1 cDNA is 3133 bp long with an ORF of 2799 bp, and the predicted amino acid sequence (895 residues) corresponds to a protein of ca . 98 kDa . All the characteristic domains known to be conserved in E(Z) homologs (subgroup I) of SET domain containing proteins are present in OsiEZ1 . In the rice genome, a 7499 bp long OsiEZ1 sequence is split into 17 exons interrupted by 16 introns . Southern analysis indicates that OsiEZ1 is represented as single copy in the rice genome . Expression studies revealed that the OsiEZ1 transcript level was highest in rice flowers, almost undetectable in developing seeds of 1-2 days post-fertilization but increased significantly in young seeds of 3-5 days post-fertilization . The OsiEZ1 transcript was barely detectable in mature zygotic embryos, but its levels were significantly higher in callus derived from rice scutellum, somatic embryos and young seedlings . The OsiEZ1/GUS recombinant protein was confined to the nucleus in living cells of particle-bombarded onion peels . The expression of OsiEZ1 complemented a set1Delta Saccharomyces cerevisiae mutant that is impaired in telomeric silencing . We suggest that the nuclear-localized OsiEZ1 has a role in regulating various aspects of plant development, and this control is most likely brought about by repressing the activity of downstream regulatory genes.

FEBS Lett, 2003 Sep 25, 552(2-3), 155 - 9
Adhesion forces measured between a calcium blocker drug and its receptor in living cells using atomic force microscope; de Souza Pereira R et al.; The adhesion force between the tip of an atomic force microscope cantilever derivatized with nimodipine (a calcium blocker, from the dihydropyridine class, currently used in clinical medicine for hypertension) and living cells of Saccharomyces cerevisiae (unicellular eukaryotes which portray ultrastructural features characteristic of higher eukaryotic cells) was measured . This methodology allowed us to locate (and visualize) pores on the cell surface which may be responsible for calcium transportation in the living cells . The interaction of the cantilever derivatized with the calcium blocker and a pore, which can be a calcium channel, is more intense than a non-derivatized cantilever and the pore . Outside the pore (on the rest of cell surface), a derivatized or a non-derivatized cantilever has the same pattern of adhesion force . The information obtained with this method is very important for the design of new, more potent and less toxic drugs for pharmacological use.

Mol Cell, 2003 Sep, 12(3), 783 - 90
BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases; Geyer R et al.; Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases . Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members . We show here that S . pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome . The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction . Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner . Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1 . We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes.

Mol Cell, 2003 Sep, 12(3), 699 - 709
A minimal RNA polymerase III transcription system from human cells reveals positive and negative regulatory roles for CK2; Hu P et al.; In higher eukaryotes, RNA polymerase (pol) III is known to use different transcription factors to recognize three basic types of promoters, but in no case have these transcription factors been completely defined . We show that a highly purified pol III complex combined with the recombinant transcription factors SNAP(c), TBP, Brf2, and Bdp1 directs multiple rounds of transcription initiation and termination from the human U6 promoter . The pol III complex contains traces of CK2, and CK2 associates with the U6 promoter region in vivo . Transcription requires CK2 phosphorylation of the pol III complex . In contrast, CK2 phosphorylation of TBP, Brf2, and Bdp1 combined is inhibitory . The results define a minimum core machinery, the ultimate target of regulatory mechanisms, capable of directing all steps of the transcription process-initiation, elongation, and termination-by a metazoan RNA polymerase, and suggest positive and negative regulatory roles for CK2 in transcription by pol III.

Mol Cell, 2003 Sep, 12(3), 663 - 73
Cytoskeletal activation of a checkpoint kinase; Hanrahan J et al.; The assembly of cytoskeletal structures is coupled to other cellular processes . We have studied the molecular mechanism by which assembly of the yeast septin cytoskeleton is monitored and coordinated with cell cycle progression by analyzing a key regulatory protein kinase, Hsl1, that becomes activated only when the septin cytoskeleton is properly assembled . We first identified a regulatory region of Hsl1 that physically associates with the kinase domain and found that it performs an autoinhibitory function both in vivo and in vitro . Several septin binding domains lie near and overlap the inhibitory domain; these are important for Hsl1 function, and binding of two septins, Cdc11 and Cdc12, relieves the autoinhibition imposed by the kinase inhibitory domain in vitro . Our results suggest that binding to multiple septins activates Hsl1 kinase activity, thereby promoting cell cycle progression . The high conservation of Hsl1 indicates that similar mechanisms may monitor cytoskeletal organization in other eukaryotes.

Mol Cell, 2003 Sep, 12(3), 615 - 25
Conserved structural motifs in intracellular trafficking pathways: structure of the gammaCOP appendage domain; Hoffman GR et al.; The formation of coated vesicles is a fundamental step in many intracellular trafficking pathways . COPI and clathrin represent two important and distinct sets of vesicle coating machinery, involved primarily in mediating intra-Golgi and endocytic transport, respectively . Here we identify an important functional region at the carboxyl terminus of the gamma subunit of the COPI complex (gammaCOP) and describe the X-ray crystal structure of this domain at 2.3 A resolution . This domain of gammaCOP exhibits unexpected structural similarity to the carboxyl-terminal appendage domains of the alpha and beta subunits of the AP2 adaptor proteins, integral components of clathrin-coated vesicles . The remarkable structural conservation exhibited by the gammaCOP appendage domain, coupled with functional data and primary sequence analysis, supports a model of COPI function with significant structural and mechanistic parallels to vesicular transport by the clathrin/AP2 system.

Plant Physiol, 2003 Oct, 133(2), 630 - 41 Epub 2003 Oct 02.
Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree; Sakr S et al.; In perennial plants, freeze-thaw cycles during the winter months can induce the formation of air bubbles in xylem vessels, leading to changes in their hydraulic conductivity . Refilling of embolized xylem vessels requires an osmotic force that is created by the accumulation of soluble sugars in the vessels . Low water potential leads to water movement from the parenchyma cells into the xylem vessels . The water flux gives rise to a positive pressure essential for the recovery of xylem hydraulic conductivity . We investigated the possible role of plasma membrane aquaporins in winter embolism recovery in walnut (Juglans regia) . First, we established that xylem parenchyma starch is converted to sucrose in the winter months . Then, from a xylem-derived cDNA library, we isolated two PIP2 aquaporin genes (JrPIP2,1 and JrPIP2,2) that encode nearly identical proteins . The water channel activity of the JrPIP2,1 protein was demonstrated by its expression in Xenopus laevis oocytes . The expression of the two PIP2 isoforms was investigated throughout the autumn-winter period . In the winter period, high levels of PIP2 mRNA and corresponding protein occurred simultaneously with the rise in sucrose . Furthermore, immunolocalization studies in the winter period show that PIP2 aquaporins were mainly localized in vessel-associated cells, which play a major role in controlling solute flux between parenchyma cells and xylem vessels . Taken together, our data suggest that PIP2 aquaporins could play a role in water transport between xylem parenchyma cells and embolized vessels.

J Biol Chem, 2003 Dec 12, 278(50), 50158 - 62 Epub 2003 Oct 02.
Tup1-Ssn6 interacts with multiple class I histone deacetylases in vivo; Davie JK et al.; The Tup1-Ssn6 corepressor complex in Saccharomyces cerevisiae represses the transcription of a diverse set of genes . Chromatin is an important component of Tup1-Ssn6-mediated repression . Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions . Here, we describe physical interactions of the corepressor complex with the class I HDACs Rpd3, Hos2, and Hos1 . In contrast, no in vivo interaction was observed between Tup-Ssn6 and Hda1, a class II HDAC . We demonstrate that Rpd3 interacts with both Tup1 and Ssn6 . Rpd3 and Hos2 interact with Ssn6 independently of Tup1 via distinct tetratricopeptide domains within Ssn6, suggesting that these two HDACs may contact the corepressor at the same time.

Genome Res, 2003 Oct, 13(10), 2242 - 51
Two distinct modes of microsatellite mutation processes: evidence from the complete genomic sequences of nine species; Dieringer D et al.; We surveyed microsatellite distribution in 10 completely sequenced genomes . Using a permutation-based statistic, we assessed for all 10 genomes whether the microsatellite distribution significantly differed from expectations . Consistent with previous reports, we observed a highly significant excess of long microsatellites . Focusing on short microsatellites containing only a few repeat units, we demonstrate that this repeat class is significantly underrepresented in most genomes . This pattern was observed across different repeat types . Computer simulations indicated that neither base substitutions nor a combination of length-dependent slippage and base substitutions could explain the observed pattern of microsatellite distribution . When we introduced one additional mutation process, a length-independent slippage (indel slippage) operating at repeats with few repetitions, our computer simulations captured the observed pattern of microsatellite distribution.

Genome Res, 2003 Oct, 13(10), 2229 - 35
Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution; Krylov DM et al.; Lineage-specific gene loss, to a large extent, accounts for the differences in gene repertoires between genomes, particularly among eukaryotes . We derived a parsimonious scenario of gene losses for eukaryotic orthologous groups (KOGs) from seven complete eukaryotic genomes . The scenario involves substantial gene loss in fungi, nematodes, and insects . Based on this evolutionary scenario and estimates of the divergence times between major eukaryotic phyla, we introduce a numerical measure, the propensity for gene loss (PGL) . We explore the connection among the propensity of a gene to be lost in evolution (PGL value), protein sequence divergence, the effect of gene knockout on fitness, the number of protein-protein interactions, and expression level for the genes in KOGs . Significant correlations between PGL and each of these variables were detected . Genes that have a lower propensity to be lost in eukaryotic evolution accumulate fewer substitutions in their protein sequences and tend to be essential for the organism viability, tend to be highly expressed, and have many interaction partners . The dependence between PGL and gene dispensability and interactivity is much stronger than that for sequence evolution rate . Thus, propensity of a gene to be lost during evolution seems to be a direct reflection of its biological importance.

BMC Evol Biol . 2003 Oct 02;3(1):21.
Apparent dependence of protein evolutionary rate on number of interactions is linked to biases in protein-protein interactions data sets; Bloom JD et al.; BACKGROUND: Several studies have suggested that proteins that interact with more partners evolve more slowly . The strength and validity of this association has been called into question . Here we investigate how biases in high-throughput protein-protein interaction studies could lead to a spurious correlation . RESULTS: We examined the correlation between evolutionary rate and the number of protein-protein interactions for sets of interactions determined by seven different high-throughput methods in Saccharomyces cerevisiae . Some methods have been shown to be biased towards counting more interactions for abundant proteins, a fact that could be important since abundant proteins are known to evolve more slowly . We show that the apparent tendency for interactive proteins to evolve more slowly varies directly with the bias towards counting more interactions for abundant proteins . Interactions studies with no bias show no correlation between evolutionary rate and the number of interactions, and the one study biased towards counting fewer interactions for abundant proteins actually suggests that interactive proteins evolve more rapidly . In all cases, controlling for protein abundance significantly decreases the observed correlation between interactions and evolutionary rate . Finally, we disprove the hypothesis that small data set size accounts for the failure of some interactions studies to show a correlation between evolutionary rate and the number of interactions . CONCLUSIONS: The only correlation supported by a careful analysis of the data is between evolutionary rate and protein abundance . The reported correlation between evolutionary rate and protein-protein interactions cannot be separated from the biases of some protein-protein interactions studies to count more interactions for abundant proteins.

Phys Rev Lett . 2003 Sep 26;91(13):138701 . Epub 2003 Sep 26.
Preferential attachment in the protein network evolution; Eisenberg E et al.; The Saccharomyces cerevisiae protein-protein interaction map, as well as many natural and man-made networks, shares the scale-free topology . The preferential attachment model was suggested as a generic network evolution model that yields this universal topology . However, it is not clear that the model assumptions hold for the protein interaction network . Using a cross-genome comparison, we show that (a) the older a protein, the better connected it is, and (b) the number of interactions a protein gains during its evolution is proportional to its connectivity . Therefore, preferential attachment governs the protein network evolution . Evolutionary mechanisms leading to such preference and some implications are discussed.

Proc Natl Acad Sci U S A, 2003 Oct 14, 100(21), 12081 - 6 Epub 2003 Oct 01.
DcpS can act in the 5'-3' mRNA decay pathway in addition to the 3'-5' pathway; van Dijk E et al.; Eukaryotic mRNA degradation proceeds through two main pathways, both involving mRNA cap breakdown . In the 3'-5' mRNA decay pathway, mRNA body degradation generates free m7GpppN that is hydrolyzed by DcpS generating m7GMP . In the 5'-3' pathway, the recently identified human Dcp2 decapping enzyme cleaves the cap of deadenylated mRNAs to produce m7GDP and 5'-phosphorylated mRNA . We investigated mRNA decay in human cell extracts by using a new assay for decapping . We observed that 5'-phosphorylated intermediates resulting from decapping appear after incubation of a substrate RNA in human cell extracts, indicating the presence of an active 5'-3' mRNA decay pathway . Surprisingly, however, the cognate m7GDP product was not detected, whereas abundant amounts of m7GMP were generated . Additional experiments revealed that m7GDP is, unexpectedly, efficiently converted to m7GMP in extracts from various organisms . The factor necessary and sufficient for this reaction was identified as DcpS in both yeast and human . m7GMP is thus a general, pathway-independent, by-product of eukaryotic mRNA decay . m7GDP breakdown should prevent misincorporation of methylated nucleotides in nucleic acids and could generate a unique indicator allowing the cell to monitor mRNA decay.

Sci Aging Knowledge Environ . 2003 Oct 01;2003(39):PE27.
Ceramide, stress, and a "LAG" in aging; Obeid LM et al.; Recent studies have implicated the longevity assurance gene LAG1 in ceramide synthesis . In light of a role for ceramide in yeast and mammalian stress responses and mammalian cellular senescence, important connections are emerging between ceramide and organismal aging . In this Perspective, we examine the evidence for these connections in yeast, Drosophila, and mammals, and speculate on their implications.

J Biol Chem, 2003 Dec 12, 278(50), 50732 - 43 Epub 2003 Sep 30.
Permease recycling and ubiquitination status reveal a particular role for Bro1 in the multivesicular body pathway; Nikko E et al.; Ubiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation . We previously identified Bro1 as a protein essential to this down-regulation . In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis . Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles . Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi . This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function.

J Biol Chem, 2003 Dec 19, 278(51), 50985 - 98 Epub 2003 Sep 30.
Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases; Jackson MD et al.; Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose . Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span . Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established . Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose . Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation) . The cellular implications are discussed . A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation . A Bronsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity . NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer . We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose . The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.

Genes Dev, 2003 Oct 1, 17(19), 2421 - 35
KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C . elegans; Desai A et al.; Segregation of the replicated genome during cell division requires kinetochores, mechanochemical organelles that assemble on mitotic chromosomes to connect them to spindle microtubules . CENP-A, a histone H3 variant, and CENP-C, a conserved structural protein, form the DNA-proximal foundation for kinetochore assembly . Using RNA interference-based genomics in Caenorhabditis elegans, we identified KNL-1, a novel kinetochore protein whose depletion, like that of CeCENP-A or CeCENP-C, leads to a "kinetochore-null" phenotype . KNL-1 is downstream of CeCENP-A and CeCENP-C in a linear assembly hierarchy . In embryonic extracts, KNL-1 exhibits substoichiometric interactions with CeCENP-C and forms a near-stoichiometric complex with CeNDC-80 and HIM-10, the C . elegans homologs of Ndc80p/HEC1p and Nuf2p-two widely conserved outer kinetochore components . However, CeNDC-80 and HIM-10 are not functionally equivalent to KNL-1 because their inhibition, although preventing formation of a mechanically stable kinetochore-microtubule interface and causing chromosome missegregation, does not result in a kinetochore-null phenotype . The greater functional importance of KNL-1 may be due to its requirement for targeting multiple components of the outer kinetochore, including CeNDC-80 and HIM-10 . Thus, KNL-1 plays a central role in translating the initiation of kinetochore assembly by CeCENP-A and CeCENP-C into the formation of a functional microtubule-binding interface.

Genes Dev, 2003 Oct 1, 17(19), 2396 - 405
mSin3-associated protein, mSds3, is essential for pericentric heterochromatin formation and chromosome segregation in mammalian cells; David G et al.; The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division . In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation . However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation . Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis . mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes . Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes . On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells.

Arch Biochem Biophys, 2003 Oct 15, 418(2), 151 - 60
5alpha-reduced C21 steroids are substrates for human cytochrome P450c17; Gupta MK et al.; The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate . We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities . hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity . hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity . Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold . Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone . We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17 . The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.

Biochem Biophys Res Commun, 2003 Oct 17, 310(2), 312 - 7
Expression of histone acetyltransferases was down-regulated in poly(ADP-ribose) polymerase-1-deficient murine cells; Ota K et al.; NF-kappaB-dependent, as well as human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR)-dependent, reporter gene expression was significantly impaired in cells derived from poly(ADP-ribose) polymerase-1 (PARP-1)-knockout (PARP-1 -/-) mice . In addition, the level of protein acetylation was markedly lower in PARP-1 -/- cells than control (PARP-1 +/+) cells . Surprisingly, the expression levels of histone acetyltransferases (HATs), p300, cAMP response element-binding protein-binding protein (CBP), and p300/CBP-associated factor (PCAF), were significantly reduced in PARP-1 -/- cells, as compared with PARP-1 +/+ cells . These results suggest that PARP-1 is required for the proper expression of particular HATs . Since p300 and CBP are coactivators of NF-kappaB, we propose here that PARP-1 participates in NF-kappaB-dependent transcription by means of maintaining the expression of HATs.

Curr Biol, 2003 Sep 30, 13(19), 1740 - 5
Cell cycle-regulated transcription through the FHA domain of Fkh2p and the coactivator Ndd1p; Darieva Z et al.; Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle . The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ({4-7}; see {8-10}for reviews) . Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression . However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown . Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p . Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p . Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity . Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex.

Curr Biol, 2003 Sep 30, 13(19), 1669 - 78
Mathematical modeling suggests cooperative interactions between a disordered polyvalent ligand and a single receptor site; Klein P et al.; BACKGROUND: The CDK inhibitor Sic1 must be phosphorylated on at least six sites in order to allow its recognition by the SCF ubiquitin ligase subunit Cdc4 . However, because Cdc4 appears to have only a single phospho-epitope binding site, the apparent cooperative dependence on the number of phosphorylation sites in Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity . RESULTS: We develop a general kinetic model, which predicts an unexpected multiplicative increase in affinity as a function of ligand sites . This effect, termed allovalency, derives from a high local concentration of interaction sites moving independently of each other . Modeling of this interaction by a first exit time approach indicates that the probability of ligand rebinding increases exponentially with the number of sites . This type of interaction is relatively immune to loss of any one site and may be easily tuned to any given threshold by adjusting the properties of individual sites . CONCLUSIONS: The allovalency model suggests that a previously undescribed mechanism may underlie certain cooperative interactions . The widespread occurrence of flexible polyvalent ligands in biological systems suggests that this principle may be broadly applicable.

J Med Chem, 2003 Oct 9, 46(21), 4609 - 24
N-hydroxy-3-phenyl-2-propenamides as novel inhibitors of human histone deacetylase with in vivo antitumor activity: discovery of (2E)-N-hydroxy-3-{4-{{(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl}amino}methyl}phenyl}-2-propenamide (NVP-LAQ824); Remiszewski SW et al.; A series of N-hydroxy-3-phenyl-2-propenamides were prepared as novel inhibitors of human histone deacetylase (HDAC) . These compounds were potent enzyme inhibitors, having IC(50)s < 400 nM in a partially purified enzyme assay . However, potency in cell growth inhibition assays ranged over 2 orders of magnitude in two human carcinoma cell lines . Selected compounds having cellular IC(50) < 750 nM were tested for maximum tolerated dose (MTD) and for efficacy in the HCT116 human colon tumor xenograft assay . Four compounds having an MTD > or = 100 mg/kg were selected for dose-response studies in the HCT116 xenograft model . One compound, 9 (NVP-LAQ824), had significant dose-related activity in the HCT116 colon and A549 lung tumor models, high MTD, and low gross toxicity . On the basis, in part, of these properties, 9 has entered human clinical trials in 2002.

Life Sci, 2003 Oct 24, 73(23), 2991 - 3004
Direct and biochemical interaction between dopamine D3 receptor and elongation factor-1Bbetagamma; Cho DI et al.; Novel signaling components of dopamine D3 receptor (D3R) were searched using yeast two-hybrid system, and the gamma subunit of elongation Factor-1B (eEF1Bgamma) was found to interact with D3R . This interaction was observed specifically between eEF1Bgamma and D3R but not with D2R or D4R . Immunocytochemical studies showed that D3R and eEF1Bgamma form clusters on the plasma membrane and their co-localization was evident in these clusters . The beta subunit of eEF1B (eEF1Bbeta), which forms a tight complex with eEF1Bgamma, was phosphorylated on serine residues in response to the stimulation of D3R . Phosphorylation of eEF1Bbeta was insensitive to pertussis toxin or wortmannin, however, stimulation of cellular protein kinase C (PKC) directly phosphorylated eEF1Bbeta and depletion of PKC abolished D3R-mediated phosphorylation of eEF1Bbeta . These results suggest the involvement of PKC, but not Gi/o proteins or phosphatidylinositol 3-kinase, in D3R-mediated phosphorylation of eEF1Bbeta . Stimulation of D3R did not activate PKC, but the activation of PKC resulted in the phosphorylation of D3R . These results show that PKC has a permissive role for the D3R-mediated phosphorylation of eEF1Bbeta, and suggest that PKC could modulate the mutual interaction between two protein by phosphorylating both D3R and eEF1Bbeta . Therefore, the cellular PKC level would be important for the D3R-mediated modulation of eEF1B, and for their cellular regulations such as protein synthesis or cellular proliferation.

Curr Opin Cell Biol, 2003 Oct, 15(5), 590 - 7
Roles of Rho-family GTPases in cell polarisation and directional migration; Fukata M et al.; Polarised cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing . Rho-family GTPases, including Cdc42, Rac1 and RhoA, play a central role in establishing cell polarisation, which requires asymmetric and ordered distribution of the signalling molecules and the cytoskeleton . Recent advances reveal that Rho GTPases, together with phosphatidylinositol 3-kinase, contribute to asymmetric phosphatidylinositol 3,4,5-trisphosphate distribution via a positive-feedback loop . Phosphatidylinositol 3,4,5-trisphosphate thereby activates the signalling cascades to the cytoskeleton as a second messenger . Rho GTPases also capture and stabilise microtubules through their effectors (e.g . IQGAP1, mDia and Par6) near the cell cortex, leading to polarised cell morphology and directional cell migration . Thus, elucidation of the signal transduction cascades from receptors to Rho GTPases and, subsequently, from Rho GTPases to microtubules has begun.

Mol Cancer Res, 2003 Sep, 1(11), 836 - 47
Decreased frequency and highly aberrant spectrum of ultraviolet-induced mutations in the hprt gene of mouse fibroblasts expressing antisense RNA to DNA polymerase zeta; Diaz M et al.; In the budding yeast Saccharomyces cerevisiae, DNA polymerase zeta (pol zeta) is responsible for the great majority of mutations generated during error-prone translesion replication of DNA that contains UV-induced lesions . The catalytic subunit of pol zeta is encoded by the Rev3 gene . The orthologue of Rev3 has been cloned from higher eukaryotic cells, including human, but its role in mutagenesis and carcinogenesis remains obscure . Investigation into the cellular function of pol zeta has been hindered by the fact that Rev3 knockout mice do not survive beyond midgestation, and embryonic stem cells used to derive these mice are not genetically stable . We have generated a transgenic mouse that expresses antisense RNA transcripts to mRev3 endogeneous RNA . These mice are viable, have greatly reduced levels of Rev3 transcript, and have reduced levels of B cells and impaired development of high-affinity memory B cells . Here, we report that exposure of fibroblasts derived from these mice to UV resulted in a 4-5-fold reduction in mutant frequency at the hprt locus at every dose examined, and the mutation spectrum was highly aberrant compared with the control cells . In the control cells, 80% of the mutations were transitions and approximately 75% of these arose from photoproducts in the putative leading strand template . Strikingly, in transgenic cells, most of the mutations were transversions and there was a complete loss of strand bias . This mutation spectrum is highly aberrant and is similar to that induced by UV in human xeroderma pigmentosum variant cells, which lack polymerase eta . These data indicate that most UV-induced mutations are dependent on DNA pol zeta, a function that has been conserved from yeast to higher eukaryotic cells . However, in mammalian cells, other DNA polymerase(s) may accomplish error-prone translesion replication and are responsible for residual UV mutagenesis observed in the absence of pol zeta . Further, these data support a central role for DNA polymerase eta in the error-free bypass of UV photoproducts . The antisense Rev3 mice should be a useful model to study mutagenic lesion bypass by pol zeta in mammalian cells and to investigate the role this polymerase plays in carcinogenesis.

Mol Biol Cell, 2003 Oct, 14(10), 4126 - 39 Epub 2003 Jul 11.
The WD-repeats of Net2p interact with Dnm1p and Fis1p to regulate division of mitochondria; Cerveny KL et al.; The Net2, Fis1, and Dnm1 proteins are required for the division of mitochondria in the yeast Saccharomyces cerevisiae . Net2p has an amino-terminal region that contains predicted coiled-coil motifs and a carboxyl-terminal domain composed of WD-40 repeats . We found that the amino-terminal part of Net2p interacts with Fis1p, whereas the carboxyl-terminal region interacts with both Dnm1p and Fis1p . Overproduction of either domain of Net2p in yeast cells poisons mitochondrial fission, and the dominant-negative effect caused by the WD-repeats of Net2p is suppressed by increased levels of Dnm1p . Point mutations in the WD-region of Net2p or in the GTPase region of Dnm1p disrupt the normal Net2p-Dnm1p interaction, causing Net2p to lose its normal punctate distribution . Our results suggest that Dnm1p interacts with the WD-repeats of Net2p and in a GTP-dependent manner recruits Net2p to sites of mitochondrial division . Furthermore, our results indicate that Net2p is required for proper assembly of the mitochondrial fission components to regulate organelle division.

Mol Biol Cell, 2003 Oct, 14(10), 4015 - 27 Epub 2003 Jul 11.
The role of mVps18p in clustering, fusion, and intracellular localization of late endocytic organelles; Poupon V et al.; Delivery of endocytosed macromolecules to mammalian cell lysosomes occurs by direct fusion of late endosomes with lysosomes, resulting in the formation of hybrid organelles from which lysosomes are reformed . The molecular mechanisms of this fusion are analogous to those of homotypic vacuole fusion in Saccharomyces cerevisiae . We report herein the major roles of the mammalian homolog of yeast Vps18p (mVps18p), a member of the homotypic fusion and vacuole protein sorting complex . When overexpressed, mVps18p caused the clustering of late endosomes/lysosomes and the recruitment of other mammalian homologs of the homotypic fusion and vacuole protein sorting complex, plus Rab7-interacting lysosomal protein . The clusters were surrounded by components of the actin cytoskeleton, including actin, ezrin, and specific unconventional myosins . Overexpression of mVps18p also overcame the effect of wortmannin treatment, which inhibits membrane traffic out of late endocytic organelles and causes their swelling . Reduction of mVps18p by RNA interference caused lysosomes to disperse away from their juxtanuclear location . Thus, mVps18p plays a critical role in endosome/lysosome tethering, fusion, intracellular localization and in the reformation of lysosomes from hybrid organelles.

Mol Cell Biol, 2003 Oct, 23(20), 7339 - 49
Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus; Wang Q et al.; Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway . Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro . Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor . A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex . The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure . U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected . Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking . Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.

EMBO J, 2003 Oct 1, 22(19), 5251 - 9
The mechanisms of {URE3} prion elimination demonstrate that large aggregates of Ure2p are dead-end products; Ripaud L et al.; The yeast prion {URE3} is a self-propagating inactive form (the propagon) of the Ure2 protein . Ure2p is composed of two domains: residues 1-93--the prion-forming domain (PFD)--and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression . Guanidine hydrochloride, and the overproduction of Ure2p 1-65 or Ure2-GFP have been shown to induce the elimination of {URE3} . We demonstrate here, two different curing mechanisms: the inhibition of {URE3} replication by guanidine hydrochloride and its destruction by Ure2p aggregation . Such aggregation is observed if PFD or Ure2-GFP are overproduced and in heterozygous URE2/URE2-GFP, {URE3} diploids . We found that the GFP foci associated with the presence of the prion were dead-end products, the propagons remaining soluble . Surprisingly, {URE3} propagated via the Ure2-GFP fusion protein alone is resistant to these two curing mechanisms and cannot promote the formation of foci . The relationship between aggregation, prion and Hsp104 gives rise to a model in which the propagon is in equilibrium with larger aggregates and functional protein.

EMBO J, 2003 Oct 1, 22(19), 5230 - 40
TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes; Siegers K et al.; The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood . Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state . TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p . In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin . GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability . These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.

EMBO J, 2003 Oct 1, 22(19), 5163 - 74
Local action of the chromatin assembly factor CAF-1 at sites of nucleotide excision repair in vivo; Green CM et al.; DNA damage and its repair can cause both local and global rearrangements of chromatin structure . In each case, the epigenetic information contained within this structure must be maintained . Using the recently developed method for the localized UV irradiation of cells, we analysed responses that occur locally to damage sites and global events triggered by local damage recognition . We thus demonstrate that, within a single cell, the recruitment of chromatin assembly factor 1 (CAF-1) to UV-induced DNA damage is a strictly local phenomenon, restricted to damage sites . Concomitantly, proliferating cell nuclear antigen (PCNA) locates to the same sites . This localized recruitment suggests that CAF-1 participates directly in chromatin structural rearrangements that occur in the vicinity of the damage . Use of nucleotide excision repair (NER)-deficient cells shows that the NER pathway--specifically dual incision--is required for recruitment of CAF-1 and PCNA . This in vivo demonstration of the local role of CAF-1, depending directly on NER, supports the hypothesis that CAF-1 ensures the maintenance of epigenetic information by acting locally at repair sites.

EMBO J, 2003 Oct 1, 22(19), 5147 - 53
Modulation of transcription factor function by an amino acid: activation of Put3p by proline; Sellick CA et al.; Saccharomyces cerevisiae are able to convert proline to glutamate so that it may be used as a source of nitrogen . Here, we show that the activator of the proline utilization genes, Put3p, is transcriptionally inert in the absence of proline but transcriptionally active in its presence . The activation of Put3p requires no additional yeast proteins and can occur in the presence of certain proline analogues: an unmodified pyrrolidine ring is able to activate Put3p as efficiently as proline itself . In addition, we show that a direct interaction occurs between Put3p and proline . These data, which represent direct control of transcriptional activator function by a metabolite, are discussed in terms of the regulation of proline-specific genes in yeast and as a general mechanism of the control of transcription.

EMBO J, 2003 Oct 1, 22(19), 5137 - 46
Zinc fingers can act as Zn2+ sensors to regulate transcriptional activation domain function; Bird AJ et al.; The yeast Zap1 transcription factor controls the expression of genes involved in zinc accumulation and storage . Zap1 is active in zinc-limited cells and repressed in replete cells . Zap1 has two activation domains, AD1 and AD2, which are both regulated by zinc . AD2 function was mapped to a region containing two Cys2His2 zinc fingers, ZF1 and ZF2, that are not involved in DNA binding . More detailed mapping placed AD2 almost precisely within the endpoints of ZF2, suggesting a role for these fingers in regulating activation domain function . Consistent with this hypothesis, ZF1 and ZF2 bound zinc in vitro but less stably than did zinc fingers involved in DNA binding . Furthermore, mutations predicted to disrupt zinc binding to ZF1 and/or ZF2 rendered AD2 constitutively active . Our results also indicate that the repressed form of AD2 requires an intramolecular interaction between ZF1 and ZF2 . These studies suggest that these zinc fingers play an unprecedented role as zinc sensors to control activation domain function.

EMBO J, 2003 Oct 1, 22(19), 5115 - 24
A common site on TBP for transcription by RNA polymerases II and III; Schroder O et al.; The TATA-binding protein (TBP) is involved in all nuclear transcription . We show that a common site on TBP is used for transcription initiation complex formation by RNA polymerases (pols) II and III . TBP, the transcription factor IIB (TFIIB)-related factor Brf1 and the pol III-specific factor Bdp1 constitute TFIIIB . A photochemical cross-linking approach was used to survey a collection of human TBP surface residue mutants for their ability to form TFIIIB-DNA complexes reliant on only the TFIIB-related part of Brf1 . Mutations impairing complex formation and transcription were identified and mapped on the surface of TBP . The most severe effects were observed for mutations in the C-terminal stirrup of TBP, which is the principal site of interaction between TBP and TFIIB . Structural modeling of the Brf1-TBP complex and comparison with its TFIIB-TBP analog further rationalizes the close resemblance of the TBP interaction with the N-proximal part of Brf1 and TFIIB, and establishes the conserved usage of a TBP surface in pol II and pol III transcription for a conserved function in the initiation of transcription.

EMBO J, 2003 Oct 1, 22(19), 4945 - 56
Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria; Mokranjac D et al.; The TIM23 translocase mediates the deltaPsi- and ATP-dependent import of proteins into mitochondria . We identified Tim14 as a novel component of the TIM23 translocase . Tim14 is an integral protein of the inner membrane with a typical J-domain exposed to the matrix space . TIM14 genes are present in the genomes of virtually all eukaryotes . In yeast, Tim14 is essential for viability . Mitochondria from cells depleted of Tim14 are deficient in the import of proteins mediated by the TIM23 complex . In particular, import of proteins that require the action of mtHsp70 is affected . Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner . A mutation in the HPD motif of the J-domain of Tim14 is lethal . Thus, Tim14 is a constituent of the mitochondrial import motor . We propose a model in which Tim14 is required for the activation of mtHsp70 and enables this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix.

EMBO J, 2003 Oct 1, 22(19), 4888 - 97
The PB1 domain and the PC motif-containing region are structurally similar protein binding modules; Yoshinaga S et al.; The PC motif is evolutionarily conserved together with the PB1 domain, a binding partner of the PC motif-containing protein . For interaction with the PB1 domain, the PC motif-containing region (PCCR) comprising the PC motif and its flanking regions is required . Because the PB1 domain and the PCCR are novel binding modules found in a variety of signaling proteins, their structural and functional characterization is crucial . Bem1p and Cdc24p interact through the PB1-PCCR interaction and regulate cell polarization in budding yeast . Here, we determined a tertiary structure of the PCCR of Cdc24p by NMR . The tertiary structure of the PCCR is similar to that of the PB1 domain of Bem1p, which is classified into a ubiquitin fold . The PC motif portion takes a compact betabetaalpha-fold, presented on the ubiquitin scaffold . Mutational studies indicate that the PB1-PCCR interaction is mainly electrostatic . Based on the structural information, we group the PB1 domains and the PCCRs into a novel family, named the PB1 family . Thus, the PB1 family proteins form a specific dimer with each other.

J Biol Chem, 2003 Dec 12, 278(50), 50428 - 34 Epub 2003 Sep 29.
Functional and structural basis of carnitine palmitoyltransferase 1A deficiency; Gobin S et al.; Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation . Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia . We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E . Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations . To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase . This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant) . Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class . They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex . By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category . They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms . This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.

Virology, 2003 Sep 15, 314(1), 460 - 7
Roles of host cell factors in circularization of retroviral dna; Kilzer JM et al.; Early during retroviral infection, a fraction of the linear reverse-transcribed viral DNA genomes become circularized by cellular enzymes, thereby inactivating the genomes for further replication . Prominent circular DNA forms include 2-long-terminal repeat (LTR) circles, made by DNA end joining, and 1-LTR circles, produced in part by homologous recombination . These reactions provide a convenient paradigm for analyzing the cellular machinery involved in DNA end joining in vertebrate cells . In previous studies, we found that inactivating components of the nonhomologous DNA end-joining (NHEJ) pathway--specifically Ku, ligase 4, or XRCC4--blocked formation of 2-LTR circles . Here we report that inactivating another NHEJ component, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), had at most modest effects on 2-LTR circle formation, providing informative parallels with other end-joining reactions . We also analyzed cells mutant in components of the RAD50/MRE11/NBS1 nuclease and found a decrease in the relative amount of 1-LTR circles, opposite to the effects of NHEJ mutants . In MRE11-mutant cells, a MRE11 gene mutant in the nuclease catalytic site failed to restore 1-LTR circle formation, supporting a model for the role of MRE11 in 1-LTR circle formation . None of the cellular mutations showed a strong effect on normal integration, consistent with the idea that the cellular pathways leading to circularization are not involved in productive integration.

J Mol Biol, 2003 Oct 10, 333(1), 201 - 9
Glucose and type 2A protein phosphatase regulate the interaction between catalytic and regulatory subunits of AMP-activated protein kinase; Gimeno-Alcaniz JV et al.; We have expressed in yeast the different subunits of AMP-activated protein kinase (AMPK) and, by using the two-hybrid system, we have found a glucose-regulated interaction between alpha 2 catalytic and gamma 1 regulatory subunits . This regulation was not affected by known regulators of the corresponding yeast orthologue, the SNF1 complex, such as Reg1 or Hxk2, but it was affected by deletion of regulatory subunits of yeast type 2A protein phosphatase (PP2A) complex . We have also found that Tpd3 and PR65 alpha, the corresponding yeast and mammalian A subunits of PP2A, interacted with AMPK alpha 2 both in yeast and mammals, respectively . This interaction occurred only through the regulatory domain of this subunit . These results suggested a direct involvement of PP2A complex in regulating the interaction between AMPK alpha 2 and gamma 1 in a glucose-dependent manner.

J Mol Biol, 2003 Oct 10, 333(1), 187 - 99
Tuning the heterogeneous early folding dynamics of phosphoglycerate kinase; Osvath S et al.; We recently reported stretched kinetics during the formation of a collapsed, long-lived intermediate state of the large two-domain enzyme phosphoglycerate kinase (PGK) . It was postulated that intrinsic roughness of the energy landscape on the way downhill to the intermediate causes the lack of a single time-scale . Here, we investigate several alternative explanations for stretched refolding dynamics in more detail: tyrosine fluorescence, multiple tryptophan probes, and rate differences between independently folding domains . To this end, we systematically simplify PGK in several steps from the full protein with two tryptophan residues and all tyrosine residues probed, to a single domain with only one tryptophan residue and no tyrosine residue probed . The kinetics in the 10 micros to 10 ms range are revealed by laser-induced temperature-jump relaxation experiments . The isolated N-terminal domain forms an intermediate by nearly single-exponential kinetics, but the isolated C-terminal domain shows strongly non-exponential kinetics . Thus, domain interaction and a cis-proline residue between the two domains are ruled out as the sole contributors to heterogeneity during the earliest folding dynamics of the C-terminal domain . We apply two limiting models for the roughness of the energy landscape . A sequential three-state model lumps all the roughness into a single trap . The "strange kinetics" model with logarithmic oscillations developed by Klafter and co-workers distributes the roughness over a larger number of states . Both models explain our data about equally well, but the coincidental values of rate constants in all of our double-exponential fits, and the absence of a spectroscopic signature distinct from the endpoints of the folding process favors more roughness than can be explained by just a single trap.

J Mol Biol, 2003 Oct 10, 333(1), 75 - 85
Correlating protein-DNA and protein-protein interaction networks; Manke T et al.; Here, we search protein-DNA binding data for prevalent pairs and higher-order tuples of co-occurring transcription factors (TF) in Saccharomyces cerevisiae . While the identification of such modules is dependent on uncertainties of genome-wide data sets, we find several biologically meaningful examples, which allow putative annotation of yet unclassified genes . For the frequently occurring transcriptional module Mcm1-Fkh2-Ndd1, we identified several new target genes involved in cell-cycle control and filament formation . Using large-scale protein interaction data, we demonstrate a significant correlation between co-occurrence of TF binding sites and the vicinity in the protein interaction network . In particular we find that directly interacting transcription factors and those which are members of a protein complex are more likely to occur together as putative DNA-binding modules.

J Microsc, 2003 Oct, 212(Pt 1), 53 - 61
Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples; Giddings TH; Specimen preparation methods based on high-pressure freezing and freeze-substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy . However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast . By contrast, some efforts to increase membrane staining may lead to excessively granular staining . No one freeze-substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis . However, one or more of the following protocols, perhaps with minor modifications, should yield satisfactory results in most cases . Freeze-substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high-resolution structural studies . Membranes are typically lightly stained but very well defined . Initial freeze-substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes . Finally, initial trials indicate that freeze-substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes . The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze-substitution reagent.

J Microsc, 2003 Oct, 212(Pt 1), 26 - 33
An oscillating cryo-knife reduces cutting-induced deformation of vitreous ultrathin sections; Al-Amoudi A et al.; A new oscillating cryo-knife for producing uncompressed vitreous sections is introduced . The knife is a modified cryo diamond knife that is driven by a piezo translator . Optimal setting for the oscillation was found to be in the inaudible frequency range of 20-25 kHz . Yeast cells and polystyrene spheres were used as model systems to describe compression in the vitreous sections . We found that compression could be reduced by a factor of about 2 when the knife was oscillating . When the oscillator was turned off, sections were compressed by 40-45% . However, only 15-25% compression was obtained when the knife was oscillating . In some cases completely uncompressed sections of yeast cells were produced . It was also found that the amount of compression depends on the specimen itself and on its embedding medium . With the results shown here, we demonstrate that the oscillating knife can produce high-quality vitreous sections with minimum cutting artefacts.

Biochem J, 2004 Jan 1, 377(Pt 1), 85 - 93
Murine bubblegum orthologue is a microsomal very long-chain acyl-CoA synthetase; Fraisl P et al.; It has been suggested that a gene termed bubblegum (Bgm), encoding an acyl-CoA synthetase, could be involved in the pathogenesis of the inherited neurodegenerative disorder X-ALD (X-linked adrenoleukodystrophy) . The precise function of the ALDP (ALD protein) still remains unclear . Aldp deficiency in mammals and Bgm deficiency in Drosophila led to accumulation of VLCFAs (very long-chain fatty acids) . As a first step towards studying this interaction in wild-type versus Aldp-deficient mice, we analysed the expression pattern of the murine orthologue of the Bgm gene . In contrast with the ubiquitously expressed Ald gene, Bgm expression is restricted to the tissues that are affected by X-ALD such as brain, testis and adrenals . During mouse brain development, Bgm mRNA was first detected by Northern-blot analysis on embryonic day 18 and increased steadily towards adulthood, whereas the highest level of Ald mRNA was found on embryonic day 12 and decreased gradually during differentiation . Protein fractionation and confocal laser imaging of Bgm-green fluorescent protein fusion proteins revealed a microsomal localization that was different from peroxisomes (where Aldp is detected), endoplasmic reticulum and Golgi . Mouse Bgm showed acyl-CoA synthetase activity towards a VLCFA substrate in addition to LCFAs, and this activity was enriched in the microsomal compartment . Speculating that Bgm expression could be regulated by Ald deficiency, we compared the abundance of Bgm mRNA in wild-type and Ald knockout mice but observed no difference . Although mouse Bgm is capable of activating VLCFA, we conclude that a direct interaction between the mouse Bgm and the Aldp seems unlikely.

J Biol Chem, 2003 Dec 19, 278(51), 51937 - 49 Epub 2003 Sep 26.
Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm; Gilchrist D et al.; The yeast karyopherin heterodimer Kap60p.Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS) . The alpha subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm . Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import . We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p.Gsp1p.GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p . Here we dissect the molecular mechanics of their KaRF activity . We show that Cse1p accelerates dissociation of Kap60p.NLS-cargo complexes and Kap60p.Nup2p complexes by increasing the affinity of Kap60p for its AIS motif . In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p.NLS complexes in a vectorial reaction mechanism . Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p.NLS-cargo complexes in the nucleoplasm of yeast . We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex.

J Biol Chem, 2003 Dec 12, 278(50), 49732 - 42 Epub 2003 Sep 26.
Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases; Bernard DG et al.; Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity . However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity . Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e . CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL . Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL . Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor . Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction.

J Biol Chem, 2003 Dec 12, 278(50), 49920 - 8 Epub 2003 Sep 26.
Coupling of the transcriptional regulation of glutathione biosynthesis to the availability of glutathione and methionine via the Met4 and Yap1 transcription factors; Wheeler GL et al.; Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest . Here, we show that the Yap1 and Met4 transcription factors regulate the expression of gamma-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion . Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered . Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response . The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1 . In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion . Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1 . The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation . These results indicate that the Yap1-dependent activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependent mechanism.

Yakugaku Zasshi, 2003 Sep, 123(9), 805 - 9
{Determination of the optical purity of N-nitrosofenfluramine found in the Chinese slimming diet}; Nakadai A et al.; From 2001 to the summer of 2002, more than 800 cases of liver damage were reported in Japan among people taking Chinese diet aids . The Japanese Ministry of Health, Labor and Welfare has recently announced that N-nitrosofenfluramine was the hepatotoxic compound contained in the diet aids based on animal experiments performed by the National Institute of Health Sciences . Although N-nitrosofenfluramine is a derivative of fenfluramine, a previously used antiobesity drug, neither pharmacologic nor toxicologic properties have been reported for N-nitroso fenfluramine . It should be noted that N-nitrosofenfluramine has two optical isomers, although it is not yet known which isomer damages the liver and other organs . The Japanese Ministry of Health, Labor and Welfare has not commented on this point . Pursuing this question, 10 types of Chinese slimming aid samples including those obtained from patients with fulminating hepatitis were analyzed by NMR, GC/MS, and a newly established HPLC method using a chiral separation column . It was found that the N-nitrosofenfluramine in all of the toxic diet aids was the (S)-isomer form . No (R)-isomer was detected . These results strongly suggest that the nitroso-compound in the diets must be prepared from pharmacologically active (S)-fenfluramine (dexfenfluramine) . Thus the pharmacologic and toxicologic properties of each isomer should be investigated.

Melanoma Res, 2003 Oct, 13(5), 503 - 9
Discrimination between gene expression patterns in the invasive margin and the tumour core of malignant melanomas; Roesch A et al.; Genes that determine the invasive capacity of the invasive front of malignant melanomas (MM) have not yet been systematically investigated in vivo . Therefore, we combined laser pressure catapulting (LPC) microdissection with cDNA microarray technology (DermArray, Research Genetics, representing about 5700 genes) to systematically analyse differences in gene expression profiles between the invasive margin and the tumour centre in nine cases of vertical growth phase MM . Signal-to-noise statistical algorithms combined with hierarchical clustering were performed to determine class-separating genes . The gene encoding phosphoenolpyruvate carboxykinase 1 (PEPCK), the Homo sapiens gene similar to Saccharomyces cerevisiae SSM4 (TEB4), the gene encoding ribosomal protein L19, the Homo sapiens gene similar to the Aspergillus nidulans SudD (a suppressor of the bimD6 homologue), the gene encoding the interleukin-3 receptor alpha subunit, the gene encoding the inositol 1,4,5-triphosphate 3-kinase isoenzyme, and three anonymous expressed sequence tags were identified as class-separating genes . These genes significantly discriminate between the invasive front and the tumour centre . Using this set of genes, 15 out of 18 LPC-dissected MM regions could be grouped correctly . We conclude that the candidate genes identified could spark further research on MM progression and may provide novel prognostic parameters.

Cell Cycle, 2003 Nov-Dec, 2(6), 568 - 72
Multiple roles of replication forks in S phase checkpoints: sensors, effectors and targets; Pasero P et al.; There is mounting evidence that replication defects are the major source of spontaneous genomic instability in the cell and that S phase checkpoints are the principle defense against such instability . In Saccharomyces cerevisiae, S phase checkpoints can be provoked by either depletion of dNTPs or DNA damage . In both cases the checkpoint kinases Mec1 and Rad53 act to suppress late origin firing, stabilize slowed or stalled replication forks and prevent S phase progression until conditions are appropriate for the resumption of DNA replication . The present review highlights recent work emphasizing the central importance of replication forks, not just as targets, but also as sensors and primary effectors of checkpoint responses, and identifies the roles played by specific fork-associated factors in these processes.

Science, 2003 Sep 26, 301(5641), 1908 - 11
An age-induced switch to a hyper-recombinational state; McMurray MA et al.; There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear . We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age . As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) occurred . Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock . In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny . Thus, nuclear genomes may be inherently unstable with age.

Science, 2003 Sep 26, 301(5641), 1859 - 60
Cell biology . An age of instability; Sinclair DA; It is well established that as we age our cancer risk increases dramatically . As Sinclair explains in his Perspective, the link between cancer and aging is now solidified by new work in budding yeast (McMurray and Gottschling) . As yeast cells age there is a marked increase in their genetic instability (a hallmark of cancer), which is independent of the mechanism that determines their life-span.

Mol Endocrinol, 2003 Dec, 17(12), 2554 - 65 Epub 2003 Sep 25.
Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation; De Cesare D et al.; Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells . In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm . In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein) . In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity . In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms . Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription . This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation . The interaction is restricted to the activator isoforms of CREM and does not require Ser117 . Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog . CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells . The two proteins also colocalize intracellularly in spermatocyte and spermatid cells . These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.

Bioinformatics, 2003 Sep 22, 19(14), 1787 - 99
CLICK and EXPANDER: a system for clustering and visualizing gene expression data; Sharan R et al.; MOTIVATION: Microarrays have become a central tool in biological research . Their applications range from functional annotation to tissue classification and genetic network inference . A key step in the analysis of gene expression data is the identification of groups of genes that manifest similar expression patterns . This translates to the algorithmic problem of clustering genes based on their expression patterns . RESULTS: We present a novel clustering algorithm, called CLICK, and its applications to gene expression analysis . The algorithm utilizes graph-theoretic and statistical techniques to identify tight groups (kernels) of highly similar elements, which are likely to belong to the same true cluster . Several heuristic procedures are then used to expand the kernels into the full clusters . We report on the application of CLICK to a variety of gene expression data sets . In all those applications it outperformed extant algorithms according to several common figures of merit . We also point out that CLICK can be successfully used for the identification of common regulatory motifs in the upstream regions of co-regulated genes . Furthermore, we demonstrate how CLICK can be used to accurately classify tissue samples into disease types, based on their expression profiles . Finally, we present a new java-based graphical tool, called EXPANDER, for gene expression analysis and visualization, which incorporates CLICK and several other popular clustering algorithms . AVAILABILITY: http://www.cs.tau.ac.il/~rshamir/expander/expander.html

J Nat Prod, 2003 Sep, 66(9), 1221 - 4
New bioactive diterpene polyesters from Euphorbia decipiens; Ahmad VU et al.; A reinvestigation with a modified extraction procedure of Euphorbia decipiens resulted in the isolation and structure elucidation of three new myrsinane-type diterpene esters (1-3) . The structures of compounds 1-3 were elucidated by spectroscopic data interpretation . Compound 1 showed inhibitory activity against prolyl endopeptidase (PEP), whereas compound 2 exhibited DNA-damaging activity in a mutant yeast bioassay.

Nucleic Acids Res Suppl, 2003, (3), 305 - 6
Sequence specific transcription factor, JDP2 interacts with histone and inhibits p300-mediated histone acetylation; Pan J et al.; Jun dimerization protein 2 (JDP2) is a novel member of AP-1 family and acts as a general repressor of a variety of transcription . JDP2 is able to bind to specific sites in target gene such as c-jun by forming homodimer or heterodimers with a Jun/ATF family member to counteract their transcriptional activity . Previously we showed that JDP2 inhibits the retinoic acid (RA) dependent transcription by recruiting a histone deacetylase 3 (HDAC3) complex to the promoter region of the target genes . We present here that JDP2 has an inhibitory activity of acetylation of all core histones mediated by histone acetyltransferase (HAT) both of p300 and PCAF in vitro . The studies of both histone-binding and HAT-inhibitory activity using a variety of recombinant JDP2(s) indicated that JDP2 might target histone itself through the histone-binding domain of JDP2, which is essential but not sufficient for the inhibition of acetylation . Therefore, our data suggested that HAT-inhibitory activity of JDP2 could in part explain the transcriptional repression of several target genes.

Curr Genet, 2003 Oct, 44(1), 19 - 25 Epub 2003 Jul 09.
Repression of transcription by Rgt1 in the absence of glucose requires Std1 and Mth1; Lakshmanan J et al.; In the yeast Saccharomyces cerevisiae, glucose induces expression of the hexose transporter ( HXT) genes by inhibiting the repressor function of the transcription factor Rgt1 . We have previously shown that Rgt1 binds to the HXT gene promoters only in the absence of glucose . In the presence of glucose, Rgt1 becomes phosphorylated and is unable to bind to the HXT promoters and repress their transcription . We report that Rgt1 interacts with Std1 and Mth1 in a yeast two-hybrid assay and co-immunoprecipitates with both proteins in vivo only when glucose is absent . In addition, we demonstrate that repression of HXT gene expression by Rgt1 is abolished in the std1 mth1 double mutant . While Rgt1 is normally phosphorylated only in the presence of high concentrations of glucose, it is constitutively modified in the std1 mth1 double mutant . Based on these data, we conclude that, in the absence of glucose, Rgt1 associates with Std1 and Mth1 to repress HXT gene expression.

Curr Genet, 2003 Dec, 44(4), 173 - 83 Epub 2003 Sep 24.
Clustered-charge to alanine scanning mutagenesis of the Mal63 MAL-activator C-terminal regulatory domain; Danzi SE et al.; The MAL-activator genes of Saccharomyces cerevisiae encode regulatory proteins required for the expression of the structural genes encoding maltose permease and maltase . Residues within the C-terminal region of the Mal63 protein required for negative regulation were previously identified . Evidence suggested that the C-terminal domain is also involved in positive regulatory functions, such as inducer responsiveness and transactivation in the context of a full-length protein . Charged-cluster to alanine scanning mutagenesis of the regulatory domain of MAL63 and the constitutive MAL43-C were undertaken to identify distinct regions within Mal63p involved in positive functions and to define their roles in induction . Mutations that affect the ability to activate transcription in the inducible MAL63 but have no effect in the constitutive MAL43-C define regions that function in induction . Those that affect both the inducible and constitutive alleles define regions involved in activation more generally . Mutations in MAL63 fell into three classes, those that have little or no impact on activity, those that decrease activity, and those that enhance function . Mutations from these classes mapped to distinct regions of the protein, identifying a region of approximately 90 residues (residues 331-423) involved in maltose sensing and an approximately 50-residue region at the extreme C-terminus (residues 420-470) required for activation, such as the formation and/or maintenance of an active state . These studies support a model for MAL-activator function which involves complex protein-protein interactions and overlapping negative and positive regulatory regions.

Biochim Biophys Acta, 2003 Sep 30, 1606(1-3), 153 - 62
A tomato alternative oxidase protein with altered regulatory properties; Holtzapffel RC et al.; We have investigated the expression and regulatory properties of the two alternative oxidase (Aox) proteins that are expressed in tomato (Lycopersicon esculentum L . Mill cv . Sweetie) after storage of green fruit at 4 degrees C . Four Aox genes were identified in the tomato genome, of which two (LeAox1a and LeAox1b) were demonstrated to be expressed in cold-treated fruit . The activity and regulatory properties of LeAox1a and LeAox1b were assayed after expression of each protein in yeast cells (Saccharomyces cerevisiae), proving that each is an active Aox protein . The LeAox1b protein was shown to have altered regulatory properties due to the substitution of a Ser for the highly conserved Cys(I) residue . LeAox1b could not form inactive disulfide-linked dimers and was activated by succinate instead of pyruvate . This is the first example of a dicot species expressing a natural Cys(I)/Ser isoform . The implications of the existence and expression of such Aox isoforms is discussed in the light of the hypothesised role for Aox in plant metabolism.

J Biol Chem, 2003 Dec 5, 278(49), 48524 - 8 Epub 2003 Sep 23.
Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis; Harvey KJ et al.; Using a plasmid competition assay, we have measured the stability of origin recognition complex (ORC) associated with sperm chromatin under physiological conditions . Under conditions in which pre-RCs are formed, both ORC and CDC6 dissociate from sperm chromatin with a relatively fast t(1/2) of 15 min . ORC dissociation from chromatin is regulated through the recruitment of CDC6 and MCM proteins as well as ATP hydrolysis . The t(1/2) for ORC alone in the absence of Cdc6 is 40 min and increases 8-fold to >2 h when Cdc6 is present . Strikingly, the presence of a non-hydrolyzable ATP derivative, ATPgammaS, not only increases both ORC and CDC6 t(1/2) but also inhibits the loading of MCM . The very stable association of ORC and Cdc6 with chromatin in this sequence-independent replication system suggests that origin selection in metazoans cannot be strictly dependent on the interaction of ORCs with specific DNA binding sequences.

J Biol Chem, 2003 Dec 12, 278(50), 49868 - 73 Epub 2003 Sep 23.
Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites; Subhi AL et al.; The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S . A., Diegelman, P., Porter, C . W., and Kruger, W . D . (2002) Cancer Res . 62, 6639-6644) . Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo . In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity . We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools . Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines . The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis . Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels . Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.

J Biol Chem, 2003 Dec 5, 278(49), 48727 - 34 Epub 2003 Sep 23.
DNA damage response-mediated degradation of Ho endonuclease via the ubiquitin system involves its nuclear export; Kaplun L et al.; Yeast mating switch Ho endonuclease is rapidly degraded by the ubiquitin system and this depends on the DNA damage response functions, MEC1, RAD9, and CHK1 . A PEST sequence marks Ho for degradation . Here we show that the novel F-box receptor, Ufo1, recruits phosphorylated Ho for degradation . Mutation of PEST residue threonine 225 stabilizes Ho, yet HoT225A still binds Ufo1 in vitro . Stable HoT225A accumulates within the nucleus, whereas HoT225E is degraded . Deletion of the nuclear exportin Msn5 traps native Ho in the nucleus and extends its half-life . These experiments suggest that Ho is degraded in the cytoplasm . In mec1 mutants stable Ho accumulates within the nucleus; Ho produced in mec1 cells does not bind Ufo1 . Thus the MEC1 pathway has functions both in phosphorylation of Thr-225 for nuclear export and in additional phosphorylations for binding Ufo1 . Cells with HO under its genomic promoter, but stabilized by deletion of the Msn5 exportin, proliferate, but are multibudded . These experiments elucidate some of the links between the DNA damage response and degradation of Ho by the ubiquitin system.

J Exp Biol, 2003 Nov, 206(Pt 21), 3869 - 75
Trypsin-modulating oostatic factor: a potential new larvicide for mosquito control; Borovsky D; Trypsin-modulating oostatic factor (TMOF), a mosquito decapeptide, terminates trypsin biosynthesis in the mosquito gut . The hormone is secreted from the ovary, starting 18 h after the blood meal, circulates in the hemolymph, binds to a gut receptor and stops trypsin biosynthesis by exerting a translational control on trypsin mRNA . Because of the unique primary amino acid sequence of the hormone (YDPAPPPPPP) and its stable three-dimensional conformation, TMOF is not degraded by gut proteolytic enzymes and can traverse the gut epithelial cells into the hemolymph of adults and larvae . Using this unique property, hormone fed to different species of mosquito larvae stops food digestion and causes larval mortality . To determine the shortest amino acid sequence that can bind to the gut receptor and still cause high larval mortality, 25 analogues of TMOF were synthesized and tested . The tetrapeptide (YDPA) was as effective as the decapeptide, indicating that the binding to the gut receptor is at the N-terminus of the molecule . Cloning and expressing the hormone on the coat protein of tobacco mosaic virus (TMV) in Chlorella sp . and Saccharomyces cerevisiae cells and feeding the recombinant cells to mosquito larvae caused larval mortality . These results indicate that TMOF can be used as a new biorational insecticide against mosquito larvae.

Insect Biochem Mol Biol, 2003 Oct, 33(10), 959 - 70
The C-terminus of the Hermes transposase contains a protein multimerization domain; Michel K et al.; Transposase activity that mediates the mobility of class II transposable elements, is most commonly initiated by the assembly of higher order synaptic complexes, called transpososomes . The formation of these complexes, that contain the transposable element's DNA as well as two or more molecules of the transposase, is dependent on interactions between transposase molecules . Using the yeast Two-Hybrid system, we were able to identify three regions mediating multimerization of the Hermes transposase, an element used for germline transformation of insects belonging to the hAT family of transposable elements . One region facilitating protein binding of Hermes transposase molecules was found within the first 252 amino acids of the transposase . The second region was located at the C-terminus of the transposase, and was found to be specific for Hermes transposase multimerization . Amino acids 551-569 were not only required for multimerization but were also necessary for transposition of the element . The third region was located between amino acids 253 and 380 and was found to eliminate the non-specific protein binding ability of the N-terminal protein interaction region but was required for the specific protein binding ability of the C-terminal region of the transposase . Five point mutations affecting the structural integrity of the C-terminal multimerization region abolished or significantly reduced transpositional activity . The same region had been previously identified to mediate dimerization in Activator (Ac), another hAT element, indicating that hAT transposase multimerization is likely to be a prerequisite for mobility of their elements.

Cell, 2003 Sep 19, 114(6), 689 - 99
AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding; Strack B et al.; HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission . The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery . We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1 . Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III . AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function . These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.

Bioprocess Biosyst Eng, 2002 Jun, 25(2), 69 - 78 Epub 2002 Apr 16.
Effect of experimental error on the efficiency of different optimization methods for bioprocess media optimization; Milavec P et al.; Four optimization methods (Simplex, Rosenbrock, iterative factorial experimental design (IFED) and genetic algorithms) for the optimization of the biotechnological media composition under conditions where the measured quantities are subjected to the experimental error were compared . The computer simulations were performed on some of the selected two- to six- parameter biotechnological models . The optimization process was modified in such a way that the experimental error was considered . The results show that the optimization efficiency increases when this new termination criteria is implemented . In addition, the method efficiency becomes independent of the experimental error . In general, Simplex and Rosenbrock methods need fewer experiments and their distribution of necessary experiments is narrower than for IFED and genetic algorithms . The increase of model parameters that need to be optimized results in a decrease in the method efficiency and in an increase of the average number of required experiments . The results were further verified on the cultivation of Saccharomyces cerevisiae and were found to be in good agreement with the results obtained from the computer simulations.

J Biol Chem, 2003 Dec 5, 278(49), 48563 - 9 Epub 2003 Sep 22.
CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding; Sherbet DP et al.; Cytochrome p450c17 (CYP17) converts the C21 steroids pregnenolone and progesterone to the C19 androgen precursors dehydroepiandrosterone (DHEA) and androstenedione, respectively, via sequential 17alpha-hydroxylase and 17,20-lyase reactions . Disabling mutations in CYP17 cause combined 17alpha-hydroxylase/17,20-lyase deficiency, but rare missense mutations cause isolated loss of 17,20-lyase activity by disrupting interactions of redox partner proteins with CYP17 . We studied an adolescent male with clinical and biochemical features of isolated 17,20-lyase deficiency, including micropenis, hypospadias, and gynecomastia, who is homozygous for CYP17 mutation E305G, which lies in the active site . When expressed in HEK-293 cells or Saccharomyces cerevisiae, mutation E305G retains 17alpha-hydroxylase activities, converting pregnenolone and progesterone to 17alpha-hydroxysteroids . However, mutation E305G lacks 17,20-lyase activity for the conversion of 17alpha-hydroxypregnenolone to DHEA, which is the dominant pathway to C19 steroids catalyzed by human CYP17 (the delta5-steroid pathway) . In contrast, mutation E305G exhibits 11-fold greater catalytic efficiency (kcat/Km) for the cleavage of 17alpha-hydroxyprogesterone to androstenedione compared with wild-type CYP17 . We conclude that mutation E305G selectively impairs 17,20-lyase activity for DHEA synthesis despite an increased capacity to form androstenedione . Mutation E305G provides genetic evidence that androstenedione formation from 17alpha-hydroxyprogesterone via the minor delta4-steroid pathway alone is not sufficient for complete formation of the male phenotype in humans.

J Biol Chem, 2003 Nov 28, 278(48), 48445 - 52 Epub 2003 Sep 22.
Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy; Tucker KA et al.; Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins . This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the delivery of proteins from the cytoplasm to the vacuole: the biosynthetic cytoplasm to vacuole targeting (Cvt) and the degradative autophagy pathways . Several components employed by these two transport routes have been identified, but their mechanistic interactions remain largely unknown . Here we report a novel gene involved in these pathways, which we have named ATG23 . Atg23 localizes to the pre-auto-phagosomal structure but also to other cytosolic punctate compartments . Our characterization of the Atg23 protein indicates that it is required for the Cvt pathway and efficient autophagy but not pexophagy . In the absence of Atg23, cargo molecules such as prApe1 are correctly recruited to a pre-autophagosomal structure that is unable to give rise to Cvt vesicles . We also demonstrate that Atg23 is a peripheral membrane protein that requires the presence of Atg9/Apg9 to be specifically targeted to lipid bilayers . Atg9 transiently interacts with Atg23 suggesting that it participates in the recruitment of this protein.

Eur J Drug Metab Pharmacokinet, 2003 Jan-Mar, 28(1), 59 - 65
In vitro metabolism of perospirone in rat, monkey and human liver microsomes; Mizuno Y et al.; In vitro metabolism of perospirone was examined with rat, monkey and human liver S9, human liver microsomes and yeast microsomes expressing human P450, using 14C labeled perospirone . With rat liver S9, the major metabolites were MX9 and ID-11614, produced by cleavage at the butylene chain . However, some butylene non-cleavage and hydration of the cyclohexane ring were found, although limited in extent . Unknown metabolites accounted for about 10% of the total . After incubation for 10 minutes with monkey liver S9, the major metabolites were ID-15036 and MX11, hydrated in the cyclohexane ring . After incubation for 60 minutes, ID-15001, i.e . the butylene chain cleavage type increased . Unknown metabolites accounted for about 20% . After incubation for 10 minutes with human liver S9, the major metabolite was ID-15036, hydrated in the cyclohexane ring . In addition, MX11 and many unknown metabolites were evident . After incubation for 60 minutes, the butylene chain cleavage type and unknown metabolites increased . Individual differences were found in the metabolic reaction rate . With human liver microsomes . MX11, ID-15001 and unknown metabolites were again the major metabolites . With yeast microsomes expressing human P450 subtypes, CYP1A1, 2C8, 2D6, 3A4 were responsible for the metabolism in particular, and CYP3A4 contributes greatly . Therefore it is unlikely that genetic polymorphism will arise a present a problem with regard to the clinical drug . The results demonstrated that the main metabolic pathway in human liver S9 and liver microsomes involve oxidation at cyclohexane, oxidative cleavage of the butylene side chain and S-oxidation . The same was the case in rat and monkey S9, but species differences were found in the proportions of the metabolites produced.

Nat Cell Biol, 2003 Oct, 5(10), 921 - 7 Epub 2003 Sep 21.
The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila; Pantalacci S et al.; Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously . The salvador (sav) gene, which encodes a scaffold protein, has been shown to restrict cell number by coordinating cell-cycle exit and apoptosis during Drosophila development . Here we identify Hippo (Hpo), the Drosophila orthologue of the mammalian MST1 and MST2 serine/threonine kinases, as a partner of Sav . Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype . Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1, overexpression of Hpo destabilizes DIAP1 in cell culture . We show that DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro . Thus, Hpo may promote apoptosis by reducing cellular amounts of DIAP1 . In addition, we show that Sav is an unstable protein that is stabilized by Hpo . We propose that Hpo and Sav function together to restrict tissue growth in vivo.

Nat Cell Biol, 2003 Oct, 5(10), 914 - 20 Epub 2003 Sep 21.
Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway; Udan RS et al.; Proliferation and apoptosis must be precisely regulated to form organs with appropriate cell numbers and to avoid tumour growth . Here we show that Hippo (Hpo), the Drosophila homologue of the mammalian Ste20-like kinases, MST1/2, promotes proper termination of cell proliferation and stimulates apoptosis during development . hpo mutant tissues are larger than normal because mutant cells continue to proliferate beyond normal tissue size and are resistant to apoptotic stimuli that usually eliminate extra cells . Hpo negatively regulates expression of Cyclin E to restrict cell proliferation, downregulates the Drosophila inhibitor of apoptosis protein DIAP1, and induces the proapoptotic gene head involution defective (hid) to promote apoptosis . The mutant phenotypes of hpo are similar to those of warts (wts), which encodes a serine/threonine kinase of the myotonic dystrophy protein kinase family, and salvador (sav), which encodes a WW domain protein that binds to Wts . We find that Sav binds to a regulatory domain of Hpo that is essential for its function, indicating that Hpo acts together with Sav and Wts in a signalling module that coordinately regulates cell proliferation and apoptosis.

Nat Cell Biol, 2003 Oct, 5(10), 928 - 35 Epub 2003 Sep 21.
S-phase checkpoint controls mitosis via an APC-independent Cdc20p function; Clarke DJ et al.; Cells divide with remarkable fidelity, allowing complex organisms to develop and possess longevity . Checkpoint controls contribute by ensuring that genome duplication and segregation occur without error so that genomic instability, associated with developmental abnormalities and a hallmark of most human cancers, is avoided . S-phase checkpoints prevent cell division while DNA is replicating . Budding yeast Mec1p and Rad53p, homologues of human checkpoint kinases ATM/ATR and Chk2, are needed for this control system . How Mec1p and Rad53p prevent mitosis in S phase is not known . Here we provide evidence that budding yeasts avoid mitosis during S phase by regulating the anaphase-promoting complex (APC) specificity factor Cdc20p: Mec1p and Rad53p repress the accumulation of Cdc20p in S phase . Because precocious Cdc20p accumulation causes anaphase onset and aneuploidy, Cdc20p concentrations must be precisely regulated during each and every cell cycle . Catastrophic mitosis induced by Cdc20p in S phase occurs even in the absence of core APC components . Thus, Cdc20p can function independently of the APC.

Science, 2003 Sep 19, 301(5640), 1728 - 31
A seven-transmembrane RGS protein that modulates plant cell proliferation; Chen JG et al.; G protein-coupled receptors (GPCRs) at the cell surface activate heterotrimeric G proteins by inducing the G protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate . Regulators of G protein signaling (RGS) proteins accelerate the deactivation of Galpha subunits to reduce GPCR signaling . Here we identified an RGS protein (AtRGS1) in Arabidopsis that has a predicted structure similar to a GPCR as well as an RGS box with GTPase accelerating activity . Expression of AtRGS1 complemented the pheromone supersensitivity phenotype of a yeast RGS mutant, sst2Delta . Loss of AtRGS1 increased the activity of the Arabidopsis Galpha subunit, resulting in increased cell elongation in hypocotyls in darkness and increased cell production in roots grown in light . These findings suggest that AtRGS1 is a critical modulator of plant cell proliferation.

Mol Endocrinol, 2003 Dec, 17(12), 2529 - 42 Epub 2003 Sep 18.
Protein inhibitor of activated signal transducer and activator of transcription 1 interacts with the N-terminal domain of mineralocorticoid receptor and represses its transcriptional activity: implication of small ubiquitin-related modifier 1 modification; Tallec LP et al.; Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators . To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASxbeta, described as SUMO (small ubiquitin-related modifier) E3-ligases . Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated . Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation . The agonist or antagonist nature of the bound ligand also determines PIAS1 corepressive action . We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo . Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties . Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status . Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications.

Infect Immun, 2003 Oct, 71(10), 5700 - 13
Analysis of immunological nonresponsiveness to the 19-kilodalton fragment of merozoite surface Protein 1 of Plasmodium yoelii: rescue by chemical conjugation to diphtheria toxoid (DT) and enhancement of immunogenicity by prior DT vaccination; Stanisic DI et al.; The Plasmodium merozoite surface protein 1 (MSP1) is a leading vaccine candidate for protecting against the blood stage of malaria . Previous studies have shown that the 19-kDa carboxyl terminus of this protein is able to induce protective immunity in some monkey and mouse strains . We show that immunization with the recombinant Plasmodium yoelii 19-kDa fragment of MSP1 (MSP1(19)) expressed in Saccharomyces cerevisiae (yMSP1(19)) can induce protective antibodies in several inbred mouse strains and one outbred mouse strain . However, mice expressing the H-2(s) major histocompatibility complex haplotype are unable to generate yMSP1(19)-specific antibodies . While synthetic peptides derived from MSP1(19) are immunogenic in B10.S mice, they cannot function as helper epitopes, and immunization with yMSP1(19) does not induce T cells that recognize the recombinant protein or synthetic peptides corresponding to its sequence . Nonresponsiveness could be overcome by using chemical linkers to conjugate yMSP1(19) to diphtheria toxoid (DT), resulting in immunogens capable of inducing protective yMSP1(19)-specific antibodies in both MSP1(19)-responsive and otherwise nonresponsive mouse strains . The ability of sera from mice immunized with the conjugate to inhibit binding of a protective monoclonal antibody (MAb 302) to yMSP1(19) correlated strongly with a delay in the prepatent period . Chemical conjugation of yMSP1(19) to DT may be a preferred method to enhance immunogenicity, as carrier priming experiments demonstrated that an existing immune response to DT enhanced a subsequent antibody response to yMSP1(19) after vaccination with yMSP1(19)-DT . These results have important implications for the development of a malaria vaccine to protect a population with diverse HLAs.

J Virol Methods, 2003 Oct, 113(1), 65 - 8
Intracellular hepatitis C virus RNA-dependent RNA polymerase activity; DeMarini DJ et al.; Hepatitis C virus (HCV) infection represents a significant health concern in over 170 million individuals worldwide . Recently, Huh7 cell-based hepatitis C virus replicon systems, which rely upon the expression and cooperation of viral nonstructural proteins to mediate replication of the entire hepatitis C virus genome, were shown to be useful for studying viral replication and antiviral agents . We report that expression of the viral RNA-dependent RNA polymerase (RdRp) in yeast cells, independent of other viral proteins, is necessary and sufficient for initiation of RNA synthesis in cis from 3'-nontranslated hepatitis C virus RNA . Furthermore, expression of the polymerase alone appears incapable of transcribing across the entire viral genome, most likely due to the secondary structure of the RNA . Other viral polypeptides, such as helicase, which are presumed to be present in the functional replicase complex, are predicted to facilitate RNA synthesis across highly structured regions.

BMC Bioinformatics . 2003 Sep 22;4(1):44.
RSEARCH: finding homologs of single structured RNA sequences; Klein RJ et al.; BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature . No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure . RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs . For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices . RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit . We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH . The primary drawback of the program is that it is slow . The C code for RSEARCH is freely available from our lab's website . CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences.

Mol Genet Genomics, 2003 Nov, 270(3), 225 - 33 Epub 2003 Sep 16.
Autocrine activation of the pheromone response pathway in matalpha2- cells is attenuated by SST2- and ASG7-dependent mechanisms; Rivers DM et al.; Yeast mat alpha2 mutants express both mating pheromones and both mating pheromone receptors . They show modest signaling in the pheromone response pathway, as revealed by increased levels of FUS1 transcript, yet are resistant to pheromone treatment . Together, these phenotypes suggest that alpha2- cells undergo autocrine activation of the pheromone response pathway, which is subsequently attenuated . We constructed a regulatable version of the alpha2 gene (GALalpha2) and showed that, upon loss of alpha2 activity, cells exhibit an initial robust response to pheromone that is attenuated within 3 h . We reasoned that the viability of alpha2- cells might be due to attenuation, and therefore performed a genome-wide synthetic lethal screen to identify potential adaptation components . We identified two genes, SST2 and ASG7 . Loss of either of these attenuation components results in activation of the pheromone pathway in alpha2- cells . Loss of both proteins causes a more severe phenotype . Sst2 functions as a GTPase activating protein (GAP) for the Galpha subunit of the trimeric G protein . Asg7 is an a -cell specific protein that acts in concert with the alpha-cell specific a -factor receptor, Ste3, to inhibit signaling by Gbetagamma . Hence, our results suggest that mat alpha2 mutants mimic the intracellular signaling events that occur in newly fused zygotes.

Curr Genet, 2003 Dec, 44(4), 195 - 201 Epub 2003 Sep 10.
Two bipartite NLSs mediate constitutive nuclear localization of Mcm10; Burich R et al.; Some components of the pre-replicative complex (pre-RC) are in the nucleus only during the G1 phase, while others are constitutively nuclear throughout the cell cycle . A nuclear localization signal (NLS) mediating the G1-specific nuclear localization has been characterized . We report here the identification of bipartite NLSs in constitutively nuclear pre-RC components, including the origin recognition complex, Mcm10 and Noc3 . Interestingly, in a subgroup of these pre-RC components, two putative NLSs were found in each protein . To examine whether multiple NLSs are required for these proteins to be nuclear throughout the cell cycle, we characterized the two NLSs in Mcm10 . We show that at least one of the two NLSs is required for directing Mcm10 into the nucleus and that either of them is sufficient to bring the green fluorescence protein into the nucleus . Thus, Mcm10 contains two functional NLSs and either is sufficient for the constitutive nuclear localization of Mcm10.

Oncogene, 2003 Sep 18, 22(40), 6129 - 41
Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38; Yustein JT et al.; The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated . Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s . Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken) . For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively . These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus . All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase . We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association . Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

Mol Biol Cell, 2003 Nov, 14(11), 4618 - 27 Epub 2003 Sep 17.
A protein complex containing Mdm10p, Mdm12p, and Mmm1p links mitochondrial membranes and DNA to the cytoskeleton-based segregation machinery; Boldogh IR et al.; Previous studies indicate that two proteins, Mmm1p and Mdm10p, are required to link mitochondria to the actin cytoskeleton of yeast and for actin-based control of mitochondrial movement, inheritance and morphology . Both proteins are integral mitochondrial outer membrane proteins . Mmm1p localizes to punctate structures in close proximity to mitochondrial DNA (mtDNA) nucleoids . We found that Mmm1p and Mdm10p exist in a complex with Mdm12p, another integral mitochondrial outer membrane protein required for mitochondrial morphology and inheritance . This interpretation is based on observations that 1) Mdm10p and Mdm12p showed the same localization as Mmm1p; 2) Mdm12p, like Mdm10p and Mmm1p, was required for mitochondrial motility; and 3) all three proteins coimmunoprecipitated with each other . Moreover, Mdm10p localized to mitochondria in the absence of the other subunits . In contrast, deletion of MMM1 resulted in mislocalization of Mdm12p, and deletion of MDM12 caused mislocalization of Mmm1p . Finally, we observed a reciprocal relationship between the Mdm10p/Mdm12p/Mmm1p complex and mtDNA . Deletion of any one of the subunits resulted in loss of mtDNA or defects in mtDNA nucleoid maintenance . Conversely, deletion of mtDNA affected mitochondrial motility: mitochondria in cells without mtDNA move 2-3 times faster than mitochondria in cells with mtDNA . These observations support a model in which the Mdm10p/Mdm12p/Mmm1p complex links the minimum heritable unit of mitochondria (mtDNA and mitochondrial outer and inner membranes) to the cytoskeletal system that drives transfer of that unit from mother to daughter cells.

J Biol Chem, 2003 Dec 5, 278(49), 49171 - 82 Epub 2003 Sep 17.
Mcm4,6,7 uses a "pump in ring" mechanism to unwind DNA by steric exclusion and actively translocate along a duplex; Kaplan DL et al.; Mcm4,6,7 is a ring-shaped heterohexamer and the putative eukaryotic replication fork helicase . In this study, we examine the mechanism of Mcm4,6,7 . Mcm4,6,7 binds to only one strand of a duplex during unwinding, corresponding to the leading strand of a replication fork . Mcm4,6,7 unwinding stops at a nick in either strand . The Mcm4,6,7 ring also actively translocates along duplex DNA, enabling the protein to drive branch migration of Holliday junctions . The Mcm4,6,7 mechanism is very similar to DnaB, except the proteins translocate with opposite polarity along DNA . Mcm4,6,7 and DnaB have different structural folds and evolved independently; thus, the similarity in mechanism is surprising . We propose a "pump in ring" mechanism for both Mcm4,6,7 and DnaB, wherein a single-stranded DNA pump is situated within the central channel of the ring-shaped helicase, and unwinding is the result of steric exclusion . In this example of convergent evolution, the "pump in ring" mechanism was probably selected by eukaryotic and bacterial replication fork helicases in order to restrict unwinding to replication fork structures, stop unwinding when the replication fork encounters a nick, and actively translocate along duplex DNA to accomplish additional activities such as DNA branch migration.

DNA Repair (Amst), 2003 Oct 7, 2(10), 1127 - 34
The Rad52-Rad59 complex interacts with Rad51 and replication protein A; Davis AP et al.; The RAD52 gene is essential for homology-dependent repair of double-strand breaks in Saccharomyces cerevisiae . Rad52 forms complexes with Rad51, replication protein A (RPA) or Rad59 and its presence is essential for the formation of Rad51-Rad52-Rad59 and RPA-Rad52-Rad59 complexes . The N-terminal region of Rad52, which is required for self-interaction to form a ring structure, is required for interaction with Rad59 . Rad59 also shows self-interaction suggesting the formation of heteromeric and homomeric rings of Rad52 and Rad59 . In wild-type cells, we propose the Rad51-Rad52-Rad59 complex is involved in conservative recombination events, including gene conversion and reciprocal recombination, whereas the Rad52-Rad59 complex participates in single-strand annealing.

Curr Biol, 2003 Sep 16, 13(18), 1583 - 95
Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability; Kanellis P et al.; BACKGROUND: Genome instability is a hallmark of cancer and plays a critical role in generating the myriad of phenotypes selected for during tumor progression . However, the mechanisms that prevent genome rearrangements remain poorly understood . RESULTS: To elucidate the mechanisms that ensure genome stability, we screened a collection of candidate genes for suppressors of gross chromosomal rearrangements (GCRs) in budding yeast . One potent suppressor gene encodes Elg1, a conserved but uncharacterized homolog of the large RFC subunit Rfc1 and the alternative RFC subunits Ctf18/Chl12 and Rad24 . Our results are consistent with the hypothesis that Elg1 forms a novel and distinct RFC-like complex in both yeast and human cells . We find that Elg1 is required for efficient S phase progression and telomere homeostasis in yeast . Elg1 interacts physically with the PCNA homolog Pol30 and the FEN-1 homolog Rad27 . The physical and genetic interactions suggest a role for Elg1 in Okazaki fragment maturation . Furthermore, Elg1 acts in concert with the alternative Rfc1-like proteins Rad24 and Ctf18 to enable Rad53 checkpoint kinase activation in response to replication stress . CONCLUSIONS: Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex . Furthermore, we propose that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation.

Antioxid Redox Signal, 2003 Aug, 5(4), 389 - 96
Cloning and initial characterization of the Arabidopsis thaliana endoplasmic reticulum oxidoreductins; Dixon DP et al.; The oxidation and isomerization of disulfide bonds is necessary for the growth of all organisms . In yeast, the oxidative folding of secretory pathway proteins is catalyzed by protein disulfide isomerase (PDI), which requires Ero1p (endoplasmic reticulum oxidoreductin) for its own oxidation . In Homo sapiens, two homologues of Ero1p, Ero1-Lalpha and Ero1-Lbeta, have been cloned . Both Ero1-Lalpha and Ero1-Lbeta interact via disulfide bonds with PDI and support the oxidation of immunoglobulin light chains . However, the function of Ero proteins in plants has not yet been analyzed . In this article, we report the cloning of the two Ero1p homologues present in Arabidopsis thaliana, demonstrating that one of the cDNAs has a shorter terminal exon than predicted and differs from the annotated sequence found in the genome database . Sequence analysis of the Arabidopsis endoplasmic reticulum oxidoreductins (AEROs) reveals that both AERO1 and AERO2 are more closely related to each other than to either of the human Eros . Both in vitro translated AERO proteins are targeted to the endoplasmic reticulum and glycosylated . The ability to use a genetically tractable multicellular organism in combination with biochemical approaches should further our understanding of redox networks and Ero function in both plants and animals.

BMC Bioinformatics . 2003 Sep 17;4(1):42.
Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals; Prag S et al.; BACKGROUND: The kelch motif is an ancient and evolutionarily-widespread sequence motif of 44-56 amino acids in length . It occurs as five to seven repeats that form a beta-propeller tertiary structure . Over 28 kelch-repeat proteins have been sequenced and functionally characterised from diverse organisms spanning from viruses, plants and fungi to mammals and it is evident from expressed sequence tag, domain and genome databases that many additional hypothetical proteins contain kelch-repeats . In general, kelch-repeat beta-propellers are involved in protein-protein interactions, however the modest sequence identity between kelch motifs, the diversity of domain architectures, and the partial information on this protein family in any single species, all present difficulties to developing a coherent view of the kelch-repeat domain and the kelch-repeat protein superfamily . To understand the complexity of this superfamily of proteins, we have analysed by bioinformatics the complement of kelch-repeat proteins encoded in the human genome and have made comparisons to the kelch-repeat proteins encoded in other sequenced genomes . RESULTS: We identified 71 kelch-repeat proteins encoded in the human genome, whereas 5 or 8 members were identified in yeasts and around 18 in C . elegans, D . melanogaster and A . gambiae . Multiple domain architectures were identified in each organism, including previously unrecognised forms . The vast majority of kelch-repeat domains are predicted to form six-bladed beta-propellers . The most prevalent domain architecture in the metazoan animal genomes studied was the BTB/kelch domain organisation and we uncovered 3 subgroups of human BTB/kelch proteins . Sequence analysis of the kelch-repeat domains of the most robustly-related subgroups identified differences in beta-propeller organisation that could provide direction for experimental study of protein-binding characteristics . CONCLUSION: The kelch-repeat superfamily constitutes a distinct and evolutionarily-widespread family of beta-propeller domain-containing proteins . Expansion of the family during the evolution of multicellular animals is mainly accounted for by a major expansion of the BTB/kelch domain architecture . BTB/kelch proteins constitute 72 % of the kelch-repeat superfamily of H . sapiens and form three subgroups, one of which appears the most-conserved during evolution . Distinctions in propeller blade organisation between subgroups 1 and 2 were identified that could provide new direction for biochemical and functional studies of novel kelch-repeat proteins.

J Protein Chem, 2003 May, 22(4), 353 - 61
Enzymatic function of loop movement in enolase: preparation and some properties of H159N, H159A, H159F, and N207A enolases; Brewer JM et al.; The hypothesis that His159 in yeast enolase moves on a polypeptide loop to protonate the phosphoryl of 2-phosphoglycerate to initiate its conversion to phosphoenolpyruvate was tested by preparing H159N, H159A, and H159F enolases . These have 0.07%-0.25% of the native activity under standard assay conditions and the pH dependence of maximum velocities of H159A and H159N mutants is markedly altered . Activation by Mg2+ is biphasic, with the smaller Mg2+ activation constant closer to that of the "catalytic" Mg2+ binding site of native enolase and the larger in the mM range in which native enolase is inhibited . A third Mg2+ may bind to the phosphoryl, functionally replacing proton donation by His159 . N207A enolase lacks an intersubunit interaction that stabilizes the closed loop(s) conformation when 2-phosphoglycerate binds . It has 21% of the native activity, also exhibits biphasic Mg2+ activation, and its reaction with the aldehyde analogue of the substrate is more strongly inhibited than is its normal enzymatic reaction . Polypeptide loop(s) closure may keep a proton from His159 interacting with the substrate phosphoryl oxygen long enough to stabilize a carbanion intermediate.

J Bioenerg Biomembr, 2003 Jun, 35(3), 243 - 56
Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid; Zeman I et al.; Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively . To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis . Only four different mutations could be identified in spite of a large number of mutants analyzed . They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA . The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments . ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier . These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions . Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p . BA complex.

Plant Mol Biol, 2003 Jul, 52(4), 801 - 15
MADS-box genes expressed during tomato seed and fruit development; Busi MV et al.; MADS-box genes in plants are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers . Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development . We have identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development . According to comparisons of their deduced amino acid sequences, they were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12) . With the exception of TAGL12, which is expressed near uniformly in every tissue, the other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs . In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity . Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role.

Pharmacopsychiatry, 2003 Jun, 36 Suppl 1, S89 - 94
Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death; Chandrasekaran K et al.; In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract EGb 761, (definition see editorial) and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death . In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils . At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons . Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA . In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death (effective concentration {EC (50)} = 5 microg/ml (12 microM) for bilobalide and 100 microg/ml for EGb 761 . These results suggest that both EGb 761 and bilobalide are protective against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.

J Clin Invest, 2003 Sep, 112(6), 824 - 6
HATs off to Hop: recruitment of a class I histone deacetylase incriminates a novel transcriptional pathway that opposes cardiac hypertrophy; Hamamori Y et al.; Histone acetylation, regulated by two antagonistic enzymes - histone acetyltransferases (HATs) and histone deacetylases (HDACs) - results in transcriptional changes and also plays a critical role in cardiac development and disease . A new study shows that overexpression of the atypical transcriptional corepressor homeodomain-only protein (Hop) causes cardiac hypertrophy via recruitment of a class I HDAC . In contrast to the body of work on transcriptional mechanisms that drive cardiac hypertrophy, including class II HDACs, this report elucidates a novel growth-suppressing transcriptional pathway in cardiac muscle that opposes hypertrophic growth.

Genes Dev, 2003 Oct 1, 17(19), 2384 - 95 Epub 2003 Sep 15.
Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends; Stellwagen AE et al.; Ku is a conserved DNA end-binding protein that plays various roles at different kinds of DNA ends . At telomeres, Ku is part of the structure that protects the chromosome end, whereas at broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway . Here, we present evidence of a new role for Ku that impacts both telomere-length maintenance and DNA repair in Saccharomyces cerevisiae . We show that Ku binds TLC1, the RNA component of telomerase . We also describe a novel separation-of-function allele of Ku that is specifically defective in TLC1 binding . In this mutant, telomeres are short and the kinetics of telomere addition are slow, but other Ku-dependent activities, such as chromosome end protection and NHEJ, are unaffected . At low frequency, yeast will use telomerase to heal DNA damage by capping the broken chromosome with telomeric DNA sequences . We show that when Ku's ability to bind TLC1 is disrupted, DNA repair via telomere healing is reduced 10- to 100-fold, and the spectrum of sequences that can acquire a telomere changes . Thus, the interaction between Ku and TLC1 RNA enables telomerase to act at both broken and normal chromosome ends.

EMBO Rep, 2003 Oct, 4(10), 959 - 63 Epub 2003 Sep 12.
Blm3 is part of nascent proteasomes and is involved in a late stage of nuclear proteasome assembly; Fehlker M et al.; Proteasomes are multisubunit proteases that are responsible for regulated proteolysis . The degradation of the proteasomal maturation factor, named Ump1 in yeast, completes the autocatalytic processing of inactive precursor complexes into the proteolytically active core particle (CP) of the proteasome . We have identified Blm3, a conserved nuclear protein, as a new component of Ump1-associated precursor complexes . A lack of Blm3 resulted in an increased rate of precursor processing and an accelerated turnover of Ump1, which suggests that Blm3 prevents premature activation of proteasomal CPs . On the basis of biochemical fractionation experiments combined with in vivo localization studies, we propose that Blm3 joins nascent CPs inside the nucleus to coordinate late stages of proteasome assembly in yeast.

Plant Cell, 2003 Oct, 15(10), 2273 - 84 Epub 2003 Sep 05.
The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress; Koiwa H et al.; Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem . Sequence comparison of the STT3a gene identified a yeast ortholog, STT3, which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N-glycosylation . NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress-responsive chaperone (BiP-GUS) and cell division (CycB1;1-GUS) genes, respectively . Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression . Interestingly, a mutation (stt3b-1) in another Arabidopsis STT3 isogene (STT3b) does not cause NaCl sensitivity . However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal . Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.

Plant Physiol, 2003 Oct, 133(2), 528 - 37 Epub 2003 Aug 14.
Diurnal and light-regulated expression of AtSTP1 in guard cells of Arabidopsis; Stadler R et al.; Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco . Therefore, guard cells depend on carbon supply from adjacent cells even during the light period . Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata . Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture . This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells . Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum . Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday . This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose . Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.

Mol Cell Biol, 2003 Oct, 23(19), 7005 - 18
Bur1 kinase is required for efficient transcription elongation by RNA polymerase II; Keogh MC et al.; The Saccharomyces cerevisiae cyclin-dependent kinase (CDK) Bur1 (Sgv1) may be homologous to mammalian Cdk9, which functions in transcriptional elongation . Although Bur1 can phosphorylate the Rpb1 carboxy-terminal domain (CTD) kinase in vitro, it has no strong specificity within the consensus heptapeptide YSPTSPS for Ser2 or Ser5 . BUR1 mutants are sensitive to the drugs 6-azauracil and mycophenolic acid and interact genetically with the elongation factors Ctk1 and Spt5 . Chromatin immunoprecipitation experiments show that Bur1 and its cyclin partner Bur2 are recruited to transcription elongation complexes, cross-linking to actively transcribing genes . Interestingly, Bur1 shows reduced cross-linking to transcribed regions downstream of polyadenylation sites . In addition, bur1 mutant strains have a reduced cross-linking ratio of RNA polymerase II at the 3' end of genes relative to promoter regions . Phosphorylation of CTD serines 2 and 5 appears normal in mutant cells, suggesting that Bur1 is not a significant source of cotranscriptional Rpb1 phosphorylation . These results show that Bur1 functions in transcription elongation but may phosphorylate a substrate other than the CTD.

Mol Cell Biol, 2003 Oct, 23(19), 6982 - 92
Rrp47p is an exosome-associated protein required for the 3' processing of stable RNAs; Mitchell P et al.; Related exosome complexes of 3'-->5' exonucleases are present in the nucleus and the cytoplasm . Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts . This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing) . Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3'-->5' exonuclease specific to the nuclear exosome fraction . Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors . Rrp47p also participates in the 3' processing of U4 and U5 small nuclear RNAs (snRNAs) . The defects in the processing of stable RNAs seen in rrp47-Delta strains closely resemble those of strains lacking Rrp6p . In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3'-extended nuclear pre-mRNAs or the cytoplasmic 3'-->5' mRNA decay pathway . We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs.

Mol Cell Biol, 2003 Oct, 23(19), 6973 - 81
A phosphomimetic mutation at Ser-138 renders iron regulatory protein 1 sensitive to iron-dependent degradation; Fillebeen C et al.; Iron regulatory protein 1 (IRP1) binds to mRNA iron-responsive elements (IREs) and thereby controls the expression of IRE-containing mRNAs . In iron-replete cells, assembly of a cubane {4Fe-4S} cluster inhibits IRE-binding activity and converts IRP1 to a cytosolic aconitase . Earlier experiments with Saccharomyces cerevisiae suggested that phosphomimetic mutations of Ser-138 negatively affect the stability of the cluster (N . M . Brown, S . A . Anderson, D . W . Steffen, T . B . Carpenter, M . C . Kennedy, W . E . Walden, and R . S . Eisenstein, Proc . Natl . Acad . Sci . USA 95:15235-15240, 1998) . Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1(S138E)) lacks aconitase activity, which is a hallmark of {4Fe-4S} cluster integrity . Similarly, IRP1(S138E) expressed in mammalian cells fails to function as aconitase . Furthermore, we demonstrate that the impairment of {4Fe-4S} cluster assembly in mammalian cells sensitizes IRP1(S138E) to iron-dependent degradation . This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin . As expected, the stability of wild-type or phosphorylation-deficient IRP1(S138A) is not affected by iron manipulations . Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution . Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.

Mol Biol Cell, 2003 Sep, 14(9), 3753 - 66 Epub 2003 May 18.
Endoplasmic reticulum export of glycosyltransferases depends on interaction of a cytoplasmic dibasic motif with Sar1; Giraudo CG et al.; Membrane proteins exit the endoplasmic reticulum (ER) in COPII-transport vesicles . ER export is a selective process in which transport signals present in the cytoplasmic tail (CT) of cargo membrane proteins must be recognized by coatomer proteins for incorporation in COPII vesicles . Two classes of ER export signals have been described for type I membrane proteins, the diacidic and the dihydrophobic motifs . Both motifs participate in the Sar1-dependent binding of Sec23p-Sec24p complex to the CTs during early steps of cargo selection . However, information concerning the amino acids in the CTs that interact with Sar1 is lacking . Herein, we describe a third class of ER export motif, {RK}(X){RK}, at the CT of Golgi resident glycosyltransferases that is required for these type II membrane proteins to exit the ER . The dibasic motif is located proximal to the transmembrane border, and experiments of cross-linking in microsomal membranes and of binding to immobilized peptides showed that it directly interacts with the COPII component Sar1 . Sar1GTP-bound to immobilized peptides binds Sec23p . Collectively, the present data suggest that interaction of the dibasic motif with Sar1 participates in early steps of selection of Golgi resident glycosyltransferases for transport in COPII vesicles.

Mol Biol Cell, 2003 Sep, 14(9), 3605 - 16 Epub 2003 May 18.
Rer1p, a retrieval receptor for ER membrane proteins, recognizes transmembrane domains in multiple modes; Sato K et al.; The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER . We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p . The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p . The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD . On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes . Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway . In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting . Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.

J Biol Chem, 2003 Dec 5, 278(49), 48997 - 9005 Epub 2003 Sep 12.
Mmm1p spans both the outer and inner mitochondrial membranes and contains distinct domains for targeting and foci formation; Kondo-Okamoto N et al.; In the yeast Saccharomyces cerevisiae, the integral membrane protein Mmm1p is required for maintenance of mitochondrial morphology and retention of mitochondrial DNA (mtDNA) . Mmm1p localizes to discrete foci on mitochondria that are adjacent to mtDNA nucleoids in the matrix, raising the possibility that this protein plays a direct role in organizing, replicating, or segregating mtDNA . Although Mmm1p has been shown to cross the outer membrane with its C terminus facing the cytoplasm, the location of the N terminus has not been resolved . Here we show that Mmm1p spans both the outer and inner mitochondrial membranes, exposing its N terminus to the matrix . Surprisingly, deletion of the N-terminal extension decreased steady-state levels of the Mmm1 protein but did not affect mitochondrial morphology or mtDNA maintenance . Moreover, expression of Neurospora crassa MMM1, which naturally lacks a long N-terminal extension, substituted for loss of Mmm1p in budding yeast . These results indicate that the matrix-exposed portion of Mmm1p is not essential for mtDNA nucleoid maintenance . Additional studies revealed that the transmembrane segment and C-terminal domain of Mmm1p are required for foci formation and mitochondrial targeting, respectively . Our data suggest that the double membrane-spanning topology of Mmm1p at the membrane contact site is critical for formation of tubular mitochondria.

Biochim Biophys Acta, 2003 Sep 23, 1642(1-2), 9 - 16
Acquisition of heat shock tolerance by regulation of intracellular redox states; Ueom J et al.; In the yeast Saccharomyces cerevisiae, a mild heat treatment strongly induces Hsp104p which provides acquisition of thermotolerance . The mechanism by which Hsp104p protects cells from the severe heat shock has not yet been completely elucidated . In this study, a pivotal role of Hsp104p as an efficient scavenger of the reactive oxygen species (ROS) is investigated . In our previous study, we reported that Hsp104p acted as a regulator in the mitochondrial respiration pathway . In this report, the recombinant wild-type and hypersensitive ras mutants (ira2Delta) with the extrachromosomal plasmids wild-type and mutant hsp104 genes were studied . The resulting strains successfully expressed both wild-type and mutant Hsp104p and showed the thermotolerance phenotype in the strain with the functional wild-type Hsp104p expressed . Upon treatment with H2O2 and menadione, the strains with the functional Hsp104p expressed showed higher survival rates than the other mutants, indicating the protective role of Hsp104p from the oxidative stress . Fluorescence measurement of the oxidation-dependent probe, 2',7'-dichlorofluoroscein diacetate (H2DCFDA), also indicated that Hsp104p significantly reduced the amount of ROS . Resistance to the oxidative stress was independent of the amount of the glutathione in the hyperactivated ras mutants . Taken all together, this study confirms that Hsp104p plays a crucial role in keeping cells from being damaged by the oxidative stress, thus acting as a modulator of the intracellular redox state.

Leukemia, 2003 Sep, 17(9), 1858 - 64
Major form of NUP98/HOXC11 fusion in adult AML with t(11;12)(p15;q13) translocation exhibits aberrant trans-regulatory activity; Gu BW et al.; Three adult patients with de novo acute myeloid leukemia of distinct subtypes harboring t(11;12)(p15;q13) have been investigated to characterize the genes involved in that translocation . Through molecular cytogenetics, a chromosome break was detected at the 3' part of nucleoporin 98 (NUP98) gene at 11p15 . Using rapid amplification of cDNA end, we identified the partner gene at 12q13, HOXC11 . Molecular analysis showed that exon 12 of NUP98 was fused in-frame to exon 2 of HOXC11 in all three cases with t(11;12)(p15;q13) . Therefore, this type of fusion may represent the major form of the NUP98-HOXC11 chimera so far reported . Moreover, two out of three cases had a confirmed deletion of the 3' part of NUP98 gene and more telomeric region of 11p harboring a group of tumor-suppressor genes . Interestingly, the NUP98-HOXC11 protein when assayed in a GAL4 reporter system, showed an aberrant trans-regulatory activity as compared to the wild-type HOXC11 in both COS-7 and HL-60 cells . Therefore, NUP98-HOXC11 may contribute to the leukemogenesis by interfering with the cellular mechanism of transcriptional regulation.

Toxicol Sci, 2003 Dec, 76(2), 299 - 309 Epub 2003 Sep 11.
Antiandrogenic activities of diesel exhaust particle extracts in PC3/AR human prostate carcinoma cells; Kizu R et al.; We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively . The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8 . PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR) . While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml . The antiandrogenic effect was greater in the following order: ET > EB > EC . Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents . The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist . The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1 . Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples . Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen . A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists . All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

Science, 2003 Oct 10, 302(5643), 279 - 82 Epub 2003 Sep 11.
The Prp19p-associated complex in spliceosome activation; Chan SP et al.; During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p . We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated . Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation . Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.

Plant Physiol, 2003 Sep, 133(1), 126 - 34
Differential metal selectivity and gene expression of two zinc transporters from rice; Ramesh SA et al.; Zinc is an essential mineral for a wide variety of physiological and biochemical processes . To understand zinc transport in cereals, we identified putative zinc transporters in gene databases . Three full-length cDNAs were identified and characterized from rice (Oryza sativa) . Two of the cDNAs partially complemented a yeast (Saccharomyces cerevisiae) mutant deficient in zinc uptake at low concentrations . The two transporters showed many similarities in function but differed in ionic selectivity and pH optimum of activity . Expression patterns also differed between the two genes . One gene was broadly expressed under all conditions, and the other gene was mainly induced by zinc deficiency to higher levels in roots than in leaves . Although the timing of expression differed between the two genes, localization of expression overlapped in roots . Comparisons of the protein sequences, ionic selectivity, and gene expression patterns of the two transporters suggest that they may play different roles in the physiology of the whole plant.

Plant Physiol, 2003 Sep, 133(1), 63 - 72
CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis; Naur P et al.; In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group . Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates . In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency . Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K(m) value . The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant . The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1 . Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines . Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.

J Clin Endocrinol Metab, 2003 Sep, 88(9), 4077 - 9
Estrogen bioactivity in fo-ti and other herbs used for their estrogen-like effects as determined by a recombinant cell bioassay; Oerter Klein K et al.; One of the most important issues in women's health concerns the risks and benefits of estrogen replacement therapy . Continual uncertainty and lack of consensus regarding estrogen replacement therapy has driven many women to seek alternative sources of estrogen, including herbal remedies . We adapted a recombinant cell bioassay to measure estrogen bioactivity in herbs . We studied, in vitro, estrogen bioactivity in red clover, dong quai, black cohosh, soy, licorice, chaste tree berry, fo-ti, and hops . Soy, clover, licorice, and hops have a large amount of measurable estrogen bioactivity, as suspected, based on previous reports using other methods . We discovered surprisingly high estrogen activity in extracts of fo-ti not previously reported . Chaste tree berry, black cohosh, and dong quai did not have measurable activity with this method . We also discovered that removal of a glycone group from soy increases its estrogen bioactivity significantly . We conclude that this recombinant cell bioassay for estradiol can be used to measure bioactivity in herbal products . The preparations of fo-ti studied had estrogen activity of 409 +/- 55 pmol/liter estradiol equivalents per microgram of herb, which is 1/300 the activity of 17 beta-estradiol . Clinical studies are underway to determine the estrogen bioactivity in women using dietary supplements containing these herbs.

EMBO J, 2003 Sep 15, 22(18), 4826 - 35
A novel eukaryotic factor for cytosolic Fe-S cluster assembly; Roy A et al.; Iron regulatory protein 1 (IRP1) is regulated through the assembly/disassembly of a {4Fe-4S} cluster, which interconverts IRP1 with cytosolic aconitase . A genetic screen to isolate Saccharomyces cerevisiae strains bearing mutations in genes required for the conversion of IRP1 to c-aconitase led to the identification of a previously uncharacterized, essential gene, which we call CFD1 (cytosolic Fe-S cluster deficient) . CFD1 encodes a highly conserved, putative P-loop ATPase . A non-lethal mutation of CFD1 (cfd1-1) reduced c-aconitase specific activity in IRP1-transformed yeast by >90%, although IRP1 in these cells could be readily converted to c-aconitase in vitro upon incubation with iron alone . IRP1-transformed cfd1-1 yeast lacked EPR-detectable Fe-S clusters in c-aconitase, pointing to a defect in Fe-S cluster assembly . The specific activity of another cytosolic Fe-S protein, Leu1p, was also inhibited by >90% in cfd1-1 yeast, whereas activity of mitochondrial Fe-S proteins was not inhibited . Consistent with a cytosolic site of activity, Cfd1p was localized in the cytoplasm . To our knowledge, Cfd1p is the first cytoplasmic Fe-S cluster assembly factor described in eukaryotes.

EMBO J, 2003 Sep 15, 22(18), 4815 - 25
Components involved in assembly and dislocation of iron-sulfur clusters on the scaffold protein Isu1p; Muhlenhoff U et al.; The mitochondrial proteins Isu1p and Isu2p play an essential role in the maturation of cellular iron-sulfur (Fe/S) proteins in eukaryotes . By radiolabelling of yeast cells with 55Fe we demonstrate that Isu1p binds an oxygen-resistant non-chelatable Fe/S cluster providing in vivo evidence for a scaffolding function of Isu1p during Fe/S cluster assembly . Depletion of the cysteine desulfurase Nfs1p, the ferredoxin Yah1p or the yeast frataxin homologue Yfh1p by regulated gene expression causes a strong decrease in the de novo synthesis of Fe/S clusters on Isu1p . In contrast, depletion of the Hsp70 chaperone Ssq1p, its co-chaperone Jac1p or the glutaredoxin Grx5p markedly increased the amount of Fe/S clusters bound to Isu1p, even though these mitochondrial proteins are crucial for maturation of Fe/S proteins . Hence Ssq1p/Jac1p and Grx5p are required in a step after Fe/S cluster synthesis on Isu1p, for instance in dissociation of preassembled Fe/S clusters from Isu1p and/or their insertion into apoproteins . We propose a model that dissects Fe/S cluster biogenesis into two major steps and assigns its central components to one of these two steps.

EMBO J, 2003 Sep 15, 22(18), 4597 - 606
Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation; Swanson KA et al.; Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles . Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins . The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies . Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity . The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit . The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.

Nature, 2003 Sep 11, 425(6954), 188 - 91
Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation; Stelter P et al.; Protein modification by ubiquitin is emerging as a signal for various biological processes in eukaryotes, including regulated proteolysis, but also for non-degradative functions such as protein localization, DNA repair and regulation of chromatin structure . A small ubiquitin-related modifier (SUMO) uses a similar conjugation system that sometimes counteracts the effects of ubiquitination . Ubiquitin and SUMO compete for modification of proliferating cell nuclear antigen (PCNA), an essential processivity factor for DNA replication and repair . Whereas multi-ubiquitination is mediated by components of the RAD6 pathway and promotes error-free repair, SUMO modification is associated with replication . Here we show that RAD6-mediated mono-ubiquitination of PCNA activates translesion DNA synthesis by the damage-tolerant polymerases eta and zeta in yeast . Moreover, polymerase zeta is differentially affected by mono-ubiquitin and SUMO modification of PCNA . Whereas ubiquitination is required for damage-induced mutagenesis, both SUMO and mono-ubiquitin contribute to spontaneous mutagenesis in the absence of DNA damage . Our findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.

J Biol Chem, 2003 Nov 21, 278(47), 46506 - 15 Epub 2003 Sep 10.
Regulators of G protein signaling and transient activation of signaling: experimental and computational analysis reveals negative and positive feedback controls on G protein activity; Hao N et al.; Cellular responses to hormones and neurotransmitters are necessarily transient . The mating pheromone signal in yeast is typical . Signal initiation requires cell surface receptors, a G protein heterotrimer, and down-stream effectors . Signal inactivation requires Sst2, a regulator of G protein signaling (RGS) protein that accelerates GTPase activity . We conducted a quantitative analysis of RGS and G protein expression and devised computational models that describe their activity in vivo . These results indicated that pheromone-dependent transcriptional induction of the RGS protein constitutes a negative feedback loop that leads to desensitization . Modeling also suggested the presence of a positive feedback loop leading to resensitization of the pathway . In confirmation of the model, we found that the RGS protein is ubiquitinated and degraded in response to pheromone stimulation . We identified and quantitated these positive and negative feedback loops, which account for the transient response to external signals observed in vivo.

DNA Repair (Amst), 2003 Sep 18, 2(9), 1007 - 13
siRNA depletion of BRCA1, but not BRCA2, causes increased genome instability in Fanconi anemia cells; Bruun D et al.; BRCA1 and BRCA2 proteins act in repair of interstrand crosslinks (ICLs) and maintenance of genome stability and are known to be part of the Fanconi anemia (FA) pathway . We have investigated the role of the BRCA1 and BRCA2 genes in genome stability following ICL damage in normal and FA cells . To circumvent cell lethality of complete disruptions in BRCA1 or BRCA2, small inhibitory RNA (siRNA) was used to transiently deplete the expression of the proteins . Using chromosomal stability after ICL damage as the end point, we find that BRCA1 functions in more than just the FA pathway for genome maintenance, whereas BRCA2 appears to act predominantly in the FA pathway . Depletion of BRCA1 causes a marked decrease, although not a complete absence of, ubiquitination of FANCD2 . In contrast to BRCA1, BRCA2 is not needed for normal ubiquitination of FANCD2 after DNA damage, a requirement for the FA pathway to function . Thus, BRCA2 is epistatic to FA genes for ICL repair, but not for damage-induced modification of FANCD2 and may act downstream form FANCD2.

Dev Cell, 2003 Sep, 5(3), 463 - 74
Synaptonemal complex assembly in C . elegans is dispensable for loading strand-exchange proteins but critical for proper completion of recombination; Colaiacovo MP et al.; Here we probe the relationships between assembly of the synaptonemal complex (SC) and progression of recombination between homologous chromosomes during Caenorhabditis elegans meiosis . We identify SYP-2 as a structural component of the SC central region and show that central region assembly depends on proper morphogenesis of chromosome axes . We find that the SC central region is dispensable for initiation of recombination and for loading of DNA strand-exchange protein RAD-51, despite the fact that extensive RAD-51 loading normally occurs in the context of assembled SC . Further, persistence of RAD-51 foci and absence of crossover products in meiotic mutants suggests that SC central region components and recombination proteins MSH-4 and MSH-5 are required to promote conversion of resected double-strand breaks into stable post-strand exchange intermediates . Our data also suggest that early prophase barriers to utilization of sister chromatids as repair templates do not depend on central region assembly.

J Biol Chem, 2003 Nov 14, 278(46), 45171 - 81 Epub 2003 Sep 08.
Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes; Lee JH et al.; The Mre11/Rad50 complex is a critical component of the cellular response to DNA double-strand breaks, in organisms ranging from archaebacteria to humans . In mammalian cells, Mre11/Rad50 (M/R) associates with a third component, Nbs1, that regulates its activities and is targeted by signaling pathways that initiate DNA damage-induced checkpoint responses . Mutations in the genes that encode Nbs1 and Mre11 are responsible for the human radiation sensitivity disorders Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder (ATLD), respectively, which are characterized by defective checkpoint responses and high levels of chromosomal abnormalities . Here we demonstrate nucleotide-dependent DNA binding by the human M/R complex that requires the Nbs1 protein and is specific for double-strand DNA duplexes . Efficient DNA binding is only observed with non-hydrolyzable analogs of ATP, suggesting that ATP hydrolysis normally effects DNA release . The alleles of MRE11 associated with ATLD and the C-terminal Nbs1 polypeptide associated with NBS were expressed with the other components and found to form triple complexes except in the case of ATLD 3/4, which exhibits variability in Nbs1 association . The ATLD 1/2, ATLD 3/4, and p70 M/R/N complexes exhibit nucleotide-dependent DNA binding and exonuclease activity equivalent to the wild-type enzyme, although the ATLD complexes both show reduced activity in endonuclease assays . Sedimentation equilibrium analysis of the recombinant human complexes indicates that Mre11 is a stable dimer, Mre11 and Nbs1 form a 1:1 complex, and both M/R and M/R/N form large multimeric assemblies of approximately 1.2 MDa . Models of M/R/N stoichiometry in light of this and previous data are discussed.

J Biol Chem, 2003 Nov 14, 278(46), 45882 - 7 Epub 2003 Sep 09.
ATO3 encoding a putative outward ammonium transporter is an RTG-independent retrograde responsive gene regulated by GCN4 and the Ssy1-Ptr3-Ssy5 amino acid sensor system; Guaragnella N et al.; Respiratory deficient yeast cells such as rhoo petites activate an inter-organelle signaling pathway called retrograde regulation . This results in changes in the expression of a subset of nuclear genes leading to major reconfigurations of metabolism that enable cells to adapt to the respiratory deficient state . Previous studies have focused on the role of three positive regulatory factors in the retrograde pathway, Rtg1p, Rtg2p, and Rtg3p, which are essential for both basal and elevated expressions of some, but not all, retrograde responsive genes . Here we characterize the retrograde regulation of one of those genes, ATO3, whose elevated expression in rhoo petites is largely independent of RTG gene function . ATO3 encodes a member of the YaaH family of proteins that is a putative outward ammonium transporter . We show that Ato3p-green fluorescent protein is preferentially localized to the plasma membrane of mother cells . rhoo petites express more Ato3p-green fluorescent protein in their plasma membrane than do rho+ cells, consistent with the elevated level of ATO3 transcripts in rhoo cells . We find that ATO3 expression has two levels of control, both of which are connected to amino acid sensing and regulation . The first involves GCN4, which is required for the bulk of ATO3 expression . The second involves the Ssy1-Ptr3-Ssy5 amino acid sensor system, which is preferentially required for elevated ATO3 expression in rhoo cells . We propose that ATO3 is induced in rhoo cells to eliminate the excess ammonia that arises because of a potential defect in ammonia assimilation in those cells.

J Biol Chem, 2003 Nov 28, 278(48), 48210 - 8 Epub 2003 Sep 08.
A copper-regulated transporter required for copper acquisition, pigmentation, and specific stages of development in Drosophila melanogaster; Zhou H et al.; The trace element copper is required for normal growth and development, serving as an essential catalytic co-factor for enzymes involved in energy generation, oxidative stress protection, neuropeptide maturation, and other fundamental processes . In yeast and mammals copper acquisition occurs through the action of the Ctr1 family of high affinity copper transporters . Here we describe studies using Drosophila melanogaster to investigate the role of copper acquisition through Ctr1 in normal growth and development . Three distinct Drosophila Ctr1 genes (Ctr1A, Ctr1B, and Ctr1C) have been identified, which have unique expression patterns over the course of development . Interestingly, Ctr1B, which is expressed exclusively during the late embryonic and larval stages of development, is transcriptionally activated in response to nutritionally induced copper deprivation and down-regulated in response to copper adequacy . The generation of Ctr1B mutant flies results in decreased larval copper accumulation, marked body pigmentation defects that parallel defects in tyrosinase activity, and specific developmental arrest under conditions of both nutritional copper limitation and excess . These studies establish that copper acquisition through the Drosophila Ctr1B transporter is crucial for normal growth and in early and specific stages of metazoan development.

FEBS Lett, 2003 Sep 11, 551(1-3), 71 - 7
The carboxyl terminal 17 amino acids within Apg7 are essential for Apg8 lipidation, but not for Apg12 conjugation; Yamazaki-Sato H et al.; In the yeast, Saccharomyces cerevisiae, two ubiquitin-like modifications, Apg12 conjugation with Apg5 and Apg8 lipidation with phosphatidylethanolamine, are essential for autophagy and the cytoplasm-to-vacuole transport of aminopeptidase I (Cvt pathway) . As a unique E1-like enzyme, Apg7 activates two modifiers (Apg12 and Apg8) in an ATP-dependent manner and, for this activity, the carboxyl terminal 40 amino acids are essential . For a better understanding of the function of the carboxyl terminus of Apg7, we performed a sequential deletion of the region . A mutant expressing Apg7DeltaC17 protein, which lacks the carboxyl 17 amino acids of Apg7, showed defects in both the Cvt pathway and autophagy . Apg8 lipidation is inhibited in the mutant, while Apg12 conjugation occurs normally . A mutant expressing Apg7DeltaC13 protein showed a defect in the Cvt pathway, but not autophagy, suggesting that the activity of Apg7 for Apg8 lipidation is more essential for the Cvt pathway than for autophagy . Mutant Apg7DeltaC17 protein is still able to interact with Apg8, Apg12 and Apg3, and forms a homodimer, indicating that the deletion of the carboxyl terminal 17 amino acids has little effect on these interactions and Apg7 dimerization . These results suggest that the carboxyl terminal 17 amino acids of Apg7 play a specific role in Apg8 lipidation indispensable for the Cvt pathway and autophagy.

Anal Chem, 2003 Jul 1, 75(13), 3145 - 52
Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis; Chen J et al.; An integrated proteome concentration/separation approach involving on-line combination of capillary isoelectric focusing (CIEF) with capillary reversed-phase liquid chromatography (CRPLC) is developed for providing significant analyte concentration and extremely high resolving power toward protein and peptide mixtures . Upon completion of analyte focusing, the self-sharpening effect greatly restricts analyte diffusion and contributes to analyte stacking in narrowly focused bands with a concentration factor of approximately 240 . In addition to analyte focusing, CIEF as the first separation dimension resolves proteins/peptides on the basis of their differences in pI and offers greater resolving power than that achieved in strong cation exchange chromatography . The grouping of two highly resolving and completely orthogonal separation techniques of CIEF and CRPLC, together with analyte focusing and concentration, significantly enhances the dynamic range and sensitivity of conventional mass spectrometry toward the identification of low-abundance proteins . The CIEF-based multidimensional separation/concentration platform enables the identification of a greater number of yeast soluble proteins than methods presented in the literature, yet requires a protein loading of only 9.6 microg . This protein loading is 2-3 orders of magnitude lower than those employed by the reported non-gel-based proteome techniques . The distribution of a codon adaptation index value for identified yeast proteins approximates to that predicted for the entire yeast proteome and supports the capability of CIEF-based proteome separation technology for achieving comprehensive proteome analysis . By reducing the inner diameter of chromatography columns from 180 microm to 100 microm, the required protein loading is further decreased from 9.6 microg to 960 ng, illustrating the potential usage of this proteome technology for the analysis of protein profiles within small cell populations or limited tissue samples.

Cell Cycle, 2003 Sep-Oct, 2(5), 479 - 83
Cell cycle-regulated centers of DNA double-strand break repair; Lisby M et al.; In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks . We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker . In response to DNA damage, Rad52 redistributes itself and forms foci specifically during S phase . We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize . Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein . In addition, we show that the two ends of a double-strand break are held tightly together in the majority of cells . Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair.

J Biol Chem, 2003 Nov 21, 278(47), 46396 - 402 Epub 2003 Sep 08.
Expression and function of the mouse V-ATPase d subunit isoforms; Nishi T et al.; We have identified a cDNA encoding a novel isoform of the mouse V-ATPase d subunit (d2) . The protein encoded is 350 amino acids in length and shows 42 and 67% identity to the yeast d subunit (Vma6p) and the mouse d1 isoform, respectively . Reverse transcriptase-PCR analysis using isoform-specific primers demonstrate that d2 is expressed mainly in kidney and at lower levels in heart, spleen, skeletal muscle, and testis . Although d1 and d2 show similar levels of sequence homology to Vma6p, only the d1 isoform can complement the phenotype of a yeast strain in which VMA6 has been disrupted when cells are grown at 30 degrees C . The d2 isoform, however, can complement the vma6Delta phenotype when cells are grown at 25 degrees C . Moreover, partial assembly of the V-ATPase complex on the vacuolar membrane can be detected under these conditions, although assembly is significantly lower than that observed for the strain expressing Vma6p . This reduced assembly is also reflected in a reduced level of concanamycin-sensitive ATPase activity and proton transport in isolated vacuoles . Comparison of the kinetic properties of V-ATPase complexes containing Vma6p and d1 demonstrate that although the Km for ATP hydrolysis is similar (0.26 and 0.31 mm, respectively), the coupling ratio (proton transport/ATP hydrolysis) is approximately 3-6-fold higher for d1-containing complexes than for Vma6p-containing complexes . These results suggest that subunit d may play a role in coupling of proton transport and ATP hydrolysis.

J Biol Chem, 2003 Oct 31, 278(44), 42733 - 6 Epub 2003 Sep 08.
Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex; Cai Y et al.; The mammalian ATM/PI 3-kinase-related TRRAP protein was previously found to be a component of a multi-protein histone acetyltransferase (HAT) complex containing the HAT TIP60 . In this report, we identify a previously uncharacterized protein encoded by the FLJ10914 ORF, which we designate MRGBP, as a new component of the TRRAP/TIP60 HAT complex . In addition, through purification of MRGBP and its associated proteins from HeLa cell nuclear extracts, we identify the thyroid receptor coactivating protein (TRCp120), DMAP1, and the related MRG15 and MRGX proteins as MRGBP-associating proteins, and we present biochemical evidence that they are previously unrecognized components of the TRRAP/TIP60 HAT complex . Taken together, our findings shed new light on the structure and function of the mammalian TRRAP/TIP60 histone acetyltransferase complex.

Cancer Lett, 2003 Sep 10, 199(1), 1 - 7
In vitro aflatoxin B1-induced p53 mutations; Chan KT et al.; The tumor suppressor gene p53 is commonly mutated with high frequencies at certain hot spots in human cancers . In liver cancers there is an especially high frequency of mutations at codon 249 . To study the impact of carcinogen targeting and the role of cytosine methylation on the mutation spectrum, a common liver cancer carcinogen aflatoxin B1 (AFB1), was studied using the p53 cDNA template to examine mutation induction . Subsequent mutations were detected with a yeast p53 functional assay and identified by DNA sequencing . The results indicated that cytosine methylation enhances AFB1-induced guanine mutations at CpG sites . However, no mutations were detected at codon 249.

Structure (Camb), 2003 Sep, 11(9), 1049 - 50
Homology among telomeric end-protection proteins; Theobald DL et al.; Telomere maintenance and end protection are essential for the survival and proliferation of eukaryotic cells, leading to the prediction that components of this system would be highly conserved . In practice, however, evidence for homology among these factors has been elusive, and, in the case of the known end-protection proteins, evolutionary relationships have been postulated largely on the basis of protein structural and functional similarity alone . Here we report support from sequence profile analyses for a significant and specific evolutionary relationship among OB-fold telomeric end-protection factors.

Biochemistry, 2003 Sep 16, 42(36), 10772 - 82
Temperature, pH, and solvent isotope dependent properties of the active sites of resting-state and cyanide-ligated recombinant cytochrome c peroxidase (H52L) revealed by proton hyperfine resonance spectra; Satterlee JD et al.; Comparative proton NMR studies have been carried out on high-spin and low-spin forms of recombinant native cytochrome c peroxidase (rCcP) and its His52 --> Leu variant . Proton NMR spectra of rCcP(H52L) (high spin) and rCcP(H52L)CN (low spin) reveal the presence of multiple enzyme forms in solution, whereas only single enzyme forms are found in spectra of wild-type and recombinant wild-type CcP and CcPCN near neutral pH . The spectroscopic behaviors of these forms have been studied in detail when pH, temperature, and solvent isotope composition were varied . For resting-state rCcP(H52L) the comparatively large NMR line widths compromise resolution, but two specific enzyme forms were found . They were interconvertible on the basis of varying temperature . For rCcP(H52L)CN four magnetically distinct enzyme forms were identified by NMR . It was found that these forms dynamically interconvert with changing pH, temperature, and solvent isotope composition (percent D(2)O) . These studies have identified the alkaline titration of His52 and essentially identical alkaline enzyme forms for natWTCcPCN and rCcP(H52L)CN . From this work we interpret an essential role of His52 in CcP function to be preservation of a single active site structure in addition to the critical role of general base catalysis.

Biochemistry, 2003 Sep 16, 42(36), 10764 - 71
Cyanide binding to cytochrome c peroxidase (H52L); Bidwai A et al.; Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods . Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.2 microM at pH 8.0 . The binding kinetics are biphasic, involving bimolecular association of the two reactants, followed by an isomerization of the enzyme/cyanide complex . The association rate constant could be determined up to pH 8.9 using pH-jump techniques . The association rate constant increases by almost 4 orders of magnitude over the pH range investigated, from 1.8 x 10(2) M(-1) s(-1) at pH 4 to 9.2 x 10(5) M(-1) s(-1) at pH 8.6 . In contrast to wild-type CcP, where the binding of HCN is the dominant binding pathway, CcP(H52L) preferentially binds the cyanide anion . Above pH 8, cyanide binding to CcP(H52L) is faster than cyanide binding to wild-type CcP . Cyanide dissociates 4 times slower from the mutant protein although the pH dependence of the dissociation rate constant is essentially identical for CcP(H52L) and CcP . Isomerization of the CcP(H52L)/cyanide complex is observed between pH 4 and 8 and stabilizes the complex . The isomerization rate constant has a similar magnitude and pH dependence as the cyanide dissociation rate constant, and the two reactions are coupled at low cyanide concentrations . This isomerization has no counterpart in the wild-type CcP/cyanide complex.

Biochemistry, 2003 Sep 16, 42(36), 10659 - 66
Evaluation of cooperative interactions between substructures of iso-1-cytochrome c using double mutant cycles; Wandschneider E et al.; A double mutant cycle has been used to evaluate interaction energies between the global stabilizer mutation asparagine 52 --> isoleucine (N52I) in iso-1-cytochrome c and mutations producing single surface histidines at positions 26, 33, 39, 54, 73, 89, and 100 . These histidine mutation sites are distributed through the four cooperative folding units of cytochrome c . The double mutant cycle starts with the iso-1-cytochrome c variant AcTM, a variant with no surface histidines and with asparagine at position 52 . Isoleucine is added singly at position 52, AcTMI52 variant, as are the surface histidines, AcHX variants, where X indicates the histidine sequence position . The double mutant variants, AcHXI52, provide the remaining corner of the double mutant cycle . The stabilities of all variants were determined by guanidine hydrochloride denaturation and interaction energies were calculated between position 52 and each histidine site . Six of the seven double mutants show additive (AcH33I52, AcH39I52, AcH54I52, AcH89I52, and AcH100I52) stability effects or weak interaction energies (AcH73I52) of the histidine mutations and the N52I mutation, consistent with cooperative effects on protein folding and stability being sparsely distributed through the protein structure . The AcH26I52 variant shows a strong favorable interaction energy, 2.0 +/- 0.5 kcal/mol, between the N52I mutation in one substructure and the addition of His 26 to an adjacent substructure . The data are consistent with an entropic stabilization of the intersubstructure hydrogen bond between His 26 and Glu 44 by the Ile 52 mutation.

Mol Biol Cell, 2003 Dec, 14(12), 4770 - 82 Epub 2003 Sep 05.
Sec3p is needed for the spatial regulation of secretion and for the inheritance of the cortical endoplasmic reticulum; Wiederkehr A et al.; Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion . Unlike all other exocyst structural genes, SEC3 is not essential for growth . Cells lacking Sec3p grow and secrete surprisingly well at 25 degrees C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud . Furthermore, sec3Delta cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to alpha factor . These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion . We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected . Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip . Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip . Sec3p may act in this process as a spatial landmark for cortical ER inheritance.

Mol Biol Cell, 2003 Nov, 14(11), 4592 - 604 Epub 2003 Sep 05.
Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit; Archambault V et al.; We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit . Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region . During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2 . Moderate overexpression of Cdc6 promotes viability of CLB2Deltadb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain . These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit . Consistent with this idea, we observed a cytokinesis defect in cdh1Delta sic1Delta cdc6Delta2-49 triple mutants . However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work . We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

Mol Biol Cell, 2003 Dec, 14(12), 4971 - 83 Epub 2003 Sep 05.
Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum; Hua Z et al.; Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family . We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin . Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles . Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex . Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature . These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway . We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.

Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10746 - 51 Epub 2003 Sep 05.
Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase; Mahajan KN et al.; Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general double-strand break (DSB) repair pathways . We report an association between TdT and a 55-kDa protein in lymphoid cells . This protein, identified as hPso4, is a homolog of the protein encoded by the PS04/PRP19 gene in Saccharomyces cerevisiae that has pleiotropic functions in DNA recombination and error-prone repair . Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA . hPso4 protein is induced 15- to 30-fold in cells by gamma radiation and chemical mutagens but not by UV treatment . Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage . We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.

Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10794 - 9 Epub 2003 Sep 05.
Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice; Cheng HL et al.; SIRT1 is a mammalian homolog of the Saccharomyces cerevisiae chromatin silencing factor Sir2 . Dominant-negative and overexpression studies have implicated a role for SIRT1 in deacetylating the p53 tumor suppressor protein to dampen apoptotic and cellular senescence pathways . To elucidate SIRT1 function in normal cells, we used gene-targeted mutation to generate mice that express either a mutant SIRT1 protein that lacks part of the catalytic domain or has no detectable SIRT1 protein at all . Both types of SIRT1 mutant mice and cells had essentially the same phenotypes . SIRT1 mutant mice were small, and exhibited notable developmental defects of the retina and heart, and only infrequently survived postnatally . Moreover, SIRT1-deficient cells exhibited p53 hyperacetylation after DNA damage and increased ionizing radiation-induced thymocyte apoptosis . In SIRT1-deficient embryonic fibroblasts, however, p53 hyperacetylation after DNA damage was not accompanied by increased p21 protein induction or DNA damage sensitivity . Together, our observations provide direct evidence that endogenous SIRT1 protein regulates p53 acetylation and p53-dependent apoptosis, and show that the function of this enzyme is required for specific developmental processes.

J Biol Chem, 2003 Nov 28, 278(48), 48051 - 8 Epub 2003 Sep 05.
Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent, and its lipidation to very low density lipoprotein occurs post-ER; Gusarova V et al.; Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL) . Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs . Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23 . Moreover, ER exit of apoB was inhibited by dominant-negative Sar1 . Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER . Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs . VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells . We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Sep, 35(9), 823 - 8
{Identification of interaction and interaction domains between neuroglobin and Na(+), K(+)-ATPase beta2 subunit}; Xu WL et al.; The pre-transformed human fetal brain cDNA library was used to screen the protein interacting with neuroglobin by using yeast two hybrid system III from ClonTech Inc . The protein encoded by one of the clones interacting with neuroglobin (NGB) was confirmed to be the C terminus of the Na(+), K(+)-ATPase beta2 subunit (NKA1b2) based on amino acid sequences . Then the full-length coding region cDNA sequence of NKA1b2 was obtained from human fetal brain cDNA library by PCR . A set of experiments were designed to test the interaction between NGB and NKA1b2 . Interaction between NGB and NKA1b2 was confirmed by binding assay in vitro . Furthermore, the interaction was also proved by co-immunoprecipitation test in vivo . Moreover, the structure integrity of neuroglobin was found to be essential for the interaction between NGB and NKA1b2 by yeast two hybrid method with a series of neuroglobin truncated mutants.

Science, 2003 Sep 5, 301(5638), 1387 - 91
Autophagy genes are essential for dauer development and life-span extension in C . elegans; Melendez A et al.; Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes . Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells . Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C . elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension . Dauer formation is associated with increased autophagy and also requires C . elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10 . Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C . elegans.

Front Biosci, 2003 Sep 01, 8, d1275 - 87
Regulation of CDC14: pathways and checkpoints of mitotic exit; Bembenek J et al.; Progression of the mitotic cell cycle is driven by fluctuations of the cyclin-dependent kinase (Cdk) activities . Entry into mitosis is promoted by the elevated activity of Cdk1 associated with B-type cyclins . Conversely, exit from mitosis requires the inactivation of Cdk1 and the dephosphorylation of at least a subset of Cdk1 substrates . The Cdc14 family of phosphatases antagonizes the action of Cdk1, and is thus a major player in controlling the mitotic exit . We review recent discoveries in several model systems that have shed light on the function of Cdc14 and propose a general framework within which Cdc14 plays conserved roles in regulating the exit from mitosis and cytokinesis.

Gene, 2003 Aug 14, 313, 17 - 42
Ribosome assembly in eukaryotes; Fromont-Racine M et al.; Ribosome synthesis is a highly complex and coordinated process that occurs not only in the nucleolus but also in the nucleoplasm and the cytoplasm of eukaryotic cells . Based on the protein composition of several ribosomal subunit precursors recently characterized in yeast, a total of more than 170 factors are predicted to participate in ribosome biogenesis and the list is still growing . So far the majority of ribosomal factors have been implicated in RNA maturation (nucleotide modification and processing) . Recent advances gave insight into the process of ribosome export and assembly . Proteomic approaches have provided the first indications for a ribosome assembly pathway in eukaryotes and confirmed the dynamic character of the whole process.

Curr Biol, 2003 Sep 2, 13(17), 1564 - 9
The actin-regulating kinase Prk1p negatively regulates Scd5p, a suppressor of clathrin deficiency, in actin organization and endocytosis; Henry KR et al.; Endocytosis is a dynamic process requiring a network of interacting proteins that assemble and disassemble during cargo capture and vesicle formation . A major mechanism for regulation of this process involves the reversible phosphorylation of endocytic factors . Recently, members of a new kinase family, the Ark/Prk kinases, which include mammalian AAK1 and GAK as well as yeast Prk1p, Ark1p, and Akl1p, were shown to regulate components of the endocytic machinery . These include animal AP-1/AP-2 mu chains and yeast Pan1p (Eps15-like), Sla1p, and epsins, but other potential targets are likely . SCD5, an essential yeast gene, was identified as a suppressor of clathrin deficiency . We also showed that Scd5p is required for normal cortical actin organization and endocytosis, possibly as a targeting subunit for protein phosphatase type 1 (PP1) . Scd5p contains a central triple repeat (3R) motif related to a known Prk1p consensus phosphorylation site L/IxxQxTG, except that Q is replaced by T . In this study we demonstrate that the Scd5p 3R sequence is phosphorylated by Prk1p to negatively regulate Scd5p . Furthermore, we show that Prk1p, Ark1p, and Akl1p have different substrate specificities and play distinct roles in actin organization and endocytosis.

Curr Biol, 2003 Sep 2, 13(17), 1549 - 56
DNA damage foci at dysfunctional telomeres; Takai H et al.; We report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells . Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, gamma-H2AX, Rad17, ATM, and Mre11 . We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF) . The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK . By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation . A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway . However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway . We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response . TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres . Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions.

Plant Mol Biol, 2003 Jun, 52(3), 569 - 78
Salt-dependent expression of a nitrate transporter and two amino acid transporter genes in Mesembryanthemum crystallinum; Popova OV et al.; Uptake and transport of inorganic nitrogen and allocation of amino acids are essential for plant growth and development . To study the effects of salinity on the regulation of transporters for nitrogenous compounds, we characterized the putative nitrate transporter McNRT1 and the amino acid transporters McAAT1 and McAAT2 from Mesembryanthemum crystallinum . By transcript analyses, McAAT1 was found in leaves, McAAT2 in roots, and McNRT1 in both tissues . By in situ PCR McNRT1 was localized, for example, to epidermal and vascular cells whereas McAAT2 was abundant in most cell types in mature roots and McAAT1 in the mesophyll and cells neighbouring xylem vessels in leaves . In response to salt stress, expression of McAAT2 and McNRT1 was stimulated in the root vasculature . In addition, McNRT1 and McAAT1 signals increased in the leaf phloem . Growth of yeast mutants deficient in histidine uptake was restored by McAAT2 whereas both McAAT1 and McAAT2 complemented a yeast mutant carrying a defect in proline uptake . The differential and cell-specific transcriptional activation of genes encoding nitrogen and amino acid transporters under salt stress suggest complex coordinated regulation of these transporter families to maintain uptake and distribution of nitrogenous compounds and amino acids under conditions of high salinity in plants.

J Chem Ecol, 2003 Aug, 29(8), 1871 - 87
Mechanisms of behavioral alterations of parasitoids reared in artificial systems; Gandolfi M et al.; A high quality of mass reared parasitoids is required for successful biological control of pest insects . Although the phenomenon of behavioral deterioration of parasitoids due to rearing in artificial conditions is well known, its significance is often underestimated, and the underlying mechanisms are poorly investigated . We quantified behavioral alterations of parasitoids reared in an artificial system vs . a natural system and elucidated some of the mechanisms involved . The model systems consisted of apple fruits (natural system) or an artificial diet devoid of apple (artificial system), the herbivore Cydia pomonella, and its larval parasitoid Hyssopus pallidus, a candidate biological control agent . Two parasitoid strains, one reared for 30 generations in the natural system and one in the artificial system, were compared by using the females' ability to respond to frass from codling moth caterpillars fed on apple fruits (apple-frass) . The searching response of parasitoids reared in the artificial system compared to those reared in the natural system was reduced by an average of 53.2% . Gas chromatography-mass spectrometry (GC-MS) analyses of the two types of caterpillars' food and of the two corresponding types of frass showed that 15 compounds were present only in apple fruits and apple-frass, three compounds only in artificial diet and artificial-diet-frass, while four compounds were present in both frass types but not in the food sources . This suggests the presence of a food-derived and a host-derived component in the frass . Results from both bioassays and chemical analyses indicate that the kairomonal activity of the frass is due to both apple fruit and host components . The reduced response of parasitoids reared in artificial conditions might, therefore, be due to a lack of recognition of the apple fruit component . In a further experiment, the two parasitoid strains were reared in the opposite system for one generation . While the response to the host frass was significantly reduced in parasitoids that emerged from the artificial system, it was fully restored in parasitoids that emerged from the natural system . This indicates that the behavioral alteration was related to a learning process during ontogenesis rather than to a selection exhibited over generations.

Cancer Immunol Immunother, 2004 Jan, 53(1), 17 - 25 Epub 2003 Sep 02.
In situ recruitment of antigen-presenting cells by intratumoral GM-CSF gene delivery; Pan PY et al.; Proper antigen presentation is paramount to the induction of effective and persistent antitumor immune responses . In a murine model of hepatic metastasis of colon cancer, we found that the numbers of in situ mature dendritic cells (DCs) and macrophages in tumor-infiltrating leukocytes (TILs) were significantly increased in mice treated with the combination therapy of herpes simplex virus thymidine kinase, interleukin 2, and GM-CSF genes when compared with control groups without GM-CSF treatment . Significantly higher levels of IFN-gamma, MIP-1 alpha, mIL-12, and GM-CSF were detected in the tumor after the combination therapy . T cells isolated from the combination therapy-treated mice exhibited higher ex vivo direct CTL activity than those from other treatment groups . Antigen-presenting cells (APCs) enriched from the TILs and liver of the combination therapy-treated mice induced higher levels of proliferation by the splenocytes from long-term surviving mice that had been cured of tumors at early time points (days 4 and 7) whereas significant APC activity was only observed in the spleen at the latter time point (day 7, 14) after the combination therapy . In contrast, APCs isolated from tk or tk + IL-2-treated mice did not induce any significant proliferation . Subcutaneous injection of fluorescence-labeled latex microspheres followed by the combination therapy showed a similar sequential trafficking of microspheres, day 4 after the combination therapy to tumor and day 14 to spleen . The results suggest that APCs recruited by intratumoral gene delivery of GM-CSF can capture antigens, mature to a stage suitable for antigen presentation, and subsequently migrate to the spleen where they can efficiently stimulate antigen-specific T cells.

Nucleic Acids Res . 2003 Sep 15;31(18):e109.
Optimizing spotting solutions for increased reproducibility of cDNA microarrays; Rickman DS et al.; The ability to extract meaningful information from transcriptome technologies such as cDNA microarrays relies on the precision, sensitivity and reproducibility of the measured values for a given gene across multiple samples . Given the lack of a 'gold standard' for the production of microarrays using current technologies, there is a high degree of variation in the quality of data derived from microarray experiments . Poor reproducibility not only adds to the cost of a given study but also leads to data sets that are difficult to interpret . For glass slide DNA microarrays, much of this variation is introduced systematically, during the spotting, or deposition, of the DNA onto the slide surface . In order to reduce this type of systematic variation we tested spotting solutions containing different detergent additives in the presence of one of two different denaturants and determined their effect on spot quality . We show that spotting cDNA in a solution consisting of the zwitterionic detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propane sulfonate (CHAPS) in the presence of formamide or dimethyl sulfoxide yields spots of superior quality in terms of morphology, size homogeneity and signal reproducibility, as well as overall intensity, when used with popular, commercially available slides.

Nucleic Acids Res, 2003 Sep 15, 31(18), 5356 - 67
Identification of Daxx interacting with p73, one of the p53 family, and its regulation of p53 activity by competitive interaction with PML; Kim EJ et al.; We performed a yeast two-hybrid screen using p73alpha, which is a member of the p53 family, as bait . We found that the p53 family members were functionally associated with Daxx, which was described originally as a cytoplasmic mediator of Fas signaling, but has been identified recently as a nuclear protein that co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription . Extensive yeast two-hybrid assays indicated a physical interaction between a region including the oligomerization domain (OD) of p73alpha (amino acids 345-380) or p53 (amino acids 319-360) and amino acids 161-311 and 667-740 (C-terminal S/P/T-rich domain) of hDaxx, which is the common binding region of Fas, ASK1 and PML . This interaction was further confirmed by in vitro GST pull-down and in vivo immunoprecipitation assays . Both Daxx and p73/p53 co-localized in nuclear dot-like structures, which are probably nuclear PML oncogenic domains (PODs) or the nuclear domain NB10 . Transient co-expression of Daxx resulted in strong inhibition of p73- and p53-mediated transcriptional activation of the synthetic p53-responsive and p21WAF1 promoters . Consequently, Gal4-Daxx repressed basal transcription in a dose-dependent manner . Treatment with trichostatin A, which is an inhibitor of histone deacetylase, or PML over-expression relieved Daxx-mediated transcriptional repression of p53 . The mechanism underlying PML-mediated derepression appears to be competitive binding between Daxx, p53 and PML . Taken together, these findings delineate a transcriptional regulatory network that is modulated by differential Daxx-p53-PML interactions in the nuclear PODs . Therefore, Daxx is implicated in the regulation of the cell cycle and apoptosis through transcriptional regulation of p53 and possibly its family members.

Nucleic Acids Res, 2003 Sep 15, 31(18), 5305 - 16
Functional dissection of the C-terminal domain of type II DNA topoisomerase from the kinetoplastid hemoflagellate Leishmania donovani; Sengupta T et al.; The amino acid sequences of the C-terminal domain (CTD) of the type II DNA topoisomerases are divergent and species specific as compared with the highly conserved N-terminal and central domains . A set of C-terminal deletion mutants of Leishmania donovani topoisomerase II was constructed . Removal of more than 178 amino acids out of 1236 amino acid residues from the C-terminus inactivates the enzyme, whereas removal of 118 amino acids or less has no apparent effect on the ability of the parasite enzyme to complement a temperature-sensitive mutation of the Saccharomyces cerevisiae topoisomerase II gene . Deletion analysis revealed a potent nuclear localization signal (NLS) within the amino acid residues 998-1058 . Immunomicroscopy results suggest that the removal of an NLS in the CTD is likely to contribute to the physiological dysfunction of these proteins . Modeling of the LdTOP2 based on the crystal structure of the yeast type II DNA topoisomerase showed that the parasite protein assumes a structure similar to its yeast counterpart harboring all the conserved residues in a structurally similar position . However, a marked difference in electrostatic potential was found in a span of 60 amino acid residues (998-1058), which also do not have any homology with topoisomerase II sequences . Such significant differences can be exploited by the structure-based design of selective inhibitors using the structure of the Leishmania enzyme as a template.

J Biol Chem, 2003 Nov 14, 278(46), 45049 - 55 Epub 2003 Sep 03.
Csg1p and newly identified Csh1p function in mannosylinositol phosphorylceramide synthesis by interacting with Csg2p; Uemura S et al.; Csg1p and Csg2p have been shown to be involved in the synthesis of mannosylinositol phosphorylceramide (MIPC) from inositol phosphorylceramide . YBR161w, termed CSH1 here, encodes a protein that exhibits a strong similarity to Csg1p . To examine whether Csh1p also functions in MIPC synthesis, we performed a {3H}dihydrosphingosine labeling experiment . Deltacsg1 cells exhibited only a reduction in the synthesis of mannosylated sphingolipids compared with wild-type cells, whereas the Deltacsg1 Deltacsh1 double deletion mutant exhibited a total loss . These results indicated that Csg1p and Csh1p have redundant functions in MIPC synthesis . Analyses using Deltacsg1 and Deltacsh1 cells in the Deltaipt1, Deltasur2, or Deltascs7 genetic background demonstrated that Csh1p has a different substrate specificity from Csg1p . We also revealed that Csg2p interacts with both Csg1p and Csh1p . Deletion of the CSG2 gene reduced the Csg1p activity and abolished the Csh1p activity . These results suggested that two distinct inositol phosphorylceramide mannosyltransferase complexes, Csg1p-Csg2p and Csh1p-Csg2p, exist.

Chem Biol, 2003 Aug, 10(8), 713 - 22
A synthetic miniprotein that binds specific DNA sequences by contacting both the major and the minor groove; Blanco JB et al.; Attachment of a slightly modified basic region of a bZIP protein (GCN4) to a distamycin-related tripyrrole provides a bivalent system capable of binding with high affinity to specific DNA sequences . Appropriate adjustment of the linker between the two units has led to a hybrid that binds a 9 base-pair-long DNA site (TTTTATGAC) with low nanomolar affinity at 4 degrees C . Circular dichroism and gel retardation studies indicate that the binding occurs by simultaneous insertion of the bZIP basic region into the DNA major groove and the tripyrrole moiety into the minor groove of the flanking sequence . Analysis of hybrids bearing alternative linkers revealed that tight, specific binding is strongly dependent on the length and nature of the connecting unit.

Biochem J, 2003 Sep 15, 374(Pt 3), e1 - 2
Trials, tribulations and finally, a transporter: the identification of the mitochondrial pyruvate transporter; Sugden MC et al.; Pyruvate occupies a central role in energy homoeostasis, and dysregulation of its cellular disposition underlies many metabolic disturbances . Although the mitochondrial membrane pyruvate transporter has been characterized, its molecular identity has proved elusive . Recent work has now identified a single candidate protein for the mitochondrial pyruvate carrier in yeast, opening the way for further studies in mammalian systems, which may have important therapeutic applications within the context of metabolic disease.

Ment Retard Dev Disabil Res Rev, 2003, 9(3), 184 - 95
A review of the biological bases of ADHD: what have we learned from imaging studies?
Durston S.
Attention Deficit Hyperactivity Disorder (ADHD) is a common and impairing neuropsychiatric disorder with onset at preschool age . Although a significant amount of progress has been made investigating the neurobiology of this disorder, its precise etiology still remains unclear . Converging evidence from studies of the neuropharmacology, genetics, neuropsychology, and neuroimaging of ADHD imply the involvement of fronto-striatal circuitry in ADHD . However, while it does appear that poor inhibitory control and the deficits in fronto-striatal circuitry associated with it are central, there is evidence to suggest that more posterior cerebral areas are also implicated in this disorder . Anatomical studies suggest widespread reductions in volume throughout the cerebrum and cerebellum, while functional imaging studies suggest that affected individuals activate more diffuse areas than controls during the performance of cognitive tasks . The future impact of new MR imaging methodologies on the field is discussed .

J Biol Chem, 2003 Dec 12, 278(50), 49812 - 8 Epub 2003 Sep 02.
The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore; Cesura AM et al.; The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention . Yet, despite detailed functional characterization, the structure of this channel remains elusive . Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target . The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A . Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, {3H}Ro 68-3400 was used as an affinity label for the identification of its protein target . In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, {3H}Ro 68-3400 predominantly labeled a protein of approximately 32 kDa . This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC) . Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites . While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.

J Biol Chem, 2003 Nov 14, 278(46), 45288 - 95 Epub 2003 Sep 01.
Multiple Nhp6 molecules are required to recruit Spt16-Pob3 to form yFACT complexes and to reorganize nucleosomes; Ruone S et al.; The Saccharomyces cerevisiae Nhp6 protein contains a DNA-binding motif that is similar to those found in the high mobility group B family of chromatin proteins . Nhp6 bound to nucleosomes and made at least two changes in them: the nucleosomal DNA became more sensitive to DNase I at specific sites, and the nucleosomes became competent to bind Spt16-Pob3 to form yFACT.nucleosome complexes . Both changes occurred at similar concentrations of Nhp6, suggesting that they reflect the same structural reorganization of the nucleosome . Nucleosomes have multiple binding sites for Nhp6, and structural reorganization was associated with a concentration of Nhp6 about 10-fold higher than that needed for simple binding . We propose that the coordinated action of multiple Nhp6 molecules is required to convert nucleosomes to an alternative form as the first step in a two-step reorganization of nucleosomes with the second step being dependent on Spt16-Pob3 . The presence of linker DNA had only subtle effects on these processes, indicating that both Nhp6 and yFACT act on core nucleosome structure rather than on the interaction between nucleosomes and adjacent DNA . These results suggest that Nhp6 and the related high mobility group B proteins may have a general role in promoting rearrangements of chromatin by initiating the destabilization of core nucleosomal structure.

Genome Res, 2003 Sep, 13(9), 2052 - 8
Asymmetric sequence divergence of duplicate genes; Conant GC et al.; Much like humans, gene duplicates may be created equal, but they do not stay that way for long . For four completely sequenced genomes we show that 20%-30% of duplicate gene pairs show asymmetric evolution in the amino acid sequence of their protein products . That is, one of the duplicates evolves much faster than the other . The greater this asymmetry, the greater the ratio Ka/Ks of amino acid substitutions (Ka) to silent substitutions (Ks) in a gene pair . This indicates that most asymmetric divergence may be caused by relaxed selective constraints on one of the duplicates . However, we also find some candidate duplicates where positive (directional) selection of beneficial mutations (Ka/Ks > 1) may play a role in asymmetric divergence . Our analysis rests on a codon-based model of molecular evolution that allows a test for asymmetric divergence in Ka . The method is also more sensitive in detecting positive selection (Ka/Ks > 1) than models relying only on pairwise gene comparisons.

Genome Biol . 2003;4(9):337 . Epub 2003 Aug 28.
An international showcase of bioinformatics research; Vision T; A report on the 11th International Conference on Intelligent Systems for Molecular Biology, Brisbane, Queensland, Australia, 29 June - 3 July 2003.

Nat Rev Genet, 2003 Sep, 4(9), 679 - 89
The development and regulation of gene repair; Liu L et al.; A technique that can direct the repair of a genetic mutation in a human chromosome using the DNA repair machinery of the cell is under development . Although this approach is not as mature as other forms of gene therapy and fundamental problems continue to arise, it promises to be the ultimate therapy for many inherited disorders . There is a continuing effort to understand the potential and the limitations of this controversial approach.

Biochem Biophys Res Commun, 2003 Sep 19, 309(2), 344 - 50
Functional expression and processing of rat choline dehydrogenase precursor; Huang S et al.; Choline dehydrogenase (CHDH, EC 1.1.99.1) was purified from rat liver mitochondria, and the amino terminal sequence was determined and used to clone a full-length cDNA encoding a protein precursor (CHDHp) of 599 amino acids (64kDa) . Sequence analysis identified a possible processing site that meets the requirements of IMP in comparison to the previously determined N-terminal sequence of mature rat CHDH . This suggested that the precursor might be processed in the intermembrane space . Confocal imaging showed that expression of the CHDHp-GFP fusion gene in NIH-3T3 cells led to fusion proteins being targeted to mitochondria . In addition, expression of a recombinant version of the CHDHp gene in Saccharomyces cerevisiae led to enrichment of the target protein in the mitochondrial inner membrane . The expressed protein conferred choline dehydrogenase activity, suggesting that both functional domains (FAD and the iron sulfur cluster) were properly assembled and that the mature CHDH was appropriately located in the inner mitochondria membrane.

Mol Microbiol, 2003 Sep, 49(6), 1595 - 603
Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis; Ju S et al.; Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder . The therapeutic mechanism of VPA has not been established . We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol-1-P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis . To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug . A decrease in intracellular inositol was apparent immediately after addition of VPA . Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol-responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase . Incorporation of 32Pi into total phospholipids was significantly decreased . Pulse labelling of CDP-DG and PG, shown previously to increase during inositol depletion, increased within 30 min . However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min . PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity . Thus, in contrast to the effect of chronic VPA treatment, short-term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.

EMBO Rep, 2003 Sep, 4(9), 872 - 6
Gcn4 occupancy of open reading frame regions results in the recruitment of chromatin-modifying complexes but not the mediator complex; Topalidou I et al.; Eukaryotic transcriptional activators usually recognize short DNA motifs, which are not only located within promoter regions, but also scattered throughout the genome . Assuming that the function of activators at non-promoter regions is wasteful and perhaps harmful, one can ask whether such binding is somehow prevented or if transcription is blocked at a downstream step . Here, we show that the yeast transcriptional activator Gcn4 is associated in vivo with several non-promoter euchromatic sites . This association results in the recruitment of the SAGA (Spt3/Ada/Gcn5/acetyltransferase) complex and the consequent activity of the Gcn5 histone acetyltransferase . The functional recruitment of the Swi/Snf nucleosome-remodelling complex was also evident at sites located in positioned nucleosomes . We show that this assemblage of coactivator complexes is not productive because of the absence of core promoter elements, other than the TATA box, that are required for stable mediator recruitment.

Biochemistry (Mosc), 2003 Aug, 68(8), 838 - 49
Fusion, fragmentation, and fission of mitochondria; Polyakov VY et al.; Individual mitochondria which form the chondriom of eucaryotic cells are highly dynamic systems capable of fusion and fragmentation . These two processes do not exclude one another and can occur concurrently . However, fragmentation and fusion of mitochondria regularly alternate in the cell cycle of some unicellular and multicellular organisms . Mitochondrial shapes are also described which are interpreted as intermediates of their "equational" division, or fission . Unlike the fragmentation, the division of mitochondria, especially synchronous division, is also accompanied by segregation of mitochondrial genomes and production of specific "dumbbell-shaped" intermediates . This review considers molecular components and possible mechanisms of fusion, fragmentation, and fission of mitochondria, and the biological significance of these processes is discussed.

Biochemistry (Mosc), 2003 Aug, 68(8), 827 - 37
Copper chaperones, intracellular copper trafficking proteins . Function, structure, and mechanism of action; Markossian KA et al.; This review summarizes findings on a new family of small cytoplasmic proteins called copper chaperones . The copper chaperones bind and deliver copper ions to intracellular compartments and insert the copper into the active sites of specific partners, copper-dependent enzymes . Three types of copper chaperones have been found in eukaryotes . Their three-dimensional structures have been determined, intracellular target proteins identified, and mechanisms of action have been revealed . The Atx1 copper chaperone binds Cu(I) and interacts directly with the copper-binding domains of a P-type ATPase copper transporter, its physiological partner . The copper chaperone CCS delivers Cu(I) to Cu,Zn-superoxide dismutase 1 . Cox17 and Cox11 proteins serve as copper chaperones for cytochrome c oxidase, a copper-dependent enzyme.

EMBO Rep, 2003 Sep, 4(9), 906 - 11 Epub 2003 Aug 15.
An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1; Gerber J et al.; Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia . The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis . Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1 . Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1 . Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1 . Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process . The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.

J Biol Chem, 2003 Nov 7, 278(45), 44178 - 87 Epub 2003 Aug 28.
Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts; Kataoka Y et al.; The development of myoblasts is regulated by various growth factors as well as by intrinsic muscle-specific transcriptional factors . In this study, we analyzed the roles for STAT3 in the growth and differentiation of myoblasts in terms of cell cycle regulation and interaction with MyoD using C2C12 cells . Here we found that STAT3 inhibited myogenic differentiation induced by low serum or MyoD as efficiently as the Ras/mitogen-activated protein kinase cascade . As for this mechanism, we found that STAT3 not only promoted cell cycle progression through the induction of c-myc but also inhibited MyoD activities through direct interaction . STAT3 inhibited not only DNA binding activities of MyoD but also its transcriptional activities . However, the inhibited transcriptional activities were restored by the supplement of p300/CBP and PCAF, suggesting that STAT3 might deprive MyoD of these transcriptional cofactors . In addition, we found that MyoD inhibited DNA binding activities of STAT3, thereby inhibiting STAT3-dependent cell growth and survival of Ba/F3 cells . These results suggest that the development of muscle cells is regulated by the coordination of cytokine signals and intrinsic transcription factors.

J Biol Chem, 2003 Nov 7, 278(45), 45027 - 33 Epub 2003 Aug 28.
Two distinct pathways for inhibiting pds1 ubiquitination in response to DNA damage; Agarwal R et al.; The presence of DNA damage activates a conserved cellular response known as the DNA damage checkpoint pathway . This pathway induces a cell cycle arrest that persists until the damage is repaired . Consequently, the failure to arrest in response to DNA damage is associated with genomic instability . In budding yeast, activation of the DNA damage checkpoint pathway leads to a mitotic cell cycle arrest . Following the detection of DNA damage, the checkpoint signal is transduced via the Mec1 kinase, which in turn activates two kinases, Rad53 and Chk1 that act in parallel pathways to bring about the cell cycle arrest . The downstream target of Rad53 is unknown . The target of Chk1 is Pds1, an inhibitor of anaphase initiation whose degradation is a prerequisite for mitotic progression . Pds1 degradation is dependent on its ubiquitination by the anaphase-promoting complex/cyclosome ubiquitin ligase, acting in conjunction with the Cdc20 protein (APC/CCdc20) . Previous studies showed that the Rad53 and Chk1 pathways independently lead to Pds1 stabilization but the mechanism for this was unknown . In the present study we show that both the Chk1 and the Rad53 pathways inhibit the APC/CCdc20-dependent ubiquitination of Pds1 but they affect different steps of the process: the Rad53 pathway inhibits the Pds1-Cdc20 interaction whereas Chk1-dependent phosphorylation of Pds1 inhibits the ubiquitination reaction itself . Finally, we show that once the DNA damage is repaired, Pds1 dephosphorylation is involved in the recovery from the checkpoint induced cell cycle arrest.

Mol Biochem Parasitol, 2003 Aug 31, 130(2), 101 - 10
Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes; Henriques C et al.; African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts . To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients . We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei . The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range . The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals . TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites . Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

Nature, 2003 Aug 28, 424(6952), 1078 - 83
S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex; Katou Y et al.; The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome . This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards . When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication . A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA . Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks . However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown . Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae . When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.

Mol Cell Biol, 2003 Sep, 23(18), 6339 - 49
Ssu72 protein mediates both poly(A)-coupled and poly(A)-independent termination of RNA polymerase II transcription; Steinmetz EJ et al.; Termination of transcription by RNA polymerase II (Pol II) is a poorly understood yet essential step in eukaryotic gene expression . Termination of pre-mRNA synthesis is coupled to recognition of RNA signals that direct cleavage and polyadenylation of the nascent transcript . Termination of nonpolyadenylated transcripts made by Pol II in the yeast Saccharomyces cerevisiae, including the small nuclear and small nucleolar RNAs, requires distinct RNA elements recognized by the Nrd1 protein and other factors . We have used genetic selection to characterize the terminator of the SNR13 snoRNA gene, revealing a bipartite structure consisting of an upstream element closely matching a Nrd1-binding sequence and a downstream element similar to a cleavage/polyadenylation signal . Genome-wide selection for factors influencing recogniton of the SNR13 terminator yielded mutations in the gene coding for the essential Pol II-binding protein Ssu72 . Ssu72 has recently been found to associate with the pre-mRNA cleavage/polyadenylation machinery, and we find that an ssu72 mutation that disrupts Nrd1-dependent termination also results in deficient poly(A)-dependent termination . These findings extend the parallels between the two termination pathways and suggest that they share a common mechanism to signal Pol II termination.

J Biol Chem, 2003 Nov 14, 278(46), 45903 - 14 Epub 2003 Aug 27.
Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like, proline-rich, and GTPase-activating protein domains of a novel Rho GTPase-activating protein, BPGAP1; Shang X et al.; RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility . Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs) . We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP . BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1 . "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins . Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells . Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1 . However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia . Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.

J Biol Chem, 2003 Nov 14, 278(46), 45730 - 6 Epub 2003 Aug 27.
Solution structure of the plant disease resistance-triggering protein NIP1 from the fungus Rhynchosporium secalis shows a novel beta-sheet fold; van't Slot KA et al.; Activation of the disease resistance response in a host plant frequently requires the interaction of a plant resistance gene product with a corresponding, pathogenderived signal encoded by an avirulence gene . The products of resistance genes from diverse plant species show remarkable structural similarity . However, due to the general paucity of information on pathogen avirulence genes the recognition process remains in most cases poorly understood . NIP1, a small protein secreted by the fungal barley pathogen Rhynchosporium secalis, is one of only a few fungal avirulence proteins identified and characterized to date . The defense-activating activity of NIP1 is mediated by barley resistance gene Rrs1 . In addition, a role of the protein in fungal virulence is suggested by its nonspecific toxicity in leaf tissues of host and non-host cereals as well as its resistance gene-independent stimulatory effect on the plant plasma membrane H+-ATPase . Four naturally occurring NIP1 isoforms are characterized by single amino acid alterations that affect the different activities in a similar way . As a step toward unraveling the signal perception/transduction mechanism, the solution structure of NIP1 was determined . The protein structure is characterized by a novel fold . It consists of two parts containing beta-sheets of two and three anti-parallel strands, respectively . Five intramolecular disulfide bonds, comprising a novel disulfide bond pattern, stabilize these parts and their position with respect to each other . A comparative analysis of the protein structure with the properties of the NIP1 isoforms suggests two loop regions to be crucial for the resistance-triggering activity of NIP1.

J Steroid Biochem Mol Biol, 2003 Jun, 85(2-5), 201 - 8
Activation function-1 domain of androgen receptor contributes to the interaction between two distinct subnuclear compartments; Goto K et al.; The nucleus contains different sets of functional compartments often called "speckles" . The splicing factor compartment (SFC) has been speculated to consist of SFs and transcription factors, which thus make transcription-splicing coupling possible at the periphery of SFC . Androgen receptor (AR), as well as glucocorticoid receptor (GR), is unique since most, if not all, of its activities are mediated via the constitutive activity of the activation function-1 (AF-1) function . Transcriptionally active AR produces 250-400 subnuclear fine speckles11 shared with GR or estrogen receptor (ER), which colocalize with chiefly activation function-2 (AF-2)-interacting p160 family- or CBP-related speckles . We herein report the isolation of ANT-1 (AR N-terminal domain (NTD) transactivating protein-1) enhancing autonomous AF-1 transactivation function of AR or GR, but not of estrogen receptor alpha (ERalpha) . The ANT-1 was identical to a binding protein of human splicing factor U5 snRNP (U5 snRNP-associated protein) . ANT-1 was compartmentalized into 15-20 coarse SFC speckles which were spatially distinct from but surrounded by the AR compartments . Our results suggest that ANT-1 may play a key role in the molecular interaction between two spatially distinct subnuclear compartments in a receptor-specific fashion, and thereby induce the strong autonomous transactivation functions either of AR- or GR-AF-1.

J Steroid Biochem Mol Biol, 2003 Jun, 85(2-5), 139 - 45
The roles of protein-protein interactions and protein methylation in transcriptional activation by nuclear receptors and their coactivators; Stallcup MR et al.; Hormone-activated nuclear receptors (NR) bind to the promoters of their target genes and recruit coactivator proteins to help activate transcription . The p160 coactivators bind directly to activated NRs and recruit secondary coactivators CBP/p300 with protein acetyltransferase activity and CARM1 with protein methyltransferase activity . To further investigate the components of the p160 coactivator complex and their mechanisms of action, we have used two guiding assumptions . First, the coactivators constitute a signal transduction pathway that convey the signal from DNA-bound NRs to the transcription machinery . Second, each coactivator has signal input and signal output domains that facilitate signal transduction . These assumptions were used to address the mechanism by which CARM1 and the N-terminal region of p160 coactivators transmit activating signals to the transcription machinery . The p160-binding activity of CARM1 is in the same centrally located structural domain as the methyltransferase activity; the p160-binding domain anchors CARM1 to the target gene promoter and thereby serves as its signal input domain . CARM1 has two signal output mechanisms: the protein methyltransferase activity, which methylates histones and other proteins in the transcription initiation complex; and a strong autonomous activation function in the C-terminal region . We identified a protein, CCCP, which binds to the C-terminal region of CARM1 and cooperates synergistically with CARM1 to enhance NR function . We also defined the N-terminal region of p160 coactivators as another signal output domain, which binds a novel coactivator called coiled-coil coactivator (CoCoA) . CoCoA acts synergistically with p160 coactivators to enhance NR function.

Biochemistry (Mosc), 2003 Jun, 68(6), 658 - 61
Trp17 and Glu20 residues in conserved WMN(D/E)PN motif are essential for Aspergillus ficuum endoinulinase (EC 3.2.1.7) activity; Park S et al.; The importance of the WMN(D/E)PN motif, which is well conserved among beta-fructofuranosidases grouped in the glycosylhydrolase family 32, in Aspergillus ficuum endoinulinase was accessed . Each mutant enzyme generated by site-directed mutagenesis of Trp17 in the conserved motif to Gln, Leu, Ser, Pro, Thr, or Met had an activity of less than 1% of the wild type . Another mutant enzyme obtained by mutation of Glu20 in the motif to Ser, Leu, Thr, Gln, Ala, or Val had an enzyme activity of less than 1% of the wild type . Furthermore, the E20D mutant enzyme, in which Glu20 in the conserved motif was replaced with Asp, had 1.1% of the wild type activity . These results clearly indicated that Trp17 and Glu20 are essential for the enzyme activity.

Ontogenez, 2003 Jul-Aug, 34(4), 267 - 72
{Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar}; Odintsova NA et al.; In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present . We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity . The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain . The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture . In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

EMBO J, 2003 Sep 1, 22(17), 4512 - 22
H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA; Larochelle M et al.; H2A.Z has been shown to regulate transcription in yeast, and that function resides in its C-terminal region as the reciprocal portion of H2A cannot substitute for the latter . We show that fusion of a transcriptional activating region to the C-terminal region of H2A, which is substituted for that of H2A.Z, can allow the chimera to fulfil the special role of H2A.Z in positive gene regulation, as well as complement growth deficiencies of htz1delta cells . We further show that the 'transcription' function of H2A.Z is linked to its ability to preferentially localize to certain intergenic DNA regions . Our results suggest that H2A.Z modulates functional interactions with transcription regulatory components, and thus increases its localization to promoters where it helps poise chromatin for gene activation.

EMBO J, 2003 Sep 1, 22(17), 4400 - 8
Import of small Tim proteins into the mitochondrial intermembrane space; Lutz T et al.; Proteins of the intermembrane space (IMS) of mitochondria are typically synthesized without presequences . Little is known about their topogenesis . We used Tim13, a member of the 'small Tim protein' family, as model protein to investigate the mechanism of translocation into the IMS . Tim13 contains four conserved cysteine residues that bind a zinc ion as cofactor . Import of Tim13 did not depend on the membrane potential or ATP hydrolysis . Upon import into mitochondria Tim13 adopted a stably folded conformation in the IMS . Mutagenesis of the cysteine residues or pretreatment with metal chelators interfered with folding of Tim13 in vitro and impaired its import into mitochondria . Upon depletion of metal ions or modification of cysteine residues, imported Tim13 diffused back out of the IMS . We propose an import pathway in which (1) Tim13 can pass through the TOM complex into and out of the IMS in an unfolded conformation, and (2) cofactor acquisition stabilizes folding on the trans side of the outer membrane and traps Tim13 in the IMS, and drives unidirectional movement of the protein across the outer membrane of mitochondria.

EMBO J, 2003 Sep 1, 22(17), 4356 - 64
The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation; Forster A et al.; The 20S proteasome is a large multisubunit assembly that performs most of the intracellular non-lysosomal proteolysis of eukaryotes . Substrates access the proteasome active sites, which are sequestered in the interior of the barrel-shaped structure, through pores that are opened by binding of activator complexes . The crystal structure of yeast proteasome in complex with an 11S activator suggested that activation results from disordering of the proteasome gate residues . Here we report further analysis of this structure, which demonstrates that, in contrast to earlier models, the activated proteasome adopts an ordered 7-fold symmetric pore conformation that is stabilized by interactions formed by a cluster of highly conserved proteasome residues (Tyr8, Asp9, Pro17 and Tyr26) . One non-canonical cluster, which appears to be mandated by the requirement that eukaryotic proteasomes also form an ordered closed conformation, explains all deviations from perfect conservation of these residues . We also demonstrate the importance of these conserved residues for proteolysis by an archaeal proteasome . Evolutionary considerations suggest that other activators might induce the same open proteasome conformation as seen with the 11S activator.

Cell, 2003 Aug 22, 114(4), 497 - 509
Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles; Miller EA et al.; We have characterized the mechanisms of cargo selection into ER-derived vesicles by the COPII subunit Sec24p . We identified a site on Sec24p that recognizes the v-SNARE Bet1p and show that packaging of a number of cargo molecules is disrupted when mutations are introduced at this site . Surprisingly, cargo proteins affected by these mutations did not share a single common sorting signal, nor were proteins sharing a putative class of signal affected to the same degree . We show that the same site is conserved as a cargo-interaction domain on the Sec24p homolog Lst1p, which only packages a subset of the cargoes recognized by Sec24p . Finally, we identified an additional mutation that defines another cargo binding domain on Sec24p, which specifically interacts with the SNARE Sec22p . Together, our data support a model whereby Sec24p proteins contain multiple independent cargo binding domains that allow for recognition of a diverse set of sorting signals.

Cell, 2003 Aug 22, 114(4), 483 - 95
SNARE selectivity of the COPII coat; Mossessova E et al.; The COPII coat buds transport vesicles from the endoplasmic reticulum that incorporate cargo and SNARE molecules . Here, we show that recognition of the ER-Golgi SNAREs Bet1, Sed5, and Sec22 occurs through three binding sites on the Sec23/24 subcomplex of yeast COPII . The A site binds to the YNNSNPF motif of Sed5 . The B site binds to Lxx-L/M-E sequences present in both the Bet1 and Sed5 molecules, as well as to the DxE cargo-sorting signal . A third, spatially distinct site binds to Sec22 . COPII selects the free v-SNARE form of Bet1 because the LxxLE sequence is sequestered in the four-helix bundle of the v-/t-SNARE complex . COPII favors Sed5 within the Sed5/Bos1/Sec22 t-SNARE complex because t-SNARE assembly removes autoinhibitory contacts to expose the YNNSNPF motif . The COPII coat seems to be a specific conductor of the fusogenic forms of these SNAREs, suggesting how vesicle fusion specificity may be programmed during budding.

Cell, 2003 Aug 22, 114(4), 457 - 67
The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis; Harvey KF et al.; Establishing and maintaining homeostasis is critical to the well-being of an organism and is determined by the balance of cell proliferation and death . Two genes that function together to regulate growth, proliferation, and apoptosis in Drosophila are warts (wts), encoding a serine/threonine kinase, and salvador (sav), encoding a WW domain containing Wts-interacting protein . However, the mechanisms by which sav and wts regulate growth and apoptosis are not well understood . Here, we describe mutations in hippo (hpo), which encodes a protein kinase most related to mammalian Mst1 and Mst2 . Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator cyclin E and apoptosis inhibitor DIAP1 . Hpo, Sav, and Wts interact physically and functionally, and regulate DIAP1 levels, likely by Hpo-mediated phosphorylation and subsequent degradation . Thus, Hpo links Sav and Wts to a key regulator of apoptosis.

Cell, 2003 Aug 22, 114(4), 445 - 56
hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts; Wu S et al.; The coordination between cell proliferation and cell death is essential to maintain homeostasis within multicellular organisms . The mechanisms underlying this regulation are yet to be completely understood . Here, we report the identification of hippo (hpo) as a gene that regulates both cell proliferation and cell death in Drosophila . hpo encodes a Ste-20 family protein kinase that binds to and phosphorylates the tumor suppressor protein Salvador (Sav), which is known to interact with the Warts (Wts) protein kinase . Loss of hpo results in elevated transcription of the cell cycle regulator cyclin E and the cell-death inhibitor diap1, leading to increased proliferation and reduced apoptosis . Further, we show that hpo, sav, and wts define a pathway that regulates diap1 at the transcriptional level . A human homolog of hpo completely rescues the overgrowth phenotype of Drosophila hpo mutants, suggesting that hpo might play a conserved role for growth control in mammals.

Cell, 2003 Aug 22, 114(4), 395 - 7
Molecular recognition of cargo by the COPII complex: a most accommodating coat; Barlowe C; The molecular mechanism by which diverse cargo proteins are recognized and exported from the ER has been unclear . Two papers in this issue of Cell add clarity by mapping multiple cargo recognition sites in the Sec24 subunit of the COPII coat complex and demonstrating roles for these sites in export of specific protein cargos from the ER.

J Am Chem Soc, 2003 Sep 3, 125(35), 10635 - 40
Mechanistic study of an improbable reaction: alkene dehydrogenation by the delta12 acetylenase of Crepis alpina; Reed DW et al.; The mechanism by which the fatty acid acetylenase of Crepis alpina catalyzes crepenynic acid ((9Z)-octadeca-9-en-12-ynoic acid) production from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements . This was accomplished by incubating appropriate mixtures of regiospecifically deuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional acetylenase . LC/MS analysis of crepenynic acid obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps, since the cleavage of the C12-H bond is very sensitive to isotopic substitution (k(H)/k(D) = 14.6 +/- 3.0) while a minimal isotope effect (k(H)/k(D) = 1.25 +/- 0.08) was observed for the C13-H bond breaking step . These data suggest that crepenynic acid is produced via initial H-atom abstraction at C12 of a linoleoyl substrate . The relationship between the mechanism of enzymatic acetylenation and epoxidation is discussed.

Pol Merkuriusz Lek, 2003 May, 14(83), 417 - 20
{Cathepsin B activity and concentration of elastase and alpha-1 proteinase inhibitor complex in non-small-cell lung cancer: 2 year follow-up study}; Passowicz-Muszynska E et al.; In 21 patients with non-small-cell lung cancer subjected to radical surgery followed by 3 cycles of chemotherapy, serum cathepsin B activity and plasma E-alpha 1IP concentration in peripheral blood and tumour arterial and venous blood were studied . Cathepsin B activity was determined by a fluorometric assay . E-alpha 1IP concentration was measured with an ELISA kit . The measurements were performed before surgery, before each chemotherapy cycle and every 60 days after chemotherapy completion, for 2 years . All the patients (n = 21) were divided into 2 subgroups: without metastases n = 16 and with metastases n = 5 . There was no significant difference between preoperative serum cathepsin B activity and E-alpha 1IP plasma values in peripheral blood and blood coming from tumour artery and vein . The surgery and chemotherapy caused a statistically significant decrease of serum cathepsin B activity and plasma E-alpha 1IP concentration both in the whole group and in the subgroup without metastases . A significant increase of cathepsin B activity in comparison to initial values was observed 2,5-4 months before cerebral metastasis appeared in the subgroup with metastases . The elevation of E-alpha 1IP concentration preceded the increase of cathepsin B activity in this subgroup . It was not statistically significant . A decrease of cathepsin B and E-alpha 1IP values was observed after cerebral metastasis excision.

J Am Soc Nephrol, 2003 Sep, 14(9 Suppl 4), S300 - 4
Vascular calcification: in vitro evidence for the role of inorganic phosphate; Giachelli CM; Uremic patients are prone to widespread ectopic extraskeletal calcification resulting from an imbalance of systemic inorganic phosphate (Pi) . There can be serious consequences of this process, particularly when it results in the calcification of the vasculature . A recent study examined the response of cultured human aortic smooth muscle cells to varying levels of extracellular Pi . Cells that were exposed to Pi levels similar to those seen in uremic patients (>1.4 mmol/L) showed dose-dependent increases in cell culture calcium deposition . The results of this study also defined the role of elevated phosphate in transforming the vascular phenotype of these cells to an osteogenic phenotype, such that a predisposition for calcification was created . Pi-induced changes included increased expression of the osteogenic markers osteocalcin and core-binding factor-1 genes, the latter of which is considered a "master gene" critical for osteoblast differentiation . These changes occur early after exposure to high phosphate levels and seem to be mediated by a sodium-dependent phosphate co-transporter, Pit-1 (Glvr-1) . Calcification of vascular cells also seems to occur in the absence of a mineral imbalance but in the presence of platelet-derived growth factor, a potent atherogenic factor . Taken together, these data suggest that calcification of vascular cells can occur early in a phosphate-rich environment similar to that seen in patients with renal failure and in a platelet-derived growth factor-rich atherosclerotic region under normal phosphorus conditions . From a clinical viewpoint, it seems that early control or prevention of hyperphosphatemia may reduce coronary calcification and its associated morbidity and mortality for patients on dialysis.

J Biol Chem, 2003 Oct 31, 278(44), 43014 - 9 Epub 2003 Aug 25.
Deacetylase activity is required for cAMP activation of a subset of CREB target genes; Fass DM et al.; Many hormones activate transcription by raising the level of cAMP within cells . In one well studied pathway, cAMP induces protein kinase A to phosphorylate the transcription factor CREB, which binds to a consensus sequence, the cAMP-regulated enhancer, found in many target genes . A generally accepted model suggests that phosphorylated CREB recruits the histone acetyltransferase CBP to activate transcription . In contrast, histone deacetylases have been linked to the cessation of CREB-dependent transcription . Here we tested this model in the regulation of endogenous CREB target genes . We used a constitutively active CREB mutant and microarray analysis to identify target genes in PC12 cells . We then tested the role of histone deacetylase activity in cAMP activation of four of these genes (c-FOS, ICER, NOR-1, and NUR77) by treating cells with the histone deacetylase inhibitor trichostatin A . Consistent with the generally accepted model, trichostatin A enhanced activation of c-FOS and NUR77 by cAMP . Surprisingly, trichostatin A blocked activation of ICER and NOR-1 . The block of ICER and NOR-1 activation persisted in the presence of cycloheximide, indicating that the trichostatin A effect did not depend on new protein synthesis . This unexpected role of histone deacetylases in transcriptional activation of certain endogenous CREB target genes was not apparent in transfected reporter genes . Chromatin immunoprecipitation analysis indicated that the differential roles of histone deacetylases in activating or repressing CREB target genes was manifested at the level of preinitiation complex recruitment . These data indicate that histone deacetylases differentially regulate CREB target genes by contributing to either activation or cessation of transcription.

Planta, 2003 Nov, 218(1), 58 - 64 Epub 2003 Aug 23.
Cloning of two contrasting high-affinity sulfate transporters from tomato induced by low sulfate and infection by the vascular pathogen Verticillium dahliae; Howarth JR et al.; Two cDNAs, LeST1-1 (AF347613) and LeST1-2 (AF347614), encoding sulfate transporters have been cloned from tomato (Lycopersicon esculentum Mill.) by reverse transcription-polymerase chain reaction and their expression characterised . Sharing 76% identity at the amino acid level, the transporters are phylogenetically associated with the Group-1, high-affinity plant sulfate transporters . Both were shown to have high affinity for sulfate by uptake kinetic analysis using a yeast (Saccharomyces cerevisiae) sulfate-transporter mutant . Km values of 11.5 microM and 9.8 microM were calculated for LeST1-1 and LeST1-2, respectively, the same order of magnitude as those previously reported for several other Group-1 high-affinity sulfate transporters . In situ hybridisation to S-deficient tomato roots showed LeST1-1 to be expressed in the epidermis and pericycle, whereas LeST1-2 expression was located to the epidermis only . Northern analysis shows that the mRNA abundances of both LeST1-1 and LeST1-2 are upregulated in the root in response to sulfate deprivation . LeST1-1 is specifically expressed in root tissue, a characteristic of Group-1 sulfate transporters . LeST1-2, however, was also detected in tomato leaves and stems and is upregulated and expressed to a similar extent in these tissues under conditions of sulfate deprivation . Induction of LeST1-2 expression was also observed in the vascular tissues of a resistant line of tomato infected with the vascular wilt pathogen Verticillium dahliae.

Chromosoma, 2003 Aug, 112(2), 77 - 86 Epub 2003 Jul 09.
Identification of novel histone post-translational modifications by peptide mass fingerprinting; Zhang L et al.; The extent and pattern of histone post-translational modifications is a key determinant dictating the structure of chromatin . We employed mass spectrometry to map the post-translational modifications present on mammalian core histones . Using accurate peptide mass fingerprinting on proteolytic digests of purified histones, we identified more than 20 novel sites of histone modification . One newly identified site of methylation, histone H4 lysine 59, maps to the surface of the nucleosome in close proximity to the site of the only identified histone core modification, histone H3 lysine 79 . Consistent with the role of histone H3 lysine 79 methylation in the formation of silent chromatin structure, histone H4 lysine 59 is essential for transcriptional silencing at the yeast silent mating loci and telomeres.

Mol Biol Cell, 2003 Oct, 14(10), 4140 - 54 Epub 2003 Aug 22.
Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii; Knechtle P et al.; We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii . The development starts with an initial isotropic growth phase followed by the emergence of germ tubes . The initial tip growth speed of 6-10 microm/h increases during early stages of development . This increase is transiently interrupted in response to the establishment of lateral branches or septa . The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae . A search for A . gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain . AgSpa2 colocalizes with sites of polarized actin . Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips . Polarization at sites of septation is transient . During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided . To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion . The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern . We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.

J Comput Biol, 2003, 10(3-4), 341 - 56
Continuous representations of time-series gene expression data; Bar-Joseph Z et al.; We present algorithms for time-series gene expression analysis that permit the principled estimation of unobserved time points, clustering, and dataset alignment . Each expression profile is modeled as a cubic spline (piecewise polynomial) that is estimated from the observed data and every time point influences the overall smooth expression curve . We constrain the spline coefficients of genes in the same class to have similar expression patterns, while also allowing for gene specific parameters . We show that unobserved time points can be reconstructed using our method with 10-15% less error when compared to previous best methods . Our clustering algorithm operates directly on the continuous representations of gene expression profiles, and we demonstrate that this is particularly effective when applied to nonuniformly sampled data . Our continuous alignment algorithm also avoids difficulties encountered by discrete approaches . In particular, our method allows for control of the number of degrees of freedom of the warp through the specification of parameterized functions, which helps to avoid overfitting . We demonstrate that our algorithm produces stable low-error alignments on real expression data and further show a specific application to yeast knock-out data that produces biologically meaningful results.

Cell Death Differ, 2003 Sep, 10(9), 940 - 5
Autophagic programmed cell death in Drosophila; Baehrecke EH; Autophagic programmed cell death occurs during the development of diverse animal groups, but the mechanisms that control this genetically regulated form of cell killing are poorly understood . Genetic studies of bulk protein degradation in yeast have provided important advances in our understanding of autophagy, and recent investigations of Drosophila autophagic cell death suggest that some of these mechanisms may be conserved . In Drosophila, several steroid-regulated genes that encode transcription regulators are required for autophagic cell death . These transcription regulators appear to activate a large number of genes that play a more direct role in cell killing, including genes that function in apoptosis such as caspases . While caspase function is required for autophagic cell death during Drosophila development, genes encoding proteins that are similar to the yeast autophagy regulators are also induced in dying salivary glands . Furthermore, numerous noncaspase proteases, cytoplasmic organizing factors, signaling molecules, and unknown factors are expressed in interesting patterns during autophagic cell death . This article reviews the current knowledge of the regulation of autophagic programmed cell death during development of Drosophila.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10370 - 5 Epub 2003 Aug 21.
Expression deconvolution: a reinterpretation of DNA microarray data reveals dynamic changes in cell populations; Lu P et al.; Cells grow in dynamically evolving populations, yet this aspect of experiments often goes unmeasured . A method is proposed for measuring the population dynamics of cells on the basis of their mRNA expression patterns . The population's expression pattern is modeled as the linear combination of mRNA expression from pure samples of cells, allowing reconstruction of the relative proportions of pure cell types in the population . Application of the method, termed expression deconvolution, to yeast grown under varying conditions reveals the population dynamics of the cells during the cell cycle, during the arrest of cells induced by DNA damage and the release of arrest in a cell cycle checkpoint mutant, during sporulation, and following environmental stress . Using expression deconvolution, cell cycle defects are detected and temporally ordered in 146 yeast deletion mutants; six of these defects are independently experimentally validated . Expression deconvolution allows a reinterpretation of the cell cycle dynamics underlying all previous microarray experiments and can be more generally applied to study most forms of cell population dynamics.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10146 - 51 Epub 2003 Aug 21.
Comparing the continuous representation of time-series expression profiles to identify differentially expressed genes; Bar-Joseph Z et al.; We present a general algorithm to detect genes differentially expressed between two nonhomogeneous time-series data sets . As increasing amounts of high-throughput biological data become available, a major challenge in genomic and computational biology is to develop methods for comparing data from different experimental sources . Time-series whole-genome expression data are a particularly valuable source of information because they can describe an unfolding biological process such as the cell cycle or immune response . However, comparisons of time-series expression data sets are hindered by biological and experimental inconsistencies such as differences in sampling rate, variations in the timing of biological processes, and the lack of repeats . Our algorithm overcomes these difficulties by using a continuous representation for time-series data and combining a noise model for individual samples with a global difference measure . We introduce a corresponding statistical method for computing the significance of this differential expression measure . We used our algorithm to compare cell-cycle-dependent gene expression in wild-type and knockout yeast strains . Our algorithm identified a set of 56 differentially expressed genes, and these results were validated by using independent protein-DNA-binding data . Unlike previous methods, our algorithm was also able to identify 22 non-cell-cycle-regulated genes as differentially expressed . This set of genes is significantly correlated in a set of independent expression experiments, suggesting additional roles for the transcription factors Fkh1 and Fkh2 in controlling cellular activity in yeast.

Science, 2003 Oct 10, 302(5643), 249 - 55 Epub 2003 Aug 21.
A gene-coexpression network for global discovery of conserved genetic modules; Stuart JM et al.; To elucidate gene function on a global scale, we identified pairs of genes that are coexpressed over 3182 DNA microarrays from humans, flies, worms, and yeast . We found 22,163 such coexpression relationships, each of which has been conserved across evolution . This conservation implies that the coexpression of these gene pairs confers a selective advantage and therefore that these genes are functionally related . Many of these relationships provide strong evidence for the involvement of new genes in core biological functions such as the cell cycle, secretion, and protein expression . We experimentally confirmed the predictions implied by some of these links and identified cell proliferation functions for several genes . By assembling these links into a gene-coexpression network, we found several components that were animal-specific as well as interrelationships between newly evolved and ancient modules.

J Biol Chem, 2003 Nov 7, 278(45), 44894 - 903 Epub 2003 Aug 21.
HADHB, HuR, and CP1 bind to the distal 3'-untranslated region of human renin mRNA and differentially modulate renin expression; Adams DJ et al.; Production of renin is critically dependent on modulation of REN mRNA stability . Here we sought to elucidate the molecular mechanisms involved . Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3'-untranslated region (3'-UTR) contributes to basal REN mRNA instability . Yeast three-hybrid screening with the REN 3'-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) beta-subunit) as a novel REN mRNA-binding protein . Recombinant HADHB bound specifically to the 3'-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1) . This required the renin stability regulatory element . Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3'-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels . Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease . Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo . Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell . Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells . In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3'-UTR of REN mRNA and regulate renin production . cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.

Nature, 2003 Aug 21, 424(6951), 961 - 5
Feedback regulation of MAPK signalling by an RNA-binding protein; Sugiura R et al.; Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes that convert extracellular signals into various outputs such as cell growth, differentiation and cell death . MAPK phosphatases selectively inactivate MAPKs by dephosphorylating critical phosphothreonine and phosphotyrosine residues . The transcriptional induction of MAPK phosphatase expression by various stimuli, including MAPK activation, has been well documented as a negative-feedback mechanism of MAPK signalling . Here we show that Rnc1, a novel K-homology-type RNA-binding protein in fission yeast, binds and stabilizes Pmp1 messenger RNA, the MAPK phosphatase for Pmk1 (refs 10, 11) . Rnc1 therefore acts as a negative regulator of Pmk1 signalling . Notably, Pmk1 phosphorylates Rnc1, causing enhancement of the RNA-binding activity of Rnc1 . Thus, Rnc1 is a component of a new negative-feedback loop that regulates the Pmk1 pathway through its binding to Pmp1 mRNA . Our findings--the post-transcriptional mRNA stabilization of a MAPK phosphatase mediated by an RNA-binding protein--provide an additional regulatory mechanism for fine-tuning of MAPK signalling pathways.

Nature, 2003 Aug 21, 424(6951), 948 - 51
Generation of prion transmission barriers by mutational control of amyloid conformations; Chien P et al.; Self-propagating beta-sheet-rich protein aggregates are implicated in a wide range of protein-misfolding phenomena, including amyloid diseases and prion-based inheritance . Two properties have emerged as common features of amyloids . Amyloid formation is ubiquitous: many unrelated proteins form such aggregates and even a single polypeptide can misfold into multiple forms--a process that is thought to underlie prion strain variation . Despite this promiscuity, amyloid propagation can be highly sequence specific: amyloid fibres often fail to catalyse the aggregation of other amyloidogenic proteins . In prions, this specificity leads to barriers that limit transmission between species . Using the yeast prion {PSI+}, we show in vitro that point mutations in Sup35p, the protein determinant of {PSI+}, alter the range of 'infectious' conformations, which in turn changes amyloid seeding specificity . We generate a new transmission barrier in vivo by using these mutations to specifically disfavour subsets of prion strains . The ability of mutations to alter the conformations of amyloid states without preventing amyloid formation altogether provides a general mechanism for the generation of prion transmission barriers and may help to explain how mutations alter toxicity in conformational diseases.

Nucleic Acids Res, 2003 Sep 1, 31(17), 4981 - 8
Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene; Clark DR et al.; In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences . Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure . To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro . Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90% . We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA . In each case, the clonogenic survival after UV was not different from that of the parental cell strains . In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA . The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.

J Biol Chem, 2003 Nov 7, 278(45), 44467 - 74 Epub 2003 Aug 20.
The Brf1 and Bdp1 subunits of transcription factor TFIIIB bind to overlapping sites in the tetratricopeptide repeats of Tfc4; Liao Y et al.; The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1 . Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1 . Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4 . Here we examine the biological importance of the second TPR array, TPRs6 -9 . Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription . Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1 . Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand . Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4 . The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1 . The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.

J Environ Sci Health B, 2003 Sep, 38(5), 571 - 9
A study on prediction of the bio-toxicity of substituted benzene based on artificial neural network; Gao DW et al.; Quantitative Structure-Activity Relationship (QSAR) between the bio-toxicity of seventy-eight kinds of substituted benzene chemicals to yeast Saccharomyces cerevisiae (1g(1/Cmiz)) and the components of vertex degree autocorrelation vectors (values of A, B, C and D) was studied by using the software of Artificial Neural Network (ANN) . The key factors of the autocorrelation descriptors for the 1g (1/Cmiz) value of yeast Saccharomyces cerevisiae, A {0}, A {1}, C {3}, C {5} and D {3} were selected from twenty-four descriptors, and were explained theoretically in this paper . The QSAR-ANN model has been used to predict the bio-toxicity of twenty-three substituted benzene chemicals . The correlation between Cmiz and LC50 was also discussed, and the liner correlation equation between them was established.

Org Biomol Chem, 2003 Jan 7, 1(1), 39 - 41
Probing the mechanism of a fungal glycosyltransferase essential for cell wall biosynthesis . UDP-chitobiose is not a substrate for chitin synthase; Chang R et al.; Chitin synthase is responsible for the biosynthesis of chitin, an essential component of the fungal cell wall . There is a long-standing question as to whether "processive" transferases such as chitin synthase operate in the same manner as non-processive transferases . The question arises from analysis of the polysaccharide structure--in chitin, for instance, each sugar residue is rotated approximately 180 degrees relative to the preceding sugar in the chain . This requires that the enzyme account for the alternating "up/down" configuration during biosynthesis . An enzyme with a single active site, analogous to the non-processive transferases--would have to accommodate a distorted glycosidic linkage at every other synthetic step . An alternative proposal is that the enzyme might assemble the disaccharide donor, addressing the "up/down" conformational problem prior to polymer synthesis . We present compelling evidence that this latter hypothesis is incorrect.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10347 - 52 Epub 2003 Aug 19.
Specific sequence changes in multiple transcript system DYT3 are associated with X-linked dystonia parkinsonism; Nolte D et al.; X-linked dystonia parkinsonism (XDP) is an X-linked recessive adult onset movement disorder characterized by both dystonia and parkinsonism . We report delineation of the disease gene within a 300-kb interval of Xq13.1 by allelic association . Sequencing of this region in a patient revealed five disease-specific single-nucleotide changes (here referred to as DSC) and a 48-bp deletion unique to XDP . One of the DSCs is located within an exon of a not previously described multiple transcript system that is composed of at least 16 exons . There is a minimum of three different transcription start sites that encode four different transcripts . Two of these transcripts include distal portions of the TAF1 gene (TATA-box binding protein-associated factor 1) and are alternatively spliced . Three exons overlap with ING2 (a putative tumor suppressor) and with a homologue of CIS4 (cytokine-inducible SH2 protein 4), both of which are encoded by the opposite strand . Although all DSCs are located within this multiple transcript system, only DSC3 lies within an exon . This exon is used by all alternative transcripts making a pathogenic role of DSC3 in XDP likely . The multiple transcript system is therefore referred to as DYT3 (disease locus in XDP).

J Biol Chem, 2003 Nov 7, 278(45), 44574 - 83 Epub 2003 Aug 19.
Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails; McBryant SJ et al.; The yeast nucleosome assembly protein 1 (yNAP1) participates in many diverse activities, such as the assembly of newly synthesized DNA into chromatin and the rearrangement of nucleosomes during transcriptional activation . yNAP1 does not require ATP hydrolysis to perform these functions and is a valuable tool for in vitro chromatin assembly . Using recombinant histone complexes, we show that yNAP1 has a preference for binding the (H3-H4)2 tetramer over the (H2A-H2B) dimer . We find that the loss of the histone tails abrogates this preference for H3 and H4, and we demonstrate a direct interaction between yNAP1 and the amino-terminal tails of H3 and H4 . yNAP1 binds to one histone fold domain, thus specifying the stoichiometry of the complexes formed with the histone dimer and tetramer . Finally, we provide evidence that the acidic carboxyl-terminal region of yNAP1, although dispensable for nucleosome assembly in vitro, contributes to binding via structure-independent electrostatic interactions . Our results are consistent with recent mechanistic investigations of NAP1 and expand our understanding of the histone chaperone family of assembly factors.

J Biol Chem, 2003 Oct 24, 278(43), 42091 - 7 Epub 2003 Aug 19.
AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes; Cadinanos J et al.; Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group . Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX) . Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1 . These proteases have different enzymatic properties, substrate specificities, and biological functions . However, a proposal has been made that plants lack a FACE-2/Rce1-type protease . Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes . Northern blot analysis demonstrates widespread expression of this gene in plant tissues . Heterologous expression of the A . thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases . The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro . These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.

J Cell Sci, 2003 Oct 1, 116(Pt 19), 3949 - 56 Epub 2003 Aug 19.
The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal; Kiesler E et al.; Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins . In a yeast two-hybrid screening we found that Hrp65 can interact with itself . Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain . We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system . These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins . Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules . Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.

J Mol Biol, 2003 Aug 29, 331(5), 1109 - 20
Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies; Tanaka T et al.; There is a major need in target validation and therapeutic applications for molecules that can interfere with protein function inside cells . Intracellular antibodies (intrabodies) can bind to specific targets in cells but isolation of intrabodies is currently difficult . Intrabodies are normally single chain Fv fragments comprising variable domains of the immunoglobulin heavy (VH) and light chains (VL) . We now demonstrate that single VH domains have excellent intracellular properties of solubility, stability and expression within the cells of higher organisms and can exhibit specific antigen recognition in vivo . We have used this intracellular single variable domain (IDab) format, based on a previously characterised intrabody consensus scaffold, to generate diverse intrabody libraries for direct in vivo screening . IDabs were isolated using two distinct antigens and affinities of isolated IDabs ranged between 20 nM and 200 nM . Moreover, IDabs selected for binding to the RAS protein could inhibit RAS-dependent oncogenic transformation of NIH3T3 cells . The IDab format is therefore ideal for in vivo intrabody use . This approach to intrabodies obviates the need for phage antibody libraries, avoids the requirement for production of antigen in vitro and allows for direct selection of intrabodies in vivo.

Radiat Res, 2003 Sep, 160(3), 309 - 17
Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A; Nazarov IB et al.; The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown . We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase . Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs . At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites . However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites . We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro . Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

Mol Biol Cell, 2003 Aug, 14(8), 3342 - 55 Epub 2003 May 03.
Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p; Shang C et al.; Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure . To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex . The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p . To identify protein-protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p . A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners . From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex . Strikingly, we found that the Dam1p-Ndc80p and Dam1p-Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.

Mol Biol Cell, 2003 Aug, 14(8), 3230 - 41 Epub 2003 May 18.
Grr1-dependent inactivation of Mth1 mediates glucose-induced dissociation of Rgt1 from HXT gene promoters; Flick KM et al.; In budding yeast, HXT genes encoding hexose permeases are induced by glucose via a mechanism in which the F box protein Grr1 antagonizes activity of the transcriptional repressor Rgt1 . Neither the mechanism of Rgt1 inactivation nor the role of Grr1 in that process has been understood . We show that glucose promotes phosphorylation of Rgt1 and its dissociation from HXT gene promoters . This cascade of events is dependent upon the F-box protein Grr1 . Inactivation of Rgt1 is sufficient to explain the requirement for Grr1 but does not involve Rgt1 proteolysis or ubiquitination . We show that inactivation of Mth1 and Std1, known negative regulators of HXT gene expression, leads to the hyperphosphorylation of Rgt1 and its dissociation from HXT promoters even in the absence of glucose . Furthermore, inactivation of Mth1 and Std1 bypasses the requirement for Grr1 for induction of these events, suggesting they are targets for inactivation by Grr1 . Consistent with that proposal, Mth1 is rapidly eliminated in response to glucose via a mechanism that requires Grr1 . Based upon these data, we propose that glucose acts via Grr1 to promote the degradation of Mth1 . Degradation of Mth1 leads to phosphorylation and dissociation of Rgt1 from HXT promoters, thereby activating HXT gene expression.

Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10164 - 9 Epub 2003 Aug 18.
Dielectrophoretic dynamic light-scattering (DDLS) spectroscopy; Halaka FG; Dielectrophoretic dynamic light-scattering (DDLS) spectroscopy is presented . DDLS identifies macromolecules based on their dielectric, or polarizability, properties . DDLS measurements are carried out in an oscillating, nonuniform electric field . The field induces macromolecules to undergo dielectrophoretic motion, which is detected by the modulation in the dynamic light-scattering autocorrelation function . The DDLS experimental setup, data analysis, and data on latex particles and yeast cells are presented.

Biochemistry, 2003 Aug 26, 42(33), 9980 - 8
Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst; Bassi GS et al.; The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins . We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein . (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA . The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM . (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA . k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex . (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h . k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM . The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing . (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites . Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit . (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers . In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.

J Biol Chem, 2003 Oct 3, 278(40), 38287 - 91 Epub 2003 Aug 15.
Translation termination factor eRF3 mediates mRNA decay through the regulation of deadenylation; Hosoda N et al.; Messenger RNA decay, which is a regulated process intimately linked to translation, begins with the deadenylation of the poly(A) tail at the 3' end . However, the precise mechanism triggering the first step of mRNA decay and its relationship to translation have not been elucidated . Here, we show that the translation termination factor eRF3 mediates mRNA deadenylation and decay in the yeast Saccharomyces cerevisiae . The N-domain of eRF3, which is not necessarily required for translation termination, interacts with the poly(A)-binding protein PABP . When this interaction is blocked by means of deletion or overexpression of the N-domain of eRF3, half-lives of all mRNAs are prolonged . The eRF3 mutant lacking the N-domain is deficient in the poly(A) shortening . Furthermore, the eRF3-mediated mRNA decay requires translation to proceed, especially ribosomal transition through the termination codon . These results indicate that the N-domain of eRF3 mediates mRNA decay by regulating deadenylation in a manner coupled to translation.

J Biol Chem, 2003 Nov 14, 278(46), 45062 - 71 Epub 2003 Aug 15.
Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity; Guo GL et al.; The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis . Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet . Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet . To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis{2-(3,5-dichlorpyridyloxy)}benzene (TCPOBOP), followed by the CA diet . A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP . In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo . Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.

Genes Dev, 2003 Sep 1, 17(17), 2162 - 76 Epub 2003 Aug 15.
Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing; Huang J et al.; Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span . rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit) . We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region . Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities . In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased . We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I . Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex . Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging.

Genes Dev, 2003 Aug 15, 17(16), 1957 - 62
ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism; Nakada D et al.; In budding yeast, TEL1 encodes a protein closely related to ATM . Xrs2 is an Nbs1 homolog and forms a complex with Mre11 and Rad50 . We show here that Tel1 associates with double-strand breaks (DSBs) through a mechanism dependent on the C terminus of Xrs2 . Although Xrs2 is required for the DNA degradation at DSBs, the C-terminal Xrs2 truncation does not affect the degradation . Tel1 and the C terminus of Xrs2 are similarly involved in cell survival and Rad53 phosphorylation after DNA damage . Our findings suggest that the Tel1 association with DNA lesions is required for the activation of DNA damage responses.

Mol Imaging, 2002 Jan-Mar, 1(1), 43 - 55
Molecular imaging of gene expression and efficacy following adenoviral-mediated brain tumor gene therapy; Rehemtulla A et al.; Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity . Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules . In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase) . Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively . In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression . MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy . Significant increases in mean tumor diffusion values were also observed during treatment with 5FC . Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass . In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression . These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.

Am J Physiol Gastrointest Liver Physiol, 2004 Jan, 286(1), G23 - 30 Epub 2003 Aug 14.
Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor; Hinnebusch BF et al.; We have examined the role that the transcription factor gut-enriched Kruppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP) . A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter . DNA-protein interactions were determined by using EMSA . Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4 . Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription . The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III . RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element . Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element . Mutant KLF4 constructs failed to fully activate IAP . We have identified the enterocyte differentiation marker IAP as a KLF4 target gene . IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

Dev Cell, 2003 Aug, 5(2), 188 - 9
Sir2 flexes its muscle; Bedalov A et al.; A new report reveals a role for the mammalian NAD-dependent deacetylase Sir2 in repressing the muscle cell differentiation program and implicates the cellular redox state as a critical determinant of transcriptional activity of differentiation-specific genes.

Genet Mol Res, 2003 Mar 31, 2(1), 136 - 47
Sex in fungi: lessons of gene regulation; Souza CA et al.; Fungi have been very useful for gene regulation studies . Mating implicates in a series of events influenced by many types of environmental input that are interpreted into regulatory pathways, including signal transduction . Although various aspects of mating and signal transduction in the yeast Saccharomyces cerevisiae have long been characterized, recent findings in filamentous fungi indicate that pheromones and pheromone receptors may be essential for mating partner recognition and also for nucleus recognition in sorting before meiosis . A brief overview on mating-type genes of ascomycete fungi and recent contributions to the understanding of their role in the regulation of multicellularity and sexual dimorphism is presented in this review.

Genet Mol Res, 2003 Mar 31, 2(1), 43 - 7
GAL4 causes developmental defects and apoptosis when expressed in the developing eye of Drosophila melanogaster; Kramer JM et al.; The UAS/GAL4 ectopic expression system is widely used in Drosophila melanogaster for the overexpression of transgenes . This system operates under the assumption that the yeast transcription factor, GAL4, is inactive in D . melanogaster . Thus, GAL4 can be expressed under the control of D . melanogaster -specific promoters with little effect upon the organism . We have shown that expression of GAL4 in the developing eye under the control of the glass multiple reporter (GMR) promoter element does have an effect on eye development . Although GMR-GAL4 heterozygotes appear normal when raised at 25 degrees C, the homozygotes have a highly disorganized ommatidial array . In addition, the levels of apoptosis in the third-instar larval eye imaginal disc (where GAL4 is expressed) are slightly higher in GMR-GAL4 heterozygotes, and much higher in GMR-GAL4 homozygotes when compared to wild type discs . The morphological eye defects caused by GMR-GAL4 are significantly enhanced when flies are raised at 29 degrees C (presumably due to the higher activity of GAL4 at this temperature); however, the levels of apoptosis appear to be similar at these two temperatures . Taken together, these data suggest that GAL4 can have adverse effects on D . melanogaster development, especially at high expression levels . In addition, GAL4 appears to induce apoptosis even in the absence of any visible morphological defects . Thus, despite the benefits of the UAS/GAL4 ectopic expression system, one must use caution in the design and interpretation of experiments.

J Gen Virol, 2003 Sep, 84(Pt 9), 2501 - 10
Herpes simplex virus type 1 tegument protein VP22 interacts with TAF-I proteins and inhibits nucleosome assembly but not regulation of histone acetylation by INHAT; van Leeuwen H et al.; Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22 . Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1 . TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones . TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF . Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I . We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I . However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro . We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome . Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

J Biol Chem, 2003 Oct 31, 278(44), 43717 - 27 Epub 2003 Aug 12.
Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber; Baxa U et al.; The {URE3} prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast . The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability . Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins . Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70 . Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis . The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change . Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages . STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case . These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.

J Biol Chem, 2003 Nov 7, 278(45), 44041 - 8 Epub 2003 Aug 12.
Interactions between the aryl hydrocarbon receptor and P-TEFb . Sequential recruitment of transcription factors and differential phosphorylation of C-terminal domain of RNA polymerase II at cyp1a1 promoter; Tian Y et al.; The expression of the cytochrome P450 1A1 gene (cyp1a1) is regulated by the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor that mediates most toxic responses induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) . In the nucleus, ligand-activated AhR binds to the xenobiotic response elements, initiating chromatin remodeling and recruitment of coregulators, leading to the formation of preinitiation complex followed by elongation . Here, we report that ligand-activated AhR recruits the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNA PII) to the cyp1a1 promoter with concomitant phosphorylation of the RNA PII carboxyl domain (CTD) . Interestingly, the serine 2 and serine 5 of the heptapeptide repeats (YSPTSPS) were sequentially phosphorylated upon TCDD treatment . Inhibition of P-TEFb kinase activity by 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) suppressed CTD phosphorylation (especially serine 2 phosphorylation) and abolished processive elongation without disrupting the assembly of the preinitiation complex at the cyp1a1 promoter . Remarkably, we found that activation of NF-kappaB by TNF-alpha selectively inhibited TCDD-induced serine 2 phosphorylation in mouse liver cells, suggesting that residue-specific phosphorylation of RNA PII CTD at the cyp1a1 promoter is an important regulatory point upon which signal "cross-talk" converges . Finally, we show that ligand-activated AhR associated with P-TEFb through the C terminus of cyclin T1, suggesting that AhR recruit the P-TEFb to the cyp1a1 promoter whereupon its kinase subunit phosphorylates the RNA PII CTD.

J Biol Chem, 2003 Nov 7, 278(45), 44758 - 68 Epub 2003 Aug 13.
A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation; Olson MR et al.; Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction . Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release . Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function . We report here that this domain, although required for IAP destabilization, is not sufficient . Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing . Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID . Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.

J Biol Chem, 2003 Oct 24, 278(43), 41947 - 53 Epub 2003 Aug 13.
Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding; Leung SW et al.; Classical protein import, mediated by the binding of a classical nuclear localization signal (NLS) to the NLS receptor, karyopherin/importin alpha, is the most well studied nuclear transport process . Classical NLSs are either monopartite sequences that contain a single cluster of basic amino acids (Lys/Arg) or bipartite sequences that contain two clusters of basic residues separated by an unconserved linker region . We have created mutations in conserved residues in each of the three NLS-binding sites/regions in Saccharomyces cerevisiae karyopherin alpha (SRP1) . For each mutant we have analyzed binding to both a monopartite and a bipartite NLS cargo in vitro . We have also expressed each karyopherin alpha mutant in vivo as the only cellular copy of the NLS receptor and examined the impact on cell growth and import of both monopartite and bipartite NLS-containing cargoes . Our results reveal the functional significance of specific residues within karyopherin alpha for NLS cargo binding . A karyopherin alpha variant with a mutation in the major NLS-binding site exhibits decreased binding to both monopartite and bipartite NLS cargoes, and this protein is not functional in vivo . However, we also find that a karyopherin alpha variant with a mutation in the minor NLS-binding site, which shows decreased binding only to bipartite NLS-containing cargoes, is also not functional in vivo . This suggests that the cell is dependent on the function of at least one bipartite NLS cargo that is imported into the nucleus by karyopherin alpha . Our experiments also reveal functional importance for the linker-binding region . This study provides insight into how changes in binding to cellular NLS sequences could impact cellular function . In addition, this work has led to the creation of conditional alleles of karyopherin alpha with well characterized defects in NLS binding that will be useful for identifying and characterizing novel NLS cargoes.

J Cell Sci, 2003 Sep 15, 116(Pt 18), 3667 - 75
The mediator coactivator complex: functional and physical roles in transcriptional regulation; Lewis BA et al.; In vivo, the DNA is packed into chromatin and transcription is dependent upon activators that recruit other factors to reverse the repressive effects of chromatin . The response to activators requires additional factors referred to as coactivators . One such coactivator, mediator, is a multi-subunit complex capable of responding to different activators . It plays an key role in activation, bridging DNA-bound activators, the general transcriptional machinery, especially RNA polymerase II, and the core promoter . Its subunits are necessary for a variety of positive and negative regulatory processes and serve as the direct targets of activators themselves . In vivo and in vitro studies support various roles for mediator in transcription initiation, while structural studies demonstrate that it engages in multiple interactions with RNA polymerase II, and adopts conformations that are activator specific.

Mol Cell Biol, 2003 Sep, 23(17), 6300 - 14
Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling; Lee SJ et al.; Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage . We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1 . Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions . These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9 . However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1 . The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade . Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.

Mol Cell Biol, 2003 Sep, 23(17), 6267 - 78
The critical cis-acting element required for IMD2 feedback regulation by GDP is a TATA box located 202 nucleotides upstream of the transcription start site; Escobar-Henriques M et al.; Guanylic nucleotides are essential cellular players, and the critical enzyme in their tightly regulated synthesis in Saccharomyces cerevisiae is encoded by the IMD2 gene . The transcription of IMD2 is subject to general repression by nutrient limitation through the cis nutrient-sensing element . It is also subject to specific feedback regulation by the end products of the guanylic nucleotide synthesis pathway . The critical cis element for this latter mechanism is the guanine response element (GRE), a TATAATA sequence which is located 202 nucleotides upstream of the transcription initiation site and which functions as the IMD2 TATA box . We show that the GRE functions in conjunction with a 52-nucleotide stretch near the transcription start site . This very unusual promoter structure ensures low, basal expression of IMD2 and the recruitment of TFIID to the GRE in response to guanylic nucleotide limitation.

Br J Cancer, 2003 Aug 18, 89(4), 713 - 9
Telomerase activity in B-cell non-Hodgkin lymphomas is regulated by hTERT transcription and correlated with telomere-binding protein expression but uncoupled from proliferation; Klapper W et al.; Telomere maintenance is a prerequisite for immortalisation, and in most malignant cells is carried out by telomerase, an enzyme that synthesis new telomeric repeats on the chromosome ends . In normal or reactive tissues with a high regenerative capacity, telomerase is regulated according to the telomere loss that occurs during proliferation . To evaluate the interaction of proliferation and telomerase activity in malignant lymphomas, we quantified telomerase expression in different non-Hodgkin lymphomas in comparison to normal or reactive lymph nodes . Surprisingly, the activity levels were the same in most of the lymphomas analysed as compared to reactive lymph nodes . Significantly higher activity was detected only in Burkitt's lymphoma . Telomerase activity correlated well with hTERT and c-myc expression, but was independent of proliferation . To evaluate interactions of telomere-binding protein expression on telomerase expression in non-Hodgkin lymphoma, the mRNA levels of TRF1, TRF2, tankyrase and hPif1 were assessed by real-time RT-PCR . We demonstrate here that the magnitude of telomerase upregulation does not necessarily reflect the requirement of telomere compensation caused by proliferation . Telomerase regulation in non-Hodgkin lymphomas is therefore uncoupled from proliferative stimuli found in reactive lymphoid tissue . We suggest that the upregulation of specific telomere-binding proteins like TRF2 may contribute to telomere maintenance in malignant lymphoma.

J Biol Chem, 2003 Oct 3, 278(40), 38109 - 12 Epub 2003 Aug 12.
An Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A; Song OK et al.; A yeast gene has been identified that encodes a novel, evolutionarily conserved Nalpha-acetyltransferase responsible for acetylation of the N-terminal residues of histones H4 and H2A . The gene has been named NAT4 . Recombinant Nat4 protein acetylated a peptide corresponding to the N-terminal tail of H4, but not an H3 peptide nor the peptide adrenocorticotropin . H4 and H2A are N-terminally acetylated in all species from yeast to mammals and hence blocked from sequencing by Edman degradation . In contrast, H4 and H2A purified from a nat4 mutant were unacetylated and could be sequenced . Analysis of yeast histones by acid-urea gel electrophoresis showed that all the H4 and H2A from the mutant migrated more rapidly than the same histones from a wild type strain, consistent with the histones from the mutant having one extra positive charge due to one less acetylated amino group . A comparison of yeast proteins from wild type and a nat4 mutant by two-dimensional gel electrophoresis showed no evidence that other yeast proteins are substrates of this acetyltransferase . Thus, Nat4 may be dedicated specifically to the N-terminal acetylation of histones H4 and H2A . Surprisingly, nat4 mutants grow at a normal rate and have no readily observable phenotypes.

Gene Expr Patterns, 2003 Aug, 3(4), 483 - 8
Expression pattern of Siaz gene during the zebrafish embryonic development; Ro H et al.; Siah is a mammalian homolog of Drosophila seven in absentia (SINA) . Here we report the identification of a new member of the SINA/Siah gene family . This new gene, designated Siaz, was found in zebrafish, and its product is predicted to share extensive amino acid sequence homology with Drosophila SINA . Siaz is maternally inherited, with zygotic expression in all blastomeres starting at the mid-blastula transition . After the 20-somite stage, Siaz expression occurs in a stage-specific manner in particular regions, including the brain, eye, cranial cavity, otic vesicle, optic chiasm and gut.

Biochim Biophys Acta, 2003 Aug 18, 1641(2-3), 145 - 56
Involvement of LMA1 and GATE-16 family members in intracellular membrane dynamics; Elazar Z et al.; Intracellular membrane fusion is conserved from yeast to man as well as among different intracellular trafficking pathways . This process can be generally divided into several well-defined biochemical reactions . First, an early recognition (or tethering) takes place between donor and acceptor membranes, mediated by ypt/rab GTPases and complexes of tethering factors . Subsequently, a closer association between the two membranes is achieved by a docking process, which involves tight association between membrane proteins termed SNAREs . The formation of such a trans-SNARE complex leads to the final membrane fusion, resulting in an accumulation of cis-SNARE complexes on the acceptor membrane . Thus, multiple rounds of transport and delivery of the donor SNARE back to its original membrane require dissociation of the SNARE complexes . SNARE dissociation, termed priming, is mediated by the AAA ATPase, N-ethylmaleimide-sensitive factor (NSF) and its partner, soluble NSF attachment protein (SNAP), in a reaction that requires ATP hydrolysis . In the present review we focus on LMA1 and GATE-16, two low-molecular-weight proteins, which assist in priming SNARE molecules in the vacuole in yeast and the Golgi complex in mammals, respectively . LMA1 and GATE-16 are suggested to keep the dissociated cis-SNAREs apart from each other, allowing multiple fusion processes to take place . GATE-16 belongs to a novel family of ubiquitin-like proteins conserved from yeast to man . We discuss here the involvement of this family in multiple intracellular trafficking pathways.

Plant Physiol, 2003 Aug, 132(4), 2248 - 55
Enhanced formaldehyde detoxification by overexpression of glutathione-dependent formaldehyde dehydrogenase from Arabidopsis; Achkor H et al.; The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes . In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde . We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Delta) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH . Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism . To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH . Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis . These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.

Plant Physiol, 2003 Aug, 132(4), 2135 - 43
PAUSED encodes the Arabidopsis exportin-t ortholog; Hunter CA et al.; Los1p/exportin-t (XPOT) mediates the nuclear export of tRNAs in yeast and mammals . The requirements for this transport pathway are unclear, however, because los1 mutations do not affect yeast growth, and the phenotype of XPOT mutations in mammals is unknown . Here, we show that PAUSED (PSD) is the Arabidopsis ortholog of LOS1/XPOT and is capable of rescuing the tRNA export defect of los1 in Brewer's yeast (Saccharomyces cerevisiae), suggesting that its function has been conserved . Putative null alleles of PSD disrupt the initiation of the shoot apical meristem and delay leaf initiation after germination, the emergence of the radicle and lateral roots, and the transition to flowering . Plants doubly mutant for psd and hasty, the Arabidopsis ortholog of exportin 5, are viable but have a more severe phenotype than either single mutant . These results suggest that PSD plays a role in tRNA export in Arabidopsis, but that at least one-and perhaps several-additional tRNA export pathways also exist . The PSD transcript is broadly expressed during development and is alternatively spliced in the 3'-untranslated region . No temporal or spatial difference in the abundance of different splice forms was observed . We propose that the mutant phenotype of psd reflects defects in developmental events and cell/tissue types that require elevated levels of protein synthesis and are therefore acutely sensitive to a reduction in tRNA export.

Plant Physiol, 2003 Aug, 132(4), 1913 - 24
PAUSED, a putative exportin-t, acts pleiotropically in Arabidopsis development but is dispensable for viability; Li J et al.; Exportin-t was first identified in humans as a protein that mediates the export of tRNAs from the nucleus to the cytoplasm . Mutations in Los1p, the Saccharomyces cerevisiae exportin-t homolog, result in nuclear accumulation of tRNAs . Because no exportin-t mutants have been reported in multicellular organisms, the developmental functions of exportin-t have not been determined . Here, we report the isolation and characterization of two Arabidopsis exportin-t mutants, paused-5 and paused-6 . The mutant phenotypes indicate that exportin-t acts pleiotropically in plant development . In particular, paused-5 and paused-6 result in delayed leaf formation during vegetative development . The two paused mutations also cause the transformation of reproductive organs into perianth organs in the hua1-1 hua2-1 background, which is partially defective in reproductive organ identity specification . The floral phenotypes of hua1-1 hua2-1 paused mutants resemble those of mutations in the floral homeotic gene AGAMOUS . Moreover, paused-5 enhances the mutant phenotypes of two floral meristem identity genes, LEAFY and APETALA1 . The developmental defects caused by paused mutations confirm the important roles of exportin-t in gene expression in multicellular organisms . In addition, a paused null allele, paused-6, is still viable, suggesting the presence of redundant tRNA export pathway(s) in Arabidopsis.

Plant Physiol, 2003 Aug, 132(4), 1884 - 91
Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals; Golovkin M et al.; U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation . In non-plant systems, it is involved in constitutive and alternative splicing . It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal . As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene . In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs . Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development . In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile . The severity of the phenotype is correlated with the level of the antisense transcript . Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development . The AP3 transcript was not detected in transgenic flowers with severe phenotype . Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities . These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants.

J Cell Biol, 2003 Aug 18, 162(4), 635 - 46 Epub 2003 Aug 11.
The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization; Gao XD et al.; Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane . Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro . We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo . Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter . Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants . Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.

Hum Mol Genet, 2003 Aug 15, 12(16), 1995 - 2001
Zinc metalloproteinase, ZMPSTE24, is mutated in mandibuloacral dysplasia; Agarwal AK et al.; Mandibuloacral dysplasia (MAD; OMIM 248370) is a rare, genetically and phenotypically heterogeneous, autosomal recessive disorder characterized by skeletal abnormalities including hypoplasia of the mandible and clavicles, acro-osteolysis, cutaneous atrophy and lipodystrophy . A homozygous missense mutation, Arg527His, in the LMNA gene which encodes nuclear lamina proteins lamins A and C has been reported in patients with MAD and partial lipodystrophy . We studied four patients with MAD who had no mutations in the LMNA gene . We now show compound heterozygous mutations, Phe361fsX379 and Trp340Arg, in the zinc metalloproteinase (ZMPSTE24) gene in one of the four patients who had severe MAD associated with progeroid appearance and generalized lipodystrophy . ZMPSTE24 is involved in post-translational proteolytic cleavage of carboxy terminal residues of farnesylated prelamin A in two steps to form mature lamin A . Deficiency of Zmpste24 in mice causes accumulation of prelamin A and phenotypic features similar to MAD . The yeast homolog, Ste24, has a parallel role in processing of prenylated mating pheromone a-factor . Since human ZMPSTE24 can also process a-factor when expressed in yeast, we assessed the functional significance of the two ZMPSTE24 mutations in the yeast to complement the mating defect of the haploid MATa yeast lacking STE24 and Ras-converting enzyme 1 (RCE1; another prenylprotein-specific endoprotease) genes . The ZMPSTE24 mutant construct, Phe361fsX379, was inactive in complementing the yeast a-factor but the mutant, Trp340Arg, was partially active compared to the wild type ZMPSTE24 construct . We conclude that mutations in ZMPSTE24 may cause MAD by affecting prelamin A processing.

J Biol Chem, 2003 Oct 31, 278(44), 43418 - 29 Epub 2003 Aug 11.
Unexpected protein families including cell defense components feature in the N-myristoylome of a higher eukaryote; Boisson B et al.; N-Myristoylation is an irreversible modification that affects the membrane binding properties of crucial cytoplasmic proteins from signal transduction cascades . We characterized the two putative N-myristoyltransferases of Arabidopsis thaliana as a means of investigating the entire N-myristoylation proteome (N-myristoylome) in a higher eukaryote . AtNMT1 compensated for the nmt1 defect in yeast, whereas AtNMT2 and chimeras of the two genes did not . Only AtNMT1 modified known N-myristoylated proteins in vitro . AtNMT1 is therefore responsible for the A . thaliana N-myristoylome, whereas AtNMT2 does not seem to have usual myristoylation activity . We began with the whole set of N-myristoylated G proteins in the A . thaliana proteome . We then used a reiterative approach, based on the in vitro N-myristoylation of more than 60 different polypeptides, to determine the substrate specificity of AtNMT1 . We found that the positive charge on residue 7 of the substrate was particularly important in substrate recognition . The A . thaliana N-myristoylome consists of 437 proteins, accounting for 1.7% of the complete proteome . We demonstrated the N-myristoylation of several unexpected protein families, including innate immunity proteins, thioredoxins, components of the protein degradation pathway, transcription factors, and a crucial regulatory enzyme of glycolysis . The role of N-myristoylation is discussed in each case; in particular, this process may underlie the "guard" hypothesis of innate immunity.

EMBO J, 2003 Aug 15, 22(16), 4325 - 36
DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1; Cobb JA et al.; To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression . In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress . Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage . Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min . This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation . A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.

EMBO J, 2003 Aug 15, 22(16), 4304 - 13
Elg1 forms an alternative RFC complex important for DNA replication and genome integrity; Bellaoui M et al.; Genome-wide synthetic genetic interaction screens with mutants in the mus81 and mms4 replication fork-processing genes identified a novel replication factor C (RFC) homolog, Elg1, which forms an alternative RFC complex with Rfc2-5 . This complex is distinct from the DNA replication RFC, the DNA damage checkpoint RFC and the sister chromatid cohesion RFC . As expected from its genetic interactions, elg1 mutants are sensitive to DNA damage . Elg1 is redundant with Rad24 in the DNA damage response and contributes to activation of the checkpoint kinase Rad53 . We find that elg1 mutants display DNA replication defects and genome instability, including increased recombination and mutation frequencies, and minichromosome maintenance defects . Mutants in elg1 show genetic interactions with pathways required for processing of stalled replication forks, and are defective in recovery from DNA damage during S phase . We propose that Elg1-RFC functions both in normal DNA replication and in the DNA damage response.

EMBO J, 2003 Aug 15, 22(16), 4223 - 36
Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase; Audhya A et al.; The essential phospholipid PI4,5P(2) is generated by a well conserved PI4P 5-kinase, Mss4, in yeast . Balanced production and turnover of PI4,5P(2) is important for normal organization of the actin cytoskeleton and cell viability . Previous studies have shown that multiple PI phosphatases can regulate PI4,5P(2) levels . We report a new, unexpected regulatory mechanism for PI4,5P(2) homeostasis, directed by nuclear-cytoplasmic shuttling of the lipid kinase . We show that Mss4 is a phosphoprotein, which contains a functional nuclear localization signal (NLS) and can shuttle between the cytoplasm and the nucleus . Temperature-conditional mss4 cells that accumulate Mss4 protein in the nucleus exhibit reduced levels of PI4,5P(2), depolarization of the actin cytoskeleton and a block in Mss4 phosphorylation, suggesting an essential role for phosphorylated Mss4 at the plasma membrane . Through the isolation of gene dosage-dependent suppressors of mss4 mutants, we identified Bcp1, a protein enriched in the nucleus, which is required for Mss4 nuclear export and is related to the mammalian BRCA2-interacting protein BCCIP . Together, these studies suggest a new mechanism for lipid kinase regulation through regulated nuclear-cytoplasmic shuttling.

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