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| EMBO J, 2003 Aug 15, 22(16), 4103 - 10 A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling; Echtay KS et al.; Oxidative stress and mitochondrial dysfunction are associated with disease and aging . Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal . Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production . We find that hydroxynonenal and structurally related compounds (such as trans-retinoic acid, trans-retinal and other 2-alkenals) specifically induce uncoupling of mitochondria through the uncoupling proteins UCP1, UCP2 and UCP3 and the adenine nucleotide translocase (ANT) . Hydroxynonenal-induced uncoupling was inhibited by potent inhibitors of ANT (carboxyatractylate and bongkrekate) and UCP (GDP) . The GDP-sensitive proton conductance induced by hydroxynonenal correlated with tissue expression of UCPs, appeared in yeast mitochondria expressing UCP1 and was absent in skeletal muscle mitochondria from UCP3 knockout mice . The carboxyatractylate-sensitive hydroxynonenal stimulation correlated with ANT content in mitochondria from Drosophila melanogaster expressing different amounts of ANT . Our findings indicate that hydroxynonenal is not merely toxic, but may be a biological signal to induce uncoupling through UCPs and ANT and thus decrease mitochondrial ROS production. EMBO J, 2003 Aug 15, 22(16), 4059 - 69 Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export; Pathre P et al.; The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER) . Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export . PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export . However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export . Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export . Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat. Traffic, 2003 Sep, 4(9), 631 - 41 Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase; Stroud WJ et al.; We investigated the relative distributional persistence of Golgi "matrix" proteins and glycosyltransferases to an endoplasmic reticulum exit block induced by expression of a GDP-restricted Sar1p . HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block endoplasmic reticulum exit and then scored for the distribution of GM130 (Golgi matrix protein of 130 kDa), a cis located golgin; p27, a member of the p24 family of proteins; giantin, a protein that interacts indirectly with GM130; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2) . All of these proteins lost their compact, juxtanuclear distribution and displayed characteristics of endoplasmic reticulum/cytoplasmic accumulation with the same dependence on plasmid concentration . The kinetics of redistribution of GM130 and GalNAcT2 were identical . Expression of Sar1pT39N displaced the COPII coat protein Sec13p from endoplasmic reticulum exit sites consistent with disruption of these sites . This occurred without disturbing the overall distribution of endoplasmic reticulum membrane . Furthermore, the reassembly of a juxtanuclear Golgi matrix as assayed by the distribution of GM130 following washout of the Golgi disrupting drug, brefeldin A, was blocked by microinjected Sar1pT39N plasmids . We conclude that the persistence, i.e . stability and maintenance, of Golgi matrix distribution and its reassembly following drug disruption are exquisitely dependent on Sar1p activity. Biochemistry, 2003 Aug 19, 42(32), 9779 - 88 Biosynthesis of docosahexaenoic acid in Euglena gracilis: biochemical and molecular evidence for the involvement of a Delta4-fatty acyl group desaturase; Meyer A et al.; Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase . We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase . For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates . Schizochytrium sp . was unable to convert exogenously supplied {2-(14)C}-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E . gracilis and Thraustochytrium sp . carried out this desaturation very efficiently . Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis . To clone the desaturase gene, a cDNA library of E . gracilis was subjected to mass sequencing . A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp . was isolated, and its function was verified by heterologous expression in yeast . The desaturase efficiently converted DPA to DHA . Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids . The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate . Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position. Biochemistry, 2003 Aug 19, 42(32), 9687 - 93 Refolding of amphioxus insulin-like peptide: implications of a bifurcating evolution of the different folding behavior of insulin and insulin-like growth factor 1; Wang S et al.; Insulin and insulin-like growth factor 1 (IGF-1) share high sequence homology, but their folding behaviors are significantly different: insulin folds into one unique thermodynamically controlled structure, while IGF-1 folds into two thermodynamically controlled disulfide isomers . However, the origin of their different folding behaviors is still elusive . The amphioxus insulin-like peptide (ILP) is thought to be the common ancestor of insulin and IGF-1 . A recombinant single-chain ILP has been expressed previously, and now its folding behavior is investigated . The folding behavior of ILP shows the characteristics of both insulin and IGF-1 . On one hand, two thermodynamically controlled disulfide isomers of ILP have been identified; on the other hand, the content of isomer 1 (its disulfides are deduced identical to those of swap IGF-1) is much less than that of isomer 2 (its disulfides are deduced identical to those of native IGF-1); that is, more than 96% of ILP folds into the native structure . The present results suggest that the different folding behaviors of insulin and IGF-1 are acquired through a bifurcating evolution: the tendency of forming the thermodynamically controlled non-native disulfide isomer is diminished during evolution from ILP to insulin, while this tendency is amplified during evolution from ILP to IGF-1 . Moreover, the N-terminal Gln residue of ILP can spontaneously form a pyroglutamate residue, and its cyclization has a significant effect on the folding behavior of ILP: the percentage of isomer 1 is approximately 2-fold that of isomer 1 of the noncyclized ILP; that is, isomer 1 becomes more favored when the N-terminal residue of ILP is cyclized . So, we deduce that the N-terminal residues have a significant effect on the folding properties of insulin, IGF-1, and ILP. Biochemistry, 2003 Aug 19, 42(32), 9609 - 18 Structure, stability, and function of hDim1 investigated by NMR, circular dichroism, and mutational analysis; Zhang YZ et al.; The 142 amino acid Dim1p protein is a component of the U4/U6.U5 tri-snRNP complex required for pre-mRNA splicing and interacts with multiple splicing-associated proteins . To gain further insight into the structural basis of its function, we determined the solution structure of the reduced form of the dominant negative human hDim1 (hDim1(1)(-)(128)) using multidimensional NMR spectroscopy . This dominant negative hDim1 assumes a thioredoxin-like fold, confirming previous NMR and crystallographic results . However, in contrast to a recent crystal structure, the NMR solution structure for the reduced form of hDim1(1)(-)(128) presented here, along with thermodynamic data, indicates that the presence of a disulfide bond between Cys38 and Cys79 is structurally and functionally unimportant . Comparison of the truncated hDim1(1)(-)(128) with the full-length protein, using NMR and circular dichroism spectroscopy, indicates that the 14 C-terminal residues can undergo a local unfolding transition and assume alternative conformations, which appear to play a functional role . Other residues essential for hDim1 function are identified by using mutational and genetic approaches . The residues thus identified are not identical with those previously shown to govern Dim1 interaction with defined protein partners. J Mol Evol, 2003 Jun, 56(6), 702 - 10 A novel strategy for analysis of gene homologues and segmental genome duplications; Noskov VN et al.; Transformation-associated recombination (TAR) cloning allows selective isolation of a desired chromosomal region or gene from complex genomes . The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae . We investigated the effect of nonhomology on the efficiency of gene capture and found that up to 15% DNA divergence did not prevent efficient gene isolation . Such tolerance to DNA divergence greatly expands the potential applications of TAR cloning for comparative genomics . In this study, we were able to use the technique to isolate nonidentical chromosomal duplications and gene homologues. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9934 - 9 Epub 2003 Aug 08. Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity; Resnick MA et al.; There are many sources of genetic diversity, ranging from programmed mutagenesis in antibody genes to random mutagenesis during species evolution or development of cancer . We propose that mutations in DNA sequence-specific transcription factors that target response elements (REs) in many genes can also provide for rapid and broad phenotypic diversity, if the mutations lead to altered binding affinities at individual REs . To test this concept, we examined the in vivo transactivation capacity of wild-type human and murine p53 and 25 partial function mutants . The p53s were expressed in yeast from a rheostatable promoter, and the transactivation capacities toward >15 promoter REs upstream of a reporter gene were measured . Surprisingly, there was wide variation in transactivation by the mutant p53s toward the various REs . This is the first study to address directly the impact of mutations in a sequence-specific transcription factor on transactivation from a wide array of REs . We propose a master gene hypothesis for phenotypic diversity where the master gene is a single transcriptional activator (or repressor) that regulates many genes through different REs . Mutations of the master gene can lead to a variety of simultaneous changes in both the selection of targets and the extent of transcriptional modulation at the individual targets, resulting in a vast number of potential phenotypes that can be created with minimal mutational changes without altering existing protein-protein interactions. J Exp Biol, 2003 Sep, 206(Pt 18), 3227 - 37 Biochemical support for the V-ATPase rotary mechanism: antibody against HA-tagged Vma7p or Vma16p but not Vma10p inhibits activity; Aviezer-Hagai K et al.; V-ATPase null mutants in yeast have a distinct, conditionally lethal phenotype that can be obtained through disruption of any one of its subunits . This enables supplementation of this mutant with the relevant subunit tagged with an epitope against which an antibody is available . In this system, the effect of antibody on the activity of the enzyme can be analyzed . Towards this end we used HA to tag subunits Vma7p, Vma10p and Vma16p, which are assumed to represent, respectively, the shaft, stator and turbine of the enzyme, and used them to supplement the corresponding yeast V-ATPase null mutants . The anti-HA epitope antibody inhibited both the ATP-dependent proton uptake and the ATPase activities of the Vma16p-HA and Vma7p-HA containing complexes, in intact vacuoles and in the detergent-solubilized enzyme . Neither of these activities was inhibited by the antibody in Vma10p-HA containing enzyme . These results support the function of Vma10p as part of the stator, while the other tagged subunits are part of the rotor apparatus . The HA-tag was attached to the N terminus of Vma16p; thus the antibody inhibition points to its accessibility outside the vacuolar membrane . This assumption is supported by the supplementation of the yeast mutant by the homologues of Vma16p isolated from Arabidopsis thaliana and lemon fruit c-DNA . Contrary to yeast, which has five predicted helices, the plant subunit Vma16p has only four . Our results confirm a recent report that only four of the yeast Vma16p complexes are actually transmembrane helices. Biochem J, 2003 Nov 1, 375(Pt 3), 673 - 80 Site-directed mutagenesis of the active site of diacylglycerol kinase alpha: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms; Abe T et al.; Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover . DAGKa is activated in vitro by Ca2+ and by acidic phospholipids . The regulatory region of DAGKa includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation . DAGKa also contains tandem C1 protein kinase C homology domains . We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKa and to determine the enzymic activities of different mutant forms of pig DAGKa in vitro . Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis . Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases . Mutation of D434 (Asp434) or D650 abolished all DAGKa activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKa . Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol . A DAGKa mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation . Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS . These results indicate that Ca2+ and PS stimulate DAGKa via distinct mechanisms. Nucleic Acids Res . 2003 Aug 15;31(16):e94. A sensitive transcriptome analysis method that can detect unknown transcripts; Fukumura R et al.; We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis . By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased . As a result we can determine the relationship between peaks and original transcripts unequivocally . This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks . This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species . The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression . Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs . Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes . Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available. Nucleic Acids Res, 2003 Aug 15, 31(16), 4888 - 98 Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus; Grainge I et al.; The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing . Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea . The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue . The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro . The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA . Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA . The purified Orc/Cdc6 homologue was also able to bind DNA . Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates . Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap . The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer. Genomics, 2003 Sep, 82(3), 401 - 5 The centrosomal proteins pericentrin and kendrin are encoded by alternatively spliced products of one gene; Flory MR et al.; Pericentrin, a critical centrosome component first identified in mouse, recruits factors required for assembly of the mitotic spindle apparatus . A similar yet larger human protein named kendrin was recently identified, but its relationship to pericentrin was not clear . Extensive sequence homology between the mouse chromosome 10 region encoding pericentrin and the human chromosome 21 region encoding kendrin indicates that these proteins are encoded by syntenic loci . However, comparison of the published mouse pericentrin cDNA sequence to mouse genomic DNA sequences revealed two important differences: the stop codon present in the published mouse pericentrin cDNA is not found in the mouse genomic sequence, and the 3' end of the published mouse pericentrin cDNA is a fragment from a different mouse chromosome . To resolve these discrepancies, we sequenced a mouse expressed sequence tag (EST) that corresponds to the 3' end for a 7.1-kb mouse pericentrin RNA encoded on chromosome 10 . Extensive northern blot analysis revealed that the pericentrin gene displays a complex expression pattern in both mouse and human: a 10-kb kendrin transcript is found in most tissues, whereas smaller transcripts are detected in a limited subset of tissues . These analyses demonstrate that pericentrin and kendrin are encoded by one gene, correct the previously published pericentrin cDNA sequence, and describe the complex expression pattern for a gene important for centrosome function in normal and transformed cells. Curr Biol, 2003 Aug 5, 13(15), R603 - 5 Meiosis: polo, FEAR and the art of dividing reductionally; Cohen-Fix O; Recent studies on the regulation of meiosis have uncovered new roles for old acquaintances: the polo-like kinase Cdc5 has been found to dictate proper kinetochore orientation during meiosis I, while the FEAR pathway is essential for some, but not all, aspects of meiosis I exit. Curr Biol, 2003 Aug 5, 13(15), R597 - 9 Microtubule dynamics: new surprises from an old MAP; McNally F; Dis1/XMAP215 family microtubule-binding proteins are essential for cell division in animals, plants and fungi, suggesting a conserved cell-division mechanism used by all eukaryotes . Two new studies, however, reveal that different family members can have very different effects on microtubule dynamics. Phys Rev Lett . 2003 Aug 1;91(5):058701 . Epub 2003 Aug 01. Probabilistic prediction in scale-free networks: diameter changes; Kim JH et al.; In complex systems, responses to small perturbations are too diverse to definitely predict how much they would be, and then such diverse responses can be predicted in a probabilistic way . Here we study such a problem in scale-free networks, for example, the diameter changes by the deletion of a single vertex for various in silico and real-world scale-free networks . We find that the diameter changes are indeed diverse and their distribution exhibits an algebraic decay with an exponent zeta asymptotically . Interestingly, the exponent zeta is robust as zeta approximately 2.2(1) for most scale-free networks and insensitive to the degree exponents gamma as long as 2<gamma</=3 . However, there is another type with zeta approximately 1.7(1) and its examples include the Internet and its related in silico model. Planta, 2003 Aug, 217(4), 668 - 75 Epub 2003 Mar 28. Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation; Zhang Y et al.; DNA homologous to the yeast ( Saccharomyces cerevisiae) protein kinase gene, GCN2, was amplified from arabidopsis { Arabidopsis thaliana (L.) Heynh.} RNA and given the name AtGCN2 . The AtGCN2 peptide sequence included adjacent protein kinase and histidyl tRNA synthetase-like domains and showed 45% sequence identity with the GCN2 peptide sequence in the protein kinase domain . AtGCN2 transcripts were detectable in RNA from roots, leaves, stems, buds, flowers, siliques and seedlings . GCN2 is required for yeast cells to respond to amino acid starvation . Expression of AtGCN2 in yeast gcn2 mutants complemented the mutation, enabling growth in the presence of sulfometuron methyl, an inhibitor of branched-chain amino acid biosynthesis, and 3-aminotriazole, an inhibitor of histidine biosynthesis. Anal Bioanal Chem, 2003 Aug, 376(7), 994 - 1005 Epub 2003 Jul 09. Acetylation of the HIV-1 Tat protein: an in vitro study; Dormeyer W et al.; In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased . It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes . Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides . In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites . We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region . Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme . Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general. Nature, 2003 Aug 28, 424(6952), 1083 - 7 Epub 2003 Aug 06. Replication of a cis-syn thymine dimer at atomic resolution; Ling H et al.; Ultraviolet light damages DNA by catalysing covalent bond formation between adjacent pyrimidines, generating cis-syn cyclobutane pyrimidine dimers (CPDs) as the most common lesion . CPDs block DNA replication by high-fidelity DNA polymerases, but they can be efficiently bypassed by the Y-family DNA polymerase pol eta . Mutations in POLH encoding pol eta are implicated in nearly 20% of xeroderma pigmentosum, a human disease characterized by extreme sensitivity to sunlight and predisposition to skin cancer . Here we have determined two crystal structures of Dpo4, an archaeal pol eta homologue, complexed with CPD-containing DNA, where the 3' and 5' thymine of the CPD separately serves as a templating base . The 3' thymine of the CPD forms a Watson-Crick base pair with the incoming dideoxyATP, but the 5' thymine forms a Hoogsteen base pair with the dideoxyATP in syn conformation . Dpo4 retains a similar tertiary structure, but each unusual DNA structure is individually fitted into the active site for catalysis . A model of the pol eta-CPD complex built from the crystal structures of Saccharomyces cerevisiae apo-pol eta and the Dpo4-CPD complex suggests unique features that allow pol eta to efficiently bypass CPDs. Microbiology, 2003 Aug, 149(Pt 8), 2039 - 48 Identification and functional expression of ctaA, a P-type ATPase gene involved in copper trafficking in Trametes versicolor; Uldschmid A et al.; Here the identification and characterization of a gene encoding a copper-trafficking enzyme, ctaA (copper-transporting ATPase), from the basidiomycete Trametes versicolor are described . This P-type copper ATPase gene has two alleles, differing primarily in the length of the second, unusually long intron, and encodes a 983 aa protein with 40 % sequence identity to yeast Ccc2p . Overexpression of ctaA in yeast grown in the presence of copper led to a 15-fold increase in laccase yields, while overexpression of ctaA and tahA, a previously identified copper homeostasis gene of T . versicolor, was additive, leading to a 20-fold increase in laccase production . In T . versicolor, overexpression of ctaA and tahA led to an eightfold increase in laccase expression, and a cotransformant still expressed laccase at 3000 micro M copper when hardly any laccase activity is detected in the wild-type strain . Apparently, at low to moderate levels of copper tahA and ctaA overexpression disturbs the normal hierarchy of copper distribution, resulting in more being directed to the Golgi, while with high copper amounts that normally switch on the copper detoxification processes, tahA and ctaA gene products seem to out-compete the metallothionein copper chaperones, meaning laccase is still supplied with copper . These results may lead to a better understanding of copper trafficking and the hierarchy of copper distribution in the cell, and possibly be useful for constructing laccase-overproducing strains for biotechnological purposes. Nucleic Acids Symp Ser, 2000, (44), 259 - 60 Histone acetyltransferase (HAT) activity of ATF-2 is necessary for the CRE-dependent transcription; Kawasaki H et al.; ATF-2 is a DNA-binding protein that binds to cAMP-response elements (CREs) and forms a hetrodimer with c-Jun, via binding of the leucine zipper motif and then stimulates the CRE-dependent transcription (1,2) . Recently, we have reported that ATF-2 has an intrinsic acetyltransferase activity that is controlled by phosphorylation . Mutant form of either p300 or ATF-2 with mutations in the HAT domain failed to stimulate the CRE-dependent transcription in response to UV irradiation . Moreover, phosphorylation of ATF-2 enhanced its HAT activity and CRE-dependent transcription . Thus, these results indicate that HAT activity of ATF-2 is important for the CRE-dependent transcription. Nucleic Acids Symp Ser, 2000, (44), 245 - 6 In vitro selection by using mutated GCN4-bZIP peptides for analysis of peptide-DNA interactions; Furusawa H et al.; In vitro selection has been used as a method to determine the optimal binding site for DNA-binding proteins . We report here in vitro selection of dsDNA sequences that bind to mutated-GCN4-bZIP peptides . The GCN4-bZIP peptide mutated from alanine to histidine on a position-14 that contacts with DNA bound to different sequence from a binding site of wild type peptide. Nucleic Acids Symp Ser, 2000, (44), 13 - 4 DNA binding of a basic leucine-zipper protein with novel folding domain; Sato S et al.; DNA-binding proteins frequently utilize short alpha-helices as their critical DNA recognition elements . In this research, we have employed the structure-based design to construct a small domain that could target the specific DNA sequences recognized by the yeast transcriptional activator GCN4 . The new DNA binding motif recognizes specific DNA sequences as a dimer with high affinity and specificity under the physiological conditions. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9668 - 73 Epub 2003 Aug 05. Shrinkage-based similarity metric for cluster analysis of microarray data; Cherepinsky V et al.; The current standard correlation coefficient used in the analysis of microarray data was introduced by M . B . Eisen, P . T . Spellman, P . O . Brown, and D . Botstein {(1998) Proc . Natl . Acad . Sci . USA 95, 14863-14868} . Its formulation is rather arbitrary . We give a mathematically rigorous correlation coefficient of two data vectors based on James-Stein shrinkage estimators . We use the assumptions described by Eisen et al., also using the fact that the data can be treated as transformed into normal distributions . While Eisen et al . use zero as an estimator for the expression vector mean mu, we start with the assumption that for each gene, mu is itself a zero-mean normal random variable {with a priori distribution N(0,tau 2)}, and use Bayesian analysis to obtain a posteriori distribution of mu in terms of the data . The shrunk estimator for mu differs from the mean of the data vectors and ultimately leads to a statistically robust estimator for correlation coefficients . To evaluate the effectiveness of shrinkage, we conducted in silico experiments and also compared similarity metrics on a biological example by using the data set from Eisen et al . For the latter, we classified genes involved in the regulation of yeast cell-cycle functions by computing clusters based on various definitions of correlation coefficients and contrasting them against clusters based on the activators known in the literature . The estimated false positives and false negatives from this study indicate that using the shrinkage metric improves the accuracy of the analysis. Genome Res, 2003 Aug, 13(8), 1863 - 72 Decay rates of human mRNAs: correlation with functional characteristics and sequence attributes; Yang E et al.; Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions . We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species . Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes . The results show statistically significant organizational principles in the variation of decay rates among functional classes . In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in "fast-decaying" mRNAs with half-lives <2 h . In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs . Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes . Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript . Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay . However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites. Trends Genet, 2003 Aug, 19(8), 439 - 46 Telomere maintenance and DNA replication: how closely are these two connected? Chakhparonian M, Wellinger RJ. The maintenance of the DNA at chromosome ends, the telomeres, depends on conventional semiconservative replication and on the action of telomerase, a specialized reverse transcriptase . Current research strongly suggests a regulatory interplay between this conventional semiconservative replication and telomerase, thus ensuring that no sequences are lost at the very ends of the telomeres during replication . Here, we describe recent findings on the interactions between the conventional replication machinery and telomere replication, and we discuss how DNA-integrity checkpoints might impinge on both the processing of the telomeric DNA ends and the establishment of the DNA end structure required for end protection and genome stability. Trends Genet, 2003 Aug, 19(8), 422 - 7 Genomic analysis of gene expression relationships in transcriptional regulatory networks; Yu H et al.; From merging several data sources, we created an extensive map of the transcriptional regulatory network in Saccharomyces cerevisiae, comprising 7419 interactions connecting 180 transcription factors (TFs) with their target genes . We integrated this network with gene-expression data, relating the expression profiles of TFs and target genes . We found that genes targeted by the same TF tend to be co-expressed, with the degree of co-expression increasing if genes share more than one TF . Moreover, shared targets of a TF tend to have similar cellular functions . By contrast, the expression relationships between the TFs and their targets are much more complicated, often exhibiting time-shifted or inverted behavior . Further information is available at http://bioinfo.mbb.yale.edu/regulation/TIG/ Trends Genet, 2003 Aug, 19(8), 415 - 7 Moonlighting proteins: old proteins learning new tricks; Jeffery CJ; Recently, several laboratories identifying proteins involved in the complex processes of replication, transcription and tumor suppression found that the 'new' protein they discovered had another, previously identified, function . A single protein with multiple functions might seem surprising, but there are actually many cases of proteins that 'moonlight', or have more than one role in an organism . As well as adding to the number and types of proteins that are known to moonlight, these new examples add to our understanding of the potential importance of moonlighting proteins. Biochem Biophys Res Commun, 2003 Aug 22, 308(2), 276 - 83 Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion; Sesaki H et al.; The dynamin-related GTPase, Mgm1p, is critical for the fusion of the mitochondrial outer membrane, maintenance of mitochondrial DNA (mtDNA), formation of normal inner membrane structures, and inheritance of mitochondria . Although there are two forms of Mgm1p, 100 and 90 kDa, their respective functions and the mechanism by which these two forms are produced are not clear . We previously isolated ugo2 mutants in a genetic screen to identify components involved in mitochondrial fusion {J . Cell Biol . 152 (2001) 1123} . In this paper, we show that ugo2 mutants are defective in PCP1, a gene encoding a rhomboid-related serine protease . Cells lacking Pcp1p are defective in the processing of Mgm1p and produce only the larger (100 kDa) form of Mgm1p . Similar to mgm1delta cells, pcp1delta cells contain partially fragmented mitochondria, instead of the long tubular branched mitochondria of wild-type cells . In addition, pcp1delta cells, like mgm1delta cells, lack mtDNA and therefore are unable to grow on nonfermentable medium . Mutations in the catalytic domain lead to complete loss of Pcp1p function . Similar to mgm1delta cells, the fragmentation of mitochondria and loss of mtDNA of pcp1delta cells were rescued when mitochondrial division was blocked by inactivating Dnm1p, a dynamin-related GTPase . Surprisingly, in contrast to mgm1delta cells, which are completely defective in mitochondrial fusion, pcp1delta cells can fuse their mitochondria after yeast cell mating . Our study demonstrates that Pcp1p is required for the processing of Mgm1p and controls normal mitochondrial shape and mtDNA maintenance by producing the 90 kDa form of Mgm1p . However, the processing of Mgm1p is not strictly required for mitochondrial fusion, indicating that the 100 kDa form is sufficient to promote fusion. Neurobiol Dis, 2003 Aug, 13(3), 238 - 45 Presenilin-1 interacts directly with the beta-site amyloid protein precursor cleaving enzyme (BACE1); Hebert SS et al.; A neuropathological hallmark of Alzheimer's disease is the presence of amyloid plaques . The major constituent of these plaques, occurring largely in brain areas important for memory and cognition, is the 40-42 amyloid residues (Abeta) . Abeta is derived from the amyloid protein precursor after cleavage by the recently identified beta-secretase (BACE1) and the putative gamma-secretase complex containing presenilin 1 (PS1) . In an attempt to develop a functional secretase enzymatic assay in yeast we demonstrate a direct binding between BACE1 and PS1 . This interaction was confirmed in vivo using coimmunoprecipitation and colocalization studies in human cultured cells . Our results show that PS1 preferably binds immature BACE1, thus possibly acting as a functional regulator of BACE1 maturation and/or activity. J Biomol Tech, 2003 Mar, 14(1), 17 - 32 A rapid method for manual or automated purification of fluorescently labeled nucleic acids for sequencing, genotyping, and microarrays; Springer AL et al.; Fluorescent dyes provide specific, sensitive, and multiplexed detection of nucleic acids . To maximize sensitivity, fluorescently labeled reaction products (e.g., cycle sequencing or primer extension products) must be purified away from residual dye-labeled precursors . Successful high-throughput analyses require that this purification be reliable, rapid, and amenable to automation . Common methods for purifying reaction products involve several steps and require processes that are not easily automated . Prolinx, Inc . has devel oped RapXtract superparamagnetic separation technology affording rapid and easy-to-perform methods that yield high-quality product and are easily automated . The technology uses superparamagnetic particles that specifically remove unincorporated dye-labeled precursors . These particles are efficiently pelleted in the presence of a magnetic field, making them ideal for purification because of the rapid separations that they allow . RapXtract-purified sequencing reactions yield data with good signal and high Phred quality scores, and they work with various sequencing dye chemistries, including BigDye and near-infrared fluorescence IRDyes . RapXtract technology can also be used to purify dye primer sequencing reactions, primer extension reactions for genotyping analysis, and nucleic acid labeling reactions for microarray hybridization . The ease of use and versatility of RapXtract technology makes it a good choice for manual or automated purification of fluorescently labeled nucleic acids. J Cell Biol, 2003 Aug 4, 162(3), 413 - 23 Vps27 recruits ESCRT machinery to endosomes during MVB sorting; Katzmann DJ et al.; Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway . The function of this pathway requires the class E vacuolar protein sorting (Vps) proteins . Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway . We demonstrate that activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27 . ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27 . A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101, the mammalian homologue of Vps23 . We propose that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin . Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes. Biochemistry, 2003 Aug 12, 42(31), 9249 - 56 Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry; Sauve AA et al.; Life span regulation and inhibition of gene silencing in yeast have been linked to nicotinamide effects on Sir2 enzymes . The Sir2 enzymes are NAD(+)-dependent protein deacetylases that influence gene expression by forming deacetylated proteins, nicotinamide and 2'-O-acetyl-ADPR . Nicotinamide is a base-exchange substrate as well as a biologically effective inhibitor . Characterization of the base-exchange reaction reveals that nicotinamide regulates sirtuins by switching between deacetylation and base exchange . Nicotinamide switching is quantitated for the Sir2s from Archeaglobus fulgidus (Sir2Af2), Saccharomyces cerevisiae (Sir2p), and mouse (Sir2alpha) . Inhibition of deacetylation was most effective for mouse Sir2 alpha, suggesting species-dependent development of this regulatory mechanism . The Sir2s are proposed to form a relatively stable covalent intermediate between ADPR and the acetyl oxygen of the acetyllysine-protein substrate . During the lifetime of this intermediate, nicotinamide occupation of the catalytic site determines the fate of the covalent complex . Saturation of the nicotinamide site for mouse, yeast, and bacterial Sir2s causes 95, 65, and 21% of the intermediate, respectively, to return to acetylated protein . The fraction of the intermediate committed to deacetylation results from competition between the nicotinamide and the neighboring 2'-hydroxyl group at the opposite stereochemical face . Nicotinamide switching supports the previously proposed Sir2 catalytic mechanism and the existence of a 1'-O-peptidyl-ADPR.Sir2 intermediate . These findings suggest a strategy for increasing Sir2 enzyme catalytic activity in vivo by inhibition of chemical exchange but not deacetylation. Recent Results Cancer Res, 2003, 164, 349 - 52 A low-calcemic vitamin D analog (Ro 25-4020) inhibits the growth of LNCaP human prostate cancer cells with increased potency by producing an active 24-oxo metabolite (Ro 29-9970); Swami S et al.; In this study, we have characterized a novel less-calcemic vitamin D analog Ro 25-4020 (1alpha, 25 dihydroxy-16-ene-5,6-trans-vitamin D3) and investigated the mechanisms underlying its enhanced growth inhibitory properties . We found that Ro 25-4020 (IC50 = 0.3 nM) exhibited greater inhibitory activity than 1,25(OH)2D3 (IC50 = 1 nM) on LNCaP human prostate cancer cell growth . However, Ro 25-4020 was tenfold less active than 1,25(OH)2D3 in receptor-binding assays, ligand-induced heterodimerization and transactivation assays using VDR . HPLC and GC-MS analyses revealed that 1,25(OH)2D3 is converted to a 24-hydroxy metabolite, which has been shown to be less potent than 1,25(OH)2D3 . In contrast, Ro 25-4020 was converted to a major 24-oxo metabolite that was more stable . Ligand-binding assays reveal that both Ro 25-4020 and its 24-oxo metabolite have similar affinity for VDR . Synthetic 24-oxo-Ro 25-4020, however, inhibited LNCaP cell proliferation as potently as 1,25(OH)2D3 and was more potent in transactivation of two out of three vitamin D target genes tested . These results suggest that conversion of Ro 25-4020 into an active and more stable 24-oxo metabolite with longer half-life contributes significantly to its potent antiproliferative actions on the LNCaP cells. J Cell Biochem, 2003 Aug 15, 89(6), 1215 - 21 alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform; Moura Lima PR et al.; The band 3 protein is the major integral protein present in the erythrocyte membrane . Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes . It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown . In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform . The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin . Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay . The confocal microscopy showed band 3 in the intercalated discs . Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane . Nat Struct Biol, 2003 Sep, 10(9), 718 - 24 Epub 2003 Aug 03. Cooperative organization in a macromolecular complex; Seeliger MA et al.; The mechanism of assembly of multiprotein complexes and the subsequent organization of activity are not well understood . Here we report the application of biophysical tools to investigate the relationship between structure and function in protein assemblies . We used as a model system the SCF(Skp2) complex that targets p27(Kip1) for ubiquitination and subsequent degradation; this process requires an adapter protein, Cks1 . By dissecting the interactions between the different subunits we show that the properties of Cks1 are highly context dependent, and its activity is acquired only when the complex is fully assembled . The results provide insights into the central role of small adapters in macromolecular assembly and explain their high sequence conservation . Simultaneous and synergistic binding of multiple subunits in a complex provides the specificity and control required before the key cell-cycle regulator p27 is committed to degradation. Plant Cell, 2003 Aug, 15(8), 1817 - 32 Cytoplasmic N-terminal protein acetylation is required for efficient photosynthesis in Arabidopsis; Pesaresi P et al.; The Arabidopsis atmak3-1 mutant was identified on the basis of a decreased effective quantum yield of photosystem II . In atmak3-1, the synthesis of the plastome-encoded photosystem II core proteins D1 and CP47 is affected, resulting in a decrease in the abundance of thylakoid multiprotein complexes . DNA array-based mRNA analysis indicated that extraplastid functions also are altered . The mutation responsible was localized to AtMAK3, which encodes a homolog of the yeast protein Mak3p . In yeast, Mak3p, together with Mak10p and Mak31p, forms the N-terminal acetyltransferase complex C (NatC) . The cytoplasmic AtMAK3 protein can functionally replace Mak3p, Mak10p, and Mak31p in acetylating N termini of endogenous proteins and the L-A virus Gag protein . This result, together with the finding that knockout of the Arabidopsis MAK10 homolog does not result in obvious physiological effects, indicates that AtMAK3 function does not require NatC complex formation, as it does in yeast . We suggest that N-acetylation of certain chloroplast precursor protein(s) is necessary for the efficient accumulation of the mature protein(s) in chloroplasts. Mol Cell Biol, 2003 Aug, 23(16), 5928 - 38 Support for a meiotic recombination initiation complex: interactions among Rec102p, Rec104p, and Spo11p; Jiao K et al.; Initiation of meiotic recombination in the yeast Saccharomyces cerevisiae requires at least 10 gene products . The initiation event creates double-strand breaks, which are then processed by other recombination enzymes . A variety of classical observations, such as the existence of recombination nodules, have suggested that the proteins catalyzing recombination form a complex . A variety of lines of evidence indicate that Rad50p, Mre11p, and Xrs2p interact, and genetic data suggesting interactions between Rec102p and Rec104p have been reported . It has recently been shown that Spo11p coimmunoprecipitates with Rec102p in meiosis as well . In this paper, we provide genetic and biochemical evidence that the meiosis-specific proteins Rec102p, Rec104p, and Spo11p all interact with each other in meiosis . Furthermore, we demonstrate that the interaction between Rec102p and Spo11p does not require Rec104p . Likewise, the interaction between Rec104p and Rec102p does not require Spo11p, although Spo11p may stabilize that association . The interactions suggest that Spo11p, Rec102p, and Rec104p may form a trimeric complex during the initiation of recombination. Mol Cell Biol, 2003 Aug, 23(16), 5857 - 66 Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1; Lee HC et al.; Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins . The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1 . To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1 . We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90 . Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90 . These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes . The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence . Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction . It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation . Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction. Mol Cell Biol, 2003 Aug, 23(16), 5572 - 80 Role of mammalian Rad54 in telomere length maintenance; Jaco I et al.; The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown . Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability . Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals . Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping . Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity. Mol Cell Biol, 2003 Aug, 23(16), 5526 - 39 p21-Activated kinase 5 (Pak5) localizes to mitochondria and inhibits apoptosis by phosphorylating BAD; Cotteret S et al.; Pak5 is the most recently identified and least understood member of the p21-activated kinase (Pak) family . This kinase is known to promote neurite outgrowth in vitro, but its localization, substrates, and effects on cell survival have not been reported . We show here that Pak5 has unique properties that distinguish it from all other members of the Pak family . First, Pak5, unlike Pak1, cannot complement an STE20 mutation in Saccharomyces cerevisiae . Second, Pak5 binds to the GTPases Cdc42 and Rac, but these GTPases do not regulate Pak5 kinase activity, which is constitutive and stronger than any other Pak . Third, Pak5 prevents apoptosis induced by camptothecin and C2-ceramide by phosphorylating BAD on Ser-112 in a protein kinase A-independent manner and prevents the localization of BAD to mitochondria, thereby inhibiting the apoptotic cascade that leads to apoptosis . Finally, we show that Pak5 itself is constitutively localized to mitochondria, and that this localization is independent of kinase activity or Cdc42 binding . These features make Pak5 unique among the Pak family and suggest that it plays an important role in apoptosis through BAD phosphorylation. Mol Cell Biol, 2003 Aug, 23(16), 5516 - 25 Identification of a unique core domain of par-4 sufficient for selective apoptosis induction in cancer cells; El-Guendy N et al.; Recent studies indicated that the leucine zipper domain protein Par-4 induces apoptosis in certain cancer cells by activation of the Fas prodeath pathway and coparallel inhibition of NF-kappaB transcriptional activity . However, the intracellular localization or functional domains of Par-4 involved in apoptosis remained unknown . In the present study, structure-function analysis indicated that inhibition of NF-kappaB activity and apoptosis is dependent on Par-4 translocation to the nucleus via a bipartite nuclear localization sequence, NLS2 . Cancer cells that were resistant to Par-4-induced apoptosis retained Par-4 in the cytoplasm . Interestingly, a 59-amino-acid core that included NLS2 but not the C-terminal leucine zipper domain was necessary and sufficient to induce Fas pathway activation, inhibition of NF-kappaB activity, and apoptosis . Most important, this core domain had an expanded target range for induction of apoptosis, extending to previously resistant cancer cells but not to normal cells . These findings have identified a unique death-inducing domain selective for apoptosis induction in cancer cells (SAC domain) which holds promise for identifying key differences between cancer and normal cells and for molecular therapy of cancer. J Biol Chem, 2003 Oct 17, 278(42), 41083 - 92 Epub 2003 Aug 01. Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest; Yanow SK et al.; We have cloned a Xenopus Dbf4-related factor named Drf1 and characterized this protein by using Xenopus egg extracts . Drf1 forms an active complex with the kinase Cdc7 . However, most of the Cdc7 in egg extracts is not associated with Drf1, which raises the possibility that some or all of the remaining Cdc7 is bound to another Dbf4-related protein . Immunodepletion of Drf1 does not prevent DNA replication in egg extracts . Consistent with this observation, Cdc45 can still associate with chromatin in Drf1-depleted extracts, albeit at significantly reduced levels . Nonetheless, Drf1 displays highly regulated binding to replicating chromatin . Treatment of egg extracts with aphidicolin results in a substantial accumulation of Drf1 on chromatin . This accumulation is blocked by addition of caffeine and by immunodepletion of either ATR or Claspin . These observations suggest that the increased binding of Drf1 to aphidicolin-treated chromatin is an active process that is mediated by a caffeine-sensitive checkpoint pathway containing ATR and Claspin . Abrogation of this pathway also leads to a large increase in the binding of Cdc45 to chromatin . This increase is substantially reduced in the absence of Drf1, which suggests that regulation of Drf1 might be involved in the suppression of Cdc45 loading during replication arrest . We also provide evidence that elimination of this checkpoint causes resumed initiation of DNA replication in both Xenopus tissue culture cells and egg extracts . Taken together, these observations argue that Drf1 is regulated by an intra-S-phase checkpoint mechanism that down-regulates the loading of Cdc45 onto chromatin containing DNA replication blocks. J Biol Chem, 2003 Oct 17, 278(42), 41462 - 71 Epub 2003 Aug 01. Targeting and assembly of mitochondrial tail-anchored protein Tom5 to the TOM complex depend on a signal distinct from that of tail-anchored proteins dispersed in the membrane; Horie C et al.; Mitochondrial outer membrane proteins are synthesized without a cleavable presequence but instead contain segments responsible for mitochondrial targeting and membrane integration within the molecule: the transmembrane segment (TMS) and N- or C-terminal flanking segment . We analyzed targeting and integration of Tom5, a C-tail anchor protein associated with the preprotein translocase of the outer membrane, to the yeast mitochondrial outer membrane in vivo using green fluorescent protein as the reporter and compared the signal with other signals for proteins dispersed in the membrane . The functional assembly of Tom5 into the TOM complex was assessed by blue native PAGE and complementation of temperature-sensitive deltatom5 cells . Correct targeting and assembly required (i) . an appropriate length TMS rather than hydrophobicity, (ii) . a proline residue located at correct position in the TMS and specific residues near the proline, and (iii) . that, in contrast to proteins dispersed in the outer membrane, the positive C-terminal segment was dispensable . Based on these findings, we constructed green fluorescent protein fusions with a C-terminal TMS in which the deduced sequences (minimum: Ser-Pro-Met) were inserted at an appropriate position within artificial Leu-Ala repeats . They were targeted to mitochondria and complemented the temperature-sensitive growth phenotype of deltatom5 yeast cells . The membrane-targeting mechanism of Tom5 appears to be distinct from that for proteins that are dispersed in the outer membrane. Anal Biochem, 2003 Sep 1, 320(1), 125 - 8 Ubiquinone binding protein used for determination of coenzyme Q; Hagerman RA et al.; The conventional method of assaying for the ubiquinone (CoQ) content of biological samples is to partition CoQ into an organic phase and separate it from contaminants by high-performance liquid chromatography (HPLC) . HPLC is an accurate method of quantifying CoQ content but is not ideal for routine clinical analyses . This paper describes the development of a rapid method for assaying the CoQ content of biological samples based on the binding of CoQ to a CoQ binding peptide . The 14-amino acid binding peptide was chemically synthesized, and conditions for immobilizing the peptide on microfuge tubes were established . CoQ could be selectively bound to the immobilized peptide, eluted, and determined spectrophotometrically . Limits of detection for the method were 0.25 to 5 nmol CoQ . To test biological samples, CoQ was isolated from cultures of Saccharomyces cerevisiae grown in oleic acid medium . The recovery of CoQ samples using the binding assay ranged from 99 to 102% of the values obtained with HPLC . The assay described here provides an inexpensive, rapid method for determining the CoQ content of large numbers of biological samples in a variety of laboratory settings. J Biochem Mol Biol, 2003 Jul 31, 36(4), 421 - 5 A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4; Xu K et al.; The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways . Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors . Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed . However, they require a generation of antibodies against the phosphorylated form of a specific substrate . We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates . Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme. J Biochem Mol Biol, 2003 Jul 31, 36(4), 344 - 8 Identification of mutations in protein kinase CKIIbeta subunit that affect its binding to ribosomal protein L41 and homodimerization; Ahn BH et al.; Protein kinase CKII is composed of two catalytic (alpha or alpha') subunits and two regulatory (beta) subunits . The CKIIbeta subunit is thought to mediate the tetramer formation and interact with other target proteins . However, its physiological function remains obscure . In this study, point mutants of CKIIbeta that are defective for the L41 binding were isolated by using the reverse two-hybrid system . A sequence analysis of the point mutants revealed that Asp-26, Met-52, and Met-78 of CKIIbeta are critical for L41 binding; Asn-67 (and/or Lys-139) and Met-52 are important for CKIIbeta homodimerization . Two point mutants, R75 and R83, of CKIIbeta interacted with L5, topoisomerase IIbeta, and CKBBP1/SAG, but not with the wild-type CKIIbeta . This indicates that CKIIbeta homodimerization is not a prerequisite for its binding to target proteins . These CKIIbeta point mutants may be useful in exploring the biochemical physiological functions of CKIIbeta. Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9873 - 7 Epub 2003 Jul 31. A SNARE required for retrograde transport to the endoplasmic reticulum; Burri L et al.; SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells . Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1) . Slt1 is an essential protein localized in the endoplasmic reticulum (ER) . It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1 . Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER . We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER . Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin. Mol Membr Biol, 2003 Jul-Sep, 20(3), 197 - 207 ER-to-Golgi transport: COP I and COP II function (Review); Duden R; COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells . These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles . COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER) . COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER . Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex. Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 632 - 9 Bone morphogenetic protein signalling in NGF-stimulated PC12 cells; Althini S et al.; Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions . Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days . NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6 . PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity . Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response . Adding the inhibitory Smad7 drastically reduced this signal . In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity . A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active . Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity . However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription . The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site . Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells. Methods, 2003 Sep, 31(1), 83 - 9 Genomewide histone acetylation microarrays; Robyr D et al.; Histone acetylation and methylation are important regulators of gene activity . Chromatin immunoprecipitation (ChIP or ChrIP) has made it possible to examine not only the state of histone acetylation at a gene but also that of histone methylation and may soon be extended to other histone modifications such as phosphorylation and ubiquitination . In principle such studies are possible as long as an antibody is available to the particular histone modification . Once a target gene is identified it is instructive to see the effect of mutating putative enzymes responsible for the modification to determine how a particular enzyme is responsible for altering chromatin of that gene . Although specific target genes have been studied that contain such modifications recent technical advances have made it possible to study histone modifications genomewide . This not only allows for alternate views of particular paradigms to be investigated, but also uncovers chromosomal patterns of histone modification that would be missed in analyzing individual genes . We describe here an approach to rapidly study histone modifications genomewide by combining chromatin immunoprecipitation and DNA microarrays. Methods, 2003 Sep, 31(1), 59 - 66 In vivo assays to study histone ubiquitylation; Kao CF et al.; The importance of histone acetylation, phosphorylation, and methylation in transcription and other DNA-mediated processes is now well established . Histones are also ubiquitylated, but in contrast to the majority of ubiquitylated proteins, ubiquitylated histones are not generally targeted for degradation and may play roles similar to those of other histone modifications . Antibodies against acetylated histones have provided unique insights into the regulation, distribution, and cellular roles of these modified histones . In this report, we describe methods to identify ubiquitylated histones in budding yeast and HeLa cells . We provide protocols to detect ubiquitylated histones that are based on a combination of in vivo genetic and immunological assays . These methods should provide relatively simple and useful tools to study the global regulation of this important but poorly understood histone modification. Curr Opin Cell Biol, 2003 Aug, 15(4), 489 - 97 Peroxisome biogenesis: advances and conundrums; Lazarow PB; Investigations of peroxisome biogenesis in diverse organisms reveal new details of this unique process and its evolutionary conservation . Interactions among soluble receptors and the membrane peroxins that catalyze protein translocation are being mapped . Ubiquitination is observed . A receptor enters the organelle carrying folded cargo and recycles back to the cytosol . Tiny peroxisome remnants - vesicles and tubules - are discovered in pex3 mutants that lack the organelle . When the mutant is transfected with a good PEX3 gene, these protoperoxisomes acquire additional membrane peroxins and then import the matrix enzymes to reform peroxisomes . Thus, de novo formation need not be postulated . Dynamic imaging of yeast reveals dynamin-dependent peroxisome division and regulated actin-dependent segregation of the organelle before cell division . These results are consistent with biogenesis by growth and division of pre-existing peroxisomes. Curr Opin Cell Biol, 2003 Aug, 15(4), 446 - 55 Protein sorting into multivesicular endosomes; Raiborg C et al.; Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes . Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation . Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes . A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process . Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation . Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding. Biochem J, 2003 Oct 15, 375(Pt 2), 329 - 37 Direct interaction of beta-dystroglycan with F-actin; Chen YJ et al.; Dystroglycans are essential transmembrane adhesion receptors for laminin . Alpha-dystroglycan is a highly glycosylated extracellular protein that interacts with laminin in the extracellular matrix and the transmembrane region of beta-dystroglycan . Beta-dystroglycan, via its cytoplasmic tail, interacts with dystrophin and utrophin and also with the actin cytoskeleton . As a part of the dystrophin-glycoprotein complex of muscles, dystroglycan is also important in maintaining sarcolemmal integrity . Mutations in dystrophin that lead to Duchenne muscular dystrophy also lead to a loss of dystroglycan from the sarcolemma, and chimaeric mice lacking muscle dystroglycan exhibit a severe muscular dystrophy phenotype . Using yeast two-hybrid analysis and biochemical and cell biological studies, we show, in the present study, that the cytoplasmic tail of beta-dystroglycan interacts directly with F-actin and, furthermore, that it bundles actin filaments and induces an aberrant actin phenotype when overexpressed in cells. Nature, 2003 Jul 31, 424(6948), 565 - 71 Machinery for protein sorting and assembly in the mitochondrial outer membrane; Wiedemann N et al.; Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes . It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination . Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM) . Mas37 forms a constituent of the SAM complex . The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15) . These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex. Nature, 2003 Jul 31, 424(6948), 549 - 52 Evolutionary capacitance as a general feature of complex gene networks; Bergman A et al.; An evolutionary capacitor buffers genotypic variation under normal conditions, thereby promoting the accumulation of hidden polymorphism . But it occasionally fails, thereby revealing this variation phenotypically . The principal example of an evolutionary capacitor is Hsp90, a molecular chaperone that targets an important set of signal transduction proteins . Experiments in Drosophila and Arabidopsis have demonstrated three key properties of Hsp90: (1) it suppresses phenotypic variation under normal conditions and releases this variation when functionally compromised; (2) its function is overwhelmed by environmental stress; and (3) it exerts pleiotropic effects on key developmental processes . But whether these properties necessarily make Hsp90 a significant and unique facilitator of adaptation is unclear . Here we use numerical simulations of complex gene networks, as well as genome-scale expression data from yeast single-gene deletion strains, to present a mechanism that extends the scope of evolutionary capacitance beyond the action of Hsp90 alone . We illustrate that most, and perhaps all, genes reveal phenotypic variation when functionally compromised, and that the availability of loss-of-function mutations accelerates adaptation to a new optimum phenotype . However, this effect does not require the mutations to be conditional on the environment . Thus, there might exist a large class of evolutionary capacitors whose effects on phenotypic variation complement the systemic, environment-induced effects of Hsp90. J Biol Chem, 2003 Oct 10, 278(41), 39517 - 26 Epub 2003 Jul 30. The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification; Nemoto T et al.; Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles . The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential . In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation . The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation . The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p . Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate . Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy . Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3 . Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis . The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate. J Biol Chem, 2003 Oct 10, 278(41), 39303 - 10 Epub 2003 Jul 30. Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90; Wegele H et al.; Hsp90 is a dimeric, ATP-regulated molecular chaperone . Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709) . To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain . To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization . We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90 . The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein . Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein . This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge . In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped . Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle . These reactions are synergistic and require Hsp90 to be in the dimeric state. Biochem Biophys Res Commun, 2003 Aug 15, 308(1), 1 - 11 Composition and function of the eukaryotic N-terminal acetyltransferase subunits; Polevoda B et al.; Saccharomyces cerevisiae contains three N-terminal acetyltransferases (NATs), NatA, NatB, and NatC, composed of the following catalytic and auxiliary subunits: Ard1p and Nat1p (NatA); Nat3p and Mdm20p (NatB); and Mak3p, Mak10, and Mak31p (NatC) . The overall patterns of N-terminally acetylated proteins and NAT orthologous genes suggest that yeast and higher eukaryotes have similar systems for N-terminal acetylation . The differential expression of certain NAT subunits during development or in carcinomas of higher eukaryotes suggests that the NATs are more highly expressed in cells undergoing rapid protein synthesis . Although Mak3p is functionally the same in yeast and plants, findings with TE2 (a human Ard1p ortholog) and Tbdn100 (a mouse Nat1p ortholog) suggest that certain of the NAT subunits may have functions other than their role in NATs or that these orthologs are not functionally equivalent . Thus, the vertebrate NATs remain to be definitively identified, and, furthermore, it remains to be seen if any of the yeast NATs contribute to other functions. Mol Microbiol, 2003 Aug, 49(4), 859 - 67 Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region; Vilela C et al.; We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region . The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function . The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs . Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF . In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it . Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known . Translation of the uORF can also control gene expression by affecting the stability of the mRNA . Finally, trans-acting factors may participate in the regulatory mechanisms . Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism. Biotechnol Lett, 2003 Jun, 25(11), 891 - 3 A screening method for detecting iron reducing wood-rot fungi; Oviedo C et al.; A plate assay using the Fe(II) selective dye, ferrozine, for detecting wood-rot fungi with Fe(III) reductive abilities, was developed . The assay is fast, simple and, in most cases, more sensitive than the corresponding liquid medium test . The brown rot fungi, Gloeophyllum trabeum and Laetiporeus sulphureus, displayed higher iron reductive capabilities than white rot fungi, Trametes versicolor, Ganoderma australe and Ceriporiopsis subvermispora. J Biol Chem, 2003 Oct 10, 278(41), 39509 - 16 Epub 2003 Jul 29. Concomitant increase of histone acetyltransferase activity and degradation of p300 during retinoic acid-induced differentiation of F9 cells; Brouillard F et al.; The p300 and closely related CBP histone acetyltransferases (HAT) function as global transcriptional co-activators that play roles in many cell differentiation and signal transduction pathways . Despite their similarities, p300 and CBP have distinct functions during retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells . F9 cells constitute a well established model system for investigating the first steps of early development and retinoic acid signaling ex vivo . p300, but not CBP, was shown to be essential for F9 differentiation . In this study we have investigated the regulation of p300 during F9 differentiation . We report a dramatic decrease of p300, but not CBP protein levels, after 48 h of retinoic acid treatment . p300 is degraded via the ubiquitin-proteasome pathway . Although the large majority of p300 is degraded, its global HAT activity stays constant during F9 differentiation, which means that its specific HAT activity increases considerably . p300 is strongly phosphorylated in both undifferentiated and differentiated F9 cells; its HAT activity, however, is independent of phosphorylation before differentiation and becomes dependent on phosphorylation during differentiation . Furthermore, we show that protein kinase A affects p300 HAT activity both in vivo and in vitro as well as p300 phosphorylation in differentiated cells . Thus, we show that p300 is differentially phosphorylated in undifferentiated versus differentiated cells and that the changes in phosphorylation affect its HAT activity . Moreover, our study suggests an explanation for the functional switch of p300-mediated repression versus activation during F9 differentiation. Nucleic Acids Res, 2003 Aug 1, 31(15), 4597 - 607 The essential transcription factor Reb1p interacts with the CLB2 UAS outside of the G2/M control region; Van Slyke C et al.; Regulation of CLB2 is important both for completion of the normal vegetative cell cycle in Saccharomyces cerevisiae and for departure from the vegetative cell cycle upon nitrogen deprivation . Cell cycle-regulated transcription of CLB2 in the G2/M phase is known to be brought about by a set of proteins including Mcm1p, Fkh2/1p and Ndd1p that associate with a 35 bp G2/M-specific sequence common to a set of co-regulated genes . CLB2 transcription is regulated by additional signals, including by nitrogen levels, by positive feedback from the Clb2-Cdc28 kinase, and by osmotic stress, but the corresponding regulatory sequences and proteins have not been identified . We have found that the essential Reb1 transcription factor binds with high affinity to a sequence upstream of CLB2, within a region implicated previously by others in regulated expression, but upstream of the known G2/M-specific site . CLB2 sequence from the region around the Reb1p site blocks activation by the Gal4 protein when positioned downstream of the Gal4-binding site . Since a mutation in the Reb1p site abrogates this effect, we suggest that Reb1p is likely to occupy this site in vivo. Nucleic Acids Res, 2003 Aug 1, 31(15), 4553 - 60 The computational analysis of scientific literature to define and recognize gene expression clusters; Raychaudhuri S et al.; A limitation of many gene expression analytic approaches is that they do not incorporate comprehensive background knowledge about the genes into the analysis . We present a computational method that leverages the peer-reviewed literature in the automatic analysis of gene expression data sets . Including the literature in the analysis of gene expression data offers an opportunity to incorporate functional information about the genes when defining expression clusters . We have created a method that associates gene expression profiles with known biological functions . Our method has two steps . First, we apply hierarchical clustering to the given gene expression data set . Secondly, we use text from abstracts about genes to (i) resolve hierarchical cluster boundaries to optimize the functional coherence of the clusters and (ii) recognize those clusters that are most functionally coherent . In the case where a gene has not been investigated and therefore lacks primary literature, articles about well-studied homologous genes are added as references . We apply our method to two large gene expression data sets with different properties . The first contains measurements for a subset of well-studied Saccharomyces cerevisiae genes with multiple literature references, and the second contains newly discovered genes in Drosophila melanogaster; many have no literature references at all . In both cases, we are able to rapidly define and identify the biologically relevant gene expression profiles without manual intervention . In both cases, we identified novel clusters that were not noted by the original investigators. Nucleic Acids Res, 2003 Aug 1, 31(15), 4425 - 33 Spurious spatial periodicity of co-expression in microarray data due to printing design; Balazsi G et al.; Global transcriptome data is increasingly combined with sophisticated mathematical analyses to extract information about the functional state of a cell . Yet the extent to which the results reflect experimental bias at the expense of true biological information remains largely unknown . Here we show that the spatial arrangement of probes on microarrays and the particulars of the printing procedure significantly affect the log-ratio data of mRNA expression levels measured during the Saccharomyces cerevisiae cell cycle . We present a numerical method that filters out these technology-derived contributions from the existing transcriptome data, leading to improved functional predictions . The example presented here underlines the need to routinely search and compensate for inherent experimental bias when analyzing systematically collected, internally consistent biological data sets. Nucleic Acids Res, 2003 Aug 1, 31(15), 4391 - 400 Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F; Meka H et al.; In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme . Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7 . We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases . The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology . We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits . To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex. Nucleic Acids Res, 2003 Aug 1, 31(15), 4326 - 31 Solution structure of the HIV-1 frameshift inducing stem-loop RNA; Staple DW et al.; The translation of reverse transcriptase and other essential viral proteins from the HIV-1 Pol mRNA requires a programmed -1 ribosomal frameshift . This frameshift is induced by two highly conserved elements within the HIV-1 mRNA: a slippery sequence comprised of a UUUUUUA heptamer, and a downstream stem-loop structure . We have determined the structure of the HIV-1 frameshift inducing RNA stem-loop, using multidimensional heteronuclear nuclear magnetic resonance (NMR) methods . The 22 nucleotide RNA solution structure {root mean squared deviation (r.m.s.d.) = 1.2 A} was determined from 475 nuclear Overhauser effect (NOE)-derived distance restrains, 20 residual dipolar couplings and direct detection of hydrogen bonds via scalar couplings . We find that the frameshift inducing stem-loop is an A-form helix capped by a structured ACAA tetraloop . The ACAA tetraloop is stabilized by an equilateral 5' and 3' stacking pattern, a sheared A-A pair and a cross-strand hydrogen bond . Unexpectedly, the ACAA tetraloop structure is nearly identical to a known tetraloop fold, previously identified in the RNase III recognition site from Saccharomyces cerevisiae. Nucleic Acids Res, 2003 Aug 1, 31(15), 4285 - 92 Mechanisms of P/CAF auto-acetylation; Santos-Rosa H et al.; P/CAF is a histone acetyltransferase enzyme which was originally identified as a CBP/p300-binding protein . In this manuscript we report that human P/CAF is acetylated in vivo . We find that P/CAF is acetylated by itself and by p300 but not by CBP . P/CAF acetylation can be an intra- or intermolecular event . The intermolecular acetylation requires the N-terminal domain of P/CAF . The intramolecular acetylation targets five lysines (416-442) at the P/CAF C-terminus, which are in the nuclear localisation signal (NLS) . Finally, we show that acetylation of P/CAF leads to an increment of its histone acetyltransferase (HAT) activity . These findings identify a new post-translation modification on P/CAF which may regulate its function. Trends Cell Biol, 2003 Aug, 13(8), 396 - 9 Cycling without CDK2? Grim JE, Clurman BE. Cyclin-dependent kinase 2 (CDK2) regulates diverse aspects of the mammalian cell cycle . Most cancer cells contain mutations in the pathways that control CDK2, and CDK2 activity has received much attention as a target for cancer therapy . However, a recent report demonstrating that some cancer cells can proliferate without CDK2 activity questions the essential role of CDK2 in cell-cycle control, as well as its suitability as a therapeutic target. Mol Cell, 2003 Jul, 12(1), 209 - 19 In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination; Sugawara N et al.; Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor . Here we used chromatin immunoprecipitation to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HML alpha donor . Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence . Rad51p binding to ssDNA requires Rad52p . The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HML alpha . Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HML alpha; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand. Mol Cell, 2003 Jul, 12(1), 147 - 55 Involvement of actin-related proteins in ATP-dependent chromatin remodeling; Shen X et al.; Actin-related proteins (Arps) and conventional actin are enigmatic components of many chromatin-remodeling enzyme complexes . The yeast INO80 ATP-dependent chromatin-remodeling complex contains stoichiometric amounts of Arp4, Arp5, Arp8, and actin . Here we have revealed functions of Arp5 and Arp8 by analysis of mutants . arp5 Delta and arp8 Delta mutants display an ino80 Delta phenotype . Purification of INO80 complexes from arp5 Delta and arp8 Delta cells shows that protein complexes remain intact but are compromised for INO80 ATPase activity, DNA binding, and nucleosome mobilization . The INO80 (arp8 Delta) complex is strikingly deficient, not only for the Arp8 subunit, but also for Arp4 and actin, suggesting an ordered assembly of Arps . Binding of Arp8 to the INO80 complex requires an N-terminal region of Ino80 adjacent to the conserved ATPase domain . GST-Arp8 binds preferentially to histones H3 and H4 in vitro, suggesting a histone chaperone function . These findings show direct involvement of Arps in the chromatin-remodeling process. Mol Cell, 2003 Jul, 12(1), 101 - 11 Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties; Kelleher DJ et al.; Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum . Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined . The roles of specific OST subunits remained enigmatic . Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p . The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity . Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity. Mol Cell, 2003 Jul, 12(1), 51 - 62 Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state; Fulco M et al.; Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span . Prompted by the observation that the {NAD(+)}/{NADH} ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation . Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation . Conversely, cells with decreased Sir2 differentiate prematurely . To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity . The {NAD(+)}/{NADH} ratio decreases as muscle cells differentiate, while an increased {NAD(+)}/{NADH} ratio inhibits muscle gene expression . Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated {NAD(+)}/{NADH} ratio . These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor . In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression. Plant J, 2003 Aug, 35(3), 295 - 304 The soybean NRAMP homologue, GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport; Kaiser BN et al.; Iron is an important nutrient in N2-fixing legume root nodules . Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain . The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol . In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters . GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules . Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules . GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4) . The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules . Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed. J Virol, 2003 Aug, 77(16), 8695 - 701 Selection of retroviral reverse transcription primer is coordinated with tRNA biogenesis; Kelly NJ et al.; Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu . To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNA(Phe) to be supplied in trans for infectivity . Wild-type yeast tRNA(Phe) expressed in mammalian cells was transported to the cytoplasm and aminoacylated . In contrast, tRNA(Phe) without the D loop (tRNA(Phe)D(-)) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNA(Phe)D(-) was directly transfected into the cytoplasm of cells . A tRNA(Phe) mutant (tRNA(Phe)UUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNA(Phe) . Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis. J Biol Chem, 2003 Oct 10, 278(41), 39402 - 12 Epub 2003 Jul 28. The coactivator p/CIP/SRC-3 facilitates retinoic acid receptor signaling via recruitment of GCN5; Brown K et al.; p/CIP/SRC-3 is a member of a family of steroid receptor coactivators/nuclear receptor coactivators (SRC/NCoA) proteins that mediate the transcriptional effects of nuclear hormone receptors (NRs) . Using deletion analysis we have mapped the location of two distinct activation domains in p/CIP (AD1 and AD2) capable of activating transcription in mammalian cells when fused to the Gal4-DNA binding domain . In addition to AD1 being coincident with the interaction domain for CBP, we demonstrate a novel in vivo interaction between the AD1 and GCN5 . Overexpression of a Gal4-AD1 fusion protein in yeast leads to growth arrest that is relieved by mutation of genes encoding components of the SAGA complex including GCN5, ADA3, and SPT7 . In addition, the AD1 of p/CIP and the ADA3 gene are shown to be essential for retinoic acid receptor alpha-dependent transcription in yeast . Transient transfection assays in mammalian cells indicate that GCN5 cooperates with p/CIP as a coactivator of RAR alpha-dependent transcription . Down-regulation of GCN5 using small interfering RNA in mammalian cells indicates that the AD1 domain and the RAR beta promoter activity are dependent, in part, on GCN5 . Mutational analysis of AD1 has identified two helical motifs that are required for interactions with GCN5 and CBP . Taken together, these results support a model by which p/CIP functions as a ligand-dependent adapter, through specific protein-protein interactions with AD1, to recruit members from at least two distinct families of acetyltransferase proteins to NRs. J Cell Biol, 2003 Aug 4, 162(3), 403 - 12 Epub 2003 Jul 28. The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER; Morsomme P et al.; Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex . Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p . However, the t-SNARE Sed5p was not required for protein sorting upon ER exit . Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM . Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature . Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins. Dev Biol, 2003 Aug 1, 260(1), 245 - 59 MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C elegans; Yang HY et al.; In most animals, successful segregation of female meiotic chromosomes involves sequential associations of the meiosis I and meiosis II spindles with the cell cortex so that extra chromosomes can be deposited in polar bodies . The resulting reduction in chromosome number is essential to prevent the generation of polyploid embryos after fertilization . Using time-lapse imaging of living Caenorhabditis elegans oocytes containing fluorescently labeled chromosomes or microtubules, we have characterized the movements of meiotic spindles relative to the cell cortex . Spindle assembly initiated several microns from the cortex . After formation of a bipolar structure, the meiosis I spindle translocated to the cortex . When microtubules were partially depleted, translocation of the bivalent chromosomes to the cortex was blocked without affecting cell cycle timing . In oocytes depleted of the microtubule-severing enzyme, MEI-1, spindles moved to the cortex, but association with the cortex was unstable . Unlike translocation of wild-type spindles, movement of MEI-1-depleted spindles was dependent on FZY-1/CDC20, a regulator of the metaphase/anaphase transition . We observed a microtubule and FZY-1/CDC20-dependent circular cytoplasmic streaming in wild-type and mei-1 mutant embryos during meiosis . We propose that, in mei-1 mutant oocytes, this cytoplasmic streaming is sufficient to drive the spindle into the cortex . Cytoplasmic streaming is not the normal spindle translocation mechanism because translocation occurred in the absence of cytoplasmic streaming in embryos depleted of either the orbit/CLASP homolog, CLS-2, or FZY-1 . These results indicate a direct role of microtubule severing in translocation of the meiotic spindle to the cortex. FEBS Lett, 2003 Jul 31, 548(1-3), 108 - 12 Differential binding of Sin3 interacting repressor domains to the PAH2 domain of Sin3A; Pang YP et al.; The Sin3 interacting domain (SID), originally described in the Mad family of repressors, is a novel transcriptional repressor domain that binds the PAH2 domain of corepressors Sin3A and Sin3B with high affinities . The conserved SID-like domains are reportedly present in five KLF proteins . However, the KLF SIDs and the Mad SIDs can be classified into two subtypes according to sequence similarity . Here, we report the finding from computational and experimental studies that the two subtypes of SID domains bind differentially to Sin3A . This finding offers insights into a mechanism of cell growth regulation by interactions of different subtypes of SID-containing repressor proteins with Sin3 . It also provides the structural basis for developing selective modulators of Sin3. FEBS Lett, 2003 Jul 31, 548(1-3), 59 - 68 Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly; Thomas CF Jr et al.; Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV . The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity . In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature . The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis . We have identified a P . carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function . The P . carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast . Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2 . PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation . Since glucan machinery is not present in mammals, a better understanding of this pathway in P . carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall. FEBS Lett, 2003 Jul 31, 548(1-3), 33 - 6 A gene-specific effect of an internal deletion in the Bdp1 subunit of the RNA polymerase III transcription initiation factor TFIIIB; Ishiguro A et al.; The Saccharomyces cerevisiae RPR1 gene encodes the RNA subunit of its RNase P, which processes RNA polymerase (pol) III primary transcripts . RPR1, which is transcribed by pol III, has been isolated as a multicopy suppressor of a specific small internal deletion (amino acids 253-269) in the Bdp1 subunit of transcription factor TFIIIB, the core pol III transcription factor . The selective effect of this Bdp1 deletion on RPR1 transcription has been analyzed in vitro . It is shown that TFIIIC-dependent assembly of TFIIIB on the RPR1 promoter is specifically sensitive to this Bdp1 deletion, leading to gene-specifically defective single-round and multiple-round transcription. Chromosoma, 2003 Aug, 112(2), 58 - 65 Epub 2003 Jul 16. CEN plasmid segregation is destabilized by tethered determinants of Ty 5 integration specificity: a role for double-strand breaks in CEN antagonism; Fuerst PG et al.; The yeast retrotransposon Ty 5 integrates preferentially into heterochromatin at the telomeres and HM loci . Target specificity is mediated by a six amino acid sequence motif (the targeting domain, TD) of integrase that interacts with Sir4p, a structural component of heterochromatin . When tethered to CEN plasmids as part of a Gal4p DNA binding domain (GBD) fusion protein, TD destabilizes plasmid segregation in a manner similar to that observed for CEN + HM or CEN +TEL antagonism . This instability is caused by the ability of TD to nucleate components of heterochromatin on the CEN plasmid, because CEN +TD antagonism is abrogated by sir2, sir3 and sir4 mutations and by TD mutations that prevent interaction with Sir4p . In strains that acquire resistance to CEN +TD antagonism, the CEN plasmid has either recombined with a 2 mu plasmid or sustained deletions in sequences required to bind GBD-TD . CEN +TD and CEN + HM antagonism is exacerbated by mutations in components of the Ku-mediated non-homologous end-joining pathway . These observations suggest that CEN antagonism is caused by DNA breaks that result from competition between CEN - and Sir-specific segregation pathways. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 210 - 7 Epub 2003 Mar 04. Cloning and characterisation of a glucoamylase gene (GlaM) from the dimorphic zygomycete Mucor circinelloides; Houghton-Larsen J et al.; This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides . The deduced 609-amino-acid enzyme (including signal peptide) is 63% identical to the Rhizopus oryzae raw starch-degrading glucoamylase and is the third glucoamylase reported to have the putative starch-binding domain placed N-terminally . The C-terminal catalytic domain is separated from the starch-binding domain by a serine/threonine-rich linker . An alignment of the cloned gene and cDNA sequences showed that the gene contains three introns . The transcriptional start site and the site of polyadenylation were defined by primer extension and 3'RACE, respectively . The atypical Kozak sequence is identical to the one used in R . oryzae in positions -1 to -4 . Northern slot blots revealed that glucoamylase transcription is induced during growth on starch and repressed by glucose . In silico analysis of the 1.9-kb promoter sequence cloned by inverse PCR revealed the presence of several putative regulatory elements, most notably a 19-bp sequence containing six overlapping copies of the Saccharomyces cerevisiae Nrg1p binding sequence. Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 202 - 9 Epub 2003 Mar 25. cDNA cloning and functional expression of alpha-glucosidase from Mortierella alliacea; Tanaka Y et al.; We recently purified an alpha-glucosidase comprising 61-kDa and 31-kDa subunits from the fungus Mortierella alliacea and characterized its soluble starch-hydrolyzing activity . Here, the cDNA coding for this enzyme was cloned, revealing that it encodes a single polypeptide of 1,053 amino acids, with a calculated molecular mass of 117 kDa . Comparison between the deduced amino acid sequence and the partial sequences of the purified enzyme suggested that an immature protein can be converted into the two subunits of mature enzyme by post-translational processing at least three cleavage sites . Heterologous expression of recombinant alpha-glucosidase in yeast gave rise to a significant increase in hydrolytic activity toward maltose and soluble starch, in both intracellular and extracellular fractions . Immunoblot analysis using antiserum against the alpha-glucosidase revealed that the active enzyme expressed in yeast is also composed of two subunits . The yeast expression system provides a model suitable for investigating the polypeptide-processing event and structure-function relationship of the alpha-glucosidase with unique substrate specificity. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 597 - 600 Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system; Ma XZ et al.; Many human genes determined by genomic sequencing have only few information about their functions . To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively . By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila . Fifty-four transgenic fly lines corresponding to 10 human genes have been established . When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes . This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila. Prog Nucleic Acid Res Mol Biol, 2003, 73, 221 - 50 The CCR4-NOT complex plays diverse roles in mRNA metabolism; Denis CL et al.; It is increasingly clear that the synthesis of eukaryotic mRNA involves an integrated series of events involving large multisubunit protein complexes . The evolutionarily conserved CCR4-NOT complex of proteins has been found to be involved in several aspects of mRNA formation, including repression and activation of mRNA initiation, control of mRNA elongation, and the deadenylation and subsequent degradation of mRNA . Its roles in such diverse processes make the CCR4-NOT complex central to the regulation of mRNA metabolism . In this review we describe the CCR4-NOT complex, its constituents, and its organization, discussing both the well characterized yeast proteins and their higher eukaryotic orthologs . The known biochemical roles of the individual components and of the complex are described with particular emphasis on the two known functions of the complex, repression of TFIID action and deadenylation of mRNA . Finally, the functional diversity of the CCR4-NOT complex is related to its mediating responses from a number of cellular signaling pathways. Aging Cell, 2002 Oct, 1(1), 47 - 56 Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster; Seroude L et al.; The relationship between gene expression and the regulation of longevity is poorly understood . Previous studies focusing on microarray or tissue-specific changes in gene expression as a function of age have provided evidence that gene expression is a dynamic process which is regulated, even late in an organism's lifespan . Using the enhancer-trap technique, a systematic analysis of the spatio-temporal regulation of gene expression in tissues of adult Drosophila is presented . As many as 80% of enhancer traps analysed displayed (some form of) transcriptional change with age . In some cases the rate of change in expression was found to correlate with changes in longevity under various conditions, suggesting that they may be indicators of 'physiological age' and therefore valuable markers for dissecting the aging process . Molecular analysis of enhancer traps that showed increased activity with age was performed to identify candidate genes that may be important in the regulation of longevity; we identified changes in reporters associated with immunity, microtubule organization and muscle function. Biotechnol Lett, 2003 Jan, 25(1), 17 - 21 Applying slow-release biocatalysis to the asymmetric reduction of ethyl 4-chloroacetoacetate; Houng JY et al.; Amberlite XAD 2 resin enhanced the asymmetric reduction of ethyl 4-chloroacetoacetate (ECA) to S-4-chloro-3-hydroxybutyric acid ethyl ester as catalyzed by Saccharomyces cerevisiae . The absorbed ECA was released slowly to the solution during the reaction so that the substrate inhibition and the spontaneous chemical hydrolysis of ECA were considerably lessened . With 75 g resin l(-1) and ECA at 74 mM, the reaction yield and the product's optical purity increased from 75% to 84% and from 88% to 93%, respectively. Plant Cell Physiol, 2003 Jul, 44(7), 726 - 34 Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice; Sonoda Y et al.; To study the regulation of ammonium uptake into rice roots, three ammonium transporter genes (OsAMT1;1, 1;2 and 1;3; Oryza sativa ammonium transporter) were isolated and examined . OsAMT1s belong to AMT1 family, containing 11 putative transmembrane-spanning domains . Southern blot analysis and screening of the rice genome database confirmed that with OsAMT1;1-1;3 the complete AMT1 family of rice had been isolated . Heterologous expression of OsAMT1s in the yeast Saccharomyces cerevisiae mutant 31019b showed that all three OsAMT1s exhibit ammonium transport activity . Northern blot analysis showed a distinct expression pattern for the three genes; more constitutive expression in shoots and roots for OsAMT1;1, root-specific and ammonium-inducible expression for OsAMT1;2, and root-specific and nitrogen-derepressible expression for OsAMT1;3 . In situ mRNA detection revealed that OsAMT1;2 is expressed in the central cylinder and cell surface of root tips . This gene expression analysis revealed a distinct nitrogen-dependent regulation for AMTs in rice, differing from that in tomato or ARABIDOPSIS: EMBO J, 2003 Aug 1, 22(15), 3792 - 802 Substrate recognition in ER-associated degradation mediated by Eps1, a member of the protein disulfide isomerase family; Wang Q et al.; Pma1-D378N is a misfolded plasma membrane protein in yeast that is prevented from delivery to the cell surface and targeted instead for ER-associated degradation (ERAD) . Degradation of Pma1-D378N is dependent on the ubiquitin ligase Doa10 and the ubiquitin chaperone Cdc48 . Recognition of Pma1-D378N by the ERAD pathway is dependent on Eps1, a transmembrane member of the protein disulfide isomerase (PDI) oxidoreductase family . Eps1 has two thioredoxin-like domains containing a CPHC and a CDKC active site . Although Eps1 interaction with wild-type Pma1 was not detected, Eps1 co-immunoprecipitates with Pma1-D378N . Eps1 interaction with Pma1-D378N requires the CPHC motif, although both thioredoxin-like domains appear to cooperate in substrate recognition . In the absence of the native transmembrane domain and cytoplasmic tail of Eps1, degradation of Pma1-D378N is slowed, suggesting that Eps1 facilitates presentation of substrate to membrane-bound components of the degradation machinery . Genetic interactions with other mutants of the ERAD machinery and induction of the unfolded protein response in eps1Delta cells support a general role for Eps1 as a recognition component of the ERAD pathway. EMBO J, 2003 Aug 1, 22(15), 3783 - 91 Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction; Friant S et al.; Serine palmitoyltransferase, the first enzyme in ceramide biosynthesis, is required for resistance to heat shock . We show that increased heat shock sensitivity in the absence of serine palmitoyltransferase activity correlates with a lack of induction of the major heat shock proteins (Hsps) at high temperature . Normal heat shock resistance can be restored, without restoration of ceramide synthesis or induction of Hsps, by overexpression of ubiquitin . This function of ubiquitin requires the proteasome . These data imply that the essential function of Hsp induction is the removal of misfolded or aggregated proteins, not their refolding . This suggests that cells stressed by heat shock do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins. Biochim Biophys Acta, 2003 Jul 23, 1622(2), 117 - 27 Cell growth selection system to detect extracellular and transmembrane protein interactions; Urech DM et al.; The interplay among extracellular and cell surface proteins, such as the interactions between ligands and receptors or between antigens and antibodies, is involved in a multitude of physiological and pathological phenomena . In the oxidizing milieu of the secretory pathway in eukaryotic cells, many extracellular proteins build disulfide bonds that significantly contribute to their correct folding and structural stability . Thus, conventional yeast two-hybrid interaction assays, which occur in the reducing intracellular environment, might not be adequate to detect extracellular protein-protein interactions . We have exploited the properties of yeast Ire1p, a type I endoplasmic reticulum (ER) membrane protein involved in the unfolded protein response (UPR) as a dimerization-activated receptor, to develop a novel system for the detection and study of interactions between extracellular and/or membrane proteins . In our system, named SCINEX-P (screening for interactions between extracellular proteins), proteins of interest were fused to truncated Ire1p so as to substitute its N-terminal lumenal domain (NLD) . Specific interaction between two partners caused dimerization of the Ire1p moiety, which, through the endogenous UPR signalling pathway, led to activation of transcription of genes that permit cell growth under selective conditions. J Nat Prod, 2003 Jul, 66(7), 1017 - 21 Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids; Cardoso CL et al.; Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine . The structural determination of 1 was based on 1D and 2D spectroscopic data . An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active. Lipids, 2003 May, 38(5), 531 - 7 Regulation by carbohydrate and clofibric acid of palmitoyl-CoA chain elongation in the liver of rats; Kudo N et al.; Regulation of palmitoyl-CoA chain elongation (PCE) and its contribution to oleic acid formation were investigated in rat liver in comparison with stearoyl-CoA desaturase (SCD) . Hepatic PCE activity was induced by the administration of 20% wt/vol glucose or fructose in the drinking water of normal rats . In streptozotocin-induced diabetic rats, the activities of both PCE and SCD were suppressed, and fructose, but not glucose, feeding caused an increase in the activities of both enzymes . Treatment of normal rats with clofibric acid in combination with carbohydrate further increased PCE, but not SCD, activity . FA analysis of hepatic lipids revealed that the proportion of oleic acid (18:1 n-9) increased upon administration of carbohydrate or clofibric acid . The treatment of rats with clofibric acid in combination with carbohydrate greatly increased the proportion of 18:1 n-9 . A significant correlation was observed between PCE activity and the hepatic proportion of 18:1 n-9 (r2 = 0.874, P < 0.01), whereas the relationship between SCD activity and the proportion of 18:1 n-9 was not significant (r2 = 0.552, P > 0.05) . Taken together, these results suggest that carbohydrate induces PCE as well as SCD activity to increase the hepatic 18:1 content in rat liver, and the increased PCE activity seems to be responsible for the further increase in 18:1 n-9 when carbohydrate is administered in combination with clofibric acid. Biochem Biophys Res Commun, 2003 Aug 8, 307(4), 900 - 6 Novel role of cytoplasmic dynein motor in maintenance of the nuclear number in conidia through organized conidiation in Aspergillus oryzae; Maruyama J et al.; Cytoplasmic dynein is a minus-end-directed, microtubule-dependent motor protein complex . DhcA, cytoplasmic dynein heavy chain in Aspergillus oryzae, contained four P-loops involved in ATP binding which were conserved as in cytoplasmic dynein heavy chains of other organisms . The amino acid sequence of A . oryzae DhcA was similar to cytoplasmic dynein heavy chains from other organisms except for the N-terminus of Saccharomyces cerevisiae Dyn1 . Disruption of dhcA gene in the region encoding four P-loop motifs resulted in a defective growth and perturbed distribution of nuclei and vacuoles . The dhcA disruptant exhibited an abnormal morphology of conidial heads and conidia with an increased nuclear number . The present study implicates a novel role of cytoplasmic dynein in maintenance of the nuclear number in conidia through an organized conidiation. J Biol Chem, 2003 Sep 12, 278(37), 34739 - 42 Epub 2003 Jul 21. The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p; Wood A et al.; Monoubiquitination of histone H2B, catalyzed by Rad6-Bre1, is required for methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively . The Paf1 protein complex, which associates with RNA polymerase II, is known to be required for these histone H3 methylation events . During the early elongation stage of transcription, the Paf1 complex is required for association of COMPASS with RNA polymerase II, but the role the Paf1 complex plays at the promoter has not been clear . We present evidence that the Paf1 complex is required for monoubiquitination of histone H2B at promoters . Strains deleted for several components of the Paf1 complex are defective in monoubiquitination of histone H2B, which results in the loss of methylation of lysines 4 and 79 of histone H3 . We also show that Paf1 complex is required for the interaction of Rad6 and COMPASS with RNA polymerase II . Finally, we show that the Paf1 complex is required for Rad6-Bre1 catalytic activity but not for the recruitment of Rad6-Bre1 to promoters . Thus, in addition to its role during the elongation phase of transcription, the Paf1 complex appears to activate the function but not the placement of the Rad6-Bre1 ubiquitin-protein ligase at the promoters of active genes. J Biol Chem, 2003 Sep 5, 278(36), 33625 - 8 Epub 2003 Jul 21. The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B; Ng HH et al.; In yeast cells, the Rtf1 and Paf1 components of the Paf1 transcriptional elongation complex are important for recruitment of Set1, the histone H3-lysine 4 (H3-Lys4) methylase, to a highly localized domain at the 5' portion of active mRNA coding regions . Here, we show that Rtf1 is essential for global methylation of H3-Lys4 and H3-Lys79, but not H3-Lys36 . This role of Rtf1 resembles that of Rad6, which mediates ubiquitination of histone H2B at lysine 123 . Indeed, Rtf1 is required for H2B ubiquitination, suggesting that its effects on H3-Lys4 and H3-Lys79 methylation are an indirect consequence of its effect on H2B ubiquitination . Rtf1 is important for telomeric silencing, with loss of H3-Lys4 and H3-Lys79 methylation synergistically reducing Sir2 association with telomeric DNA . Dot1, the H3-Lys79 methylase, associates with transcriptionally active genes, but unlike the association of Set1 and Set2 (the H3-Lys36 methylase), this association is largely independent of Rtf1 . We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor. J Cell Biol, 2003 Jul 21, 162(2), 211 - 22 Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel; Bayer MJ et al.; Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits . In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models . We tested the role of V0 sectors in vacuole fusion . V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies . Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient . Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse . The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release . Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity . The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release . These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing. J Cell Sci, 2003 Sep 1, 116(Pt 17), 3623 - 34 Epub 2003 Jul 22. Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome; Eckert JH et al.; The protein import machinery of the peroxisome consists of many proteins, collectively called the peroxins . By applying the split-ubiquitin technique we systematically tested the pair-wise interactions between the Nub- and Cub-labeled peroxins for the first time in the living cells of the yeast Saccharomyces cerevisiae . We found that Pex10p plays a central role in the protein interaction network by connecting the ubiquitin conjugation enzyme Pex4p to the other members of the protein import machinery . A yeast strain harboring a deletion of PEX3 enabled us to estimate the influence of the peroxisomal membrane on the formation of a subset of the investigated protein-protein interactions. Brain, 2003 Oct, 126(Pt 10), 2191 - 202 Epub 2003 Jul 22. Cannabinoids inhibit neurodegeneration in models of multiple sclerosis; Pryce G et al.; Multiple sclerosis is increasingly being recognized as a neurodegenerative disease that is triggered by inflammatory attack of the CNS . As yet there is no satisfactory treatment . Using experimental allergic encephalo myelitis (EAE), an animal model of multiple sclerosis, we demonstrate that the cannabinoid system is neuroprotective during EAE . Mice deficient in the cannabinoid receptor CB1 tolerate inflammatory and excitotoxic insults poorly and develop substantial neurodegeneration following immune attack in EAE . In addition, exogenous CB1 agonists can provide significant neuroprotection from the consequences of inflammatory CNS disease in an experimental allergic uveitis model . Therefore, in addition to symptom management, cannabis may also slow the neurodegenerative processes that ultimately lead to chronic disability in multiple sclerosis and probably other diseases. J Mol Biol, 2003 Aug 1, 331(1), 155 - 65 MDL1 is a high copy suppressor of ATM1: evidence for a role in resistance to oxidative stress; Chloupkova M et al.; The yeast ATM1 gene is essential for normal cellular iron homeostasis . Deletion of ATM1 results in mitochondrial iron accumulation and increased sensitivity to oxidative stress and transition metal toxicity . Atm1p is an ATP-binding cassette (ABC) transporter localized to the mitochondrial inner membrane . The specific function of Atm1p has not been determined, though roles in both mitochondrial iron export and cytosolic Fe-S cluster assembly have been proposed . We undertook a screen for yeast genes capable of suppressing the abnormalities of cellular iron metabolism demonstrated by Deltaatm1 cells . One of the genes we identified was MDL1, which like ATM1, encodes a mitochondrial inner membrane ABC transporter . Mdl1p has previously been shown to function in the export of peptides from the mitochondrial matrix . We demonstrate that over-expression of MDL1 in Deltaatm1 cells results in a reduction of mitochondrial iron content, and decreased sensitivity to H(2)O(2) and transition metal toxicity . Additionally, in studies of the effect of over-expression and deletion of MDL1, we have identified a novel role for Mdl1p in the regulation of cellular resistance to oxidative stress. J Mol Biol, 2003 Aug 1, 331(1), 123 - 38 Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair; Kijas AW et al.; In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches . The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins . Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR . Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR . Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding . Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding . The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain . DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1 . In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1 . These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components. Cancer Res, 2003 Jul 15, 63(14), 3909 - 12 Recapitulation of the cellular xeroderma pigmentosum-variant phenotypes using short interfering RNA for DNA polymerase H; Laposa RR et al.; The lesion-specific DNA polymerase POLH gene is mutated in xeroderma pigmentosum variant (XP-V) patients who exhibit an increased skin cancer incidence from UV exposure . Normal cells in which POLH expression was reduced using short interfering RNAs (siRNAs) were compared with the XP-V cellular phenotype that results from naturally occurring inactivating mutations . Stable clones expressing siRNA had partially reduced POLH protein levels, and intermediate levels of UV sensitivity and S phase checkpoint activation, but similar levels of Mre11 foci as in XP-V cells . Therefore, suppression of POLH expression levels by siRNA recapitulates most of the phenotypes seen in cells from XP-V patients with inactivating mutations in POLH. Neuron, 2003 Jul 17, 39(2), 255 - 67 Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction; Haghighi AP et al.; Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity . A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release . Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) . Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release . Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones . Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII . Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function . Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila. Mol Cells, 2003 Jun 30, 15(3), 301 - 6 Cloning and expression of the isoflavone synthase gene (IFS-Tp) from Trifolium pratense; Kim BG et al.; Isoflavones are secondary metabolites found mainly in leguminous plants . Their synthesis from flavanones is catalyzed by isoflavone synthase (IFS) . We have cloned a isoflavone synthase gene (IFS-Tp) from Trifolium pratense that encodes a predicted 525 amino acids protein, molecular weight 59 kDa, with strong homology to IFS's from other legumes . IFS-Tp was expressed in all the tissues examined, and addition of glutathione and UV irradiation enhanced its expression . Microsomes from yeast transformed with IFS-Tp were able to convert naringenin to genistein, indicating that IFS-Tp has isoflavone synthase activity. Nat Biotechnol, 2003 Aug, 21(8), 914 - 9 Epub 2003 Jul 20. Engineering tolerance and accumulation of lead and cadmium in transgenic plants; Song WY et al.; We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination . We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast . DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium . Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals . These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium. Nat Biotechnol, 2003 Aug, 21(8), 921 - 6 Epub 2003 Jul 20. A proteomics approach to understanding protein ubiquitination; Peng J et al.; There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale . We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate . Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination . We identified 1,075 proteins from the sample . In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates . Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo . The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination. Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9156 - 61 Epub 2003 Jul 18. Pheromone gland-specific fatty-acyl reductase of the silkmoth, Bombyx mori; Moto K et al.; The C10-C18 unsaturated, acyclic, aliphatic compounds that contain an oxygenated functional group (alcohol, aldehyde, or acetate ester) are a major class of sex pheromones produced by female moths . In the biosynthesis of these pheromone components, the key enzyme required to produce the oxygenated functional groups is fatty-acyl reductase (FAR) . This enzyme converts fatty-acyl pheromone precursors to their corresponding alcohols, which, depending on the moth species, can then be acetylated or oxidized to the corresponding aldehydes . Despite the significant role this enzyme has in generating the species-specific oxygenated constituents of lepidopteran sex pheromones, the enzyme has yet to be fully characterized and identified . In experiments designed to characterize a pheromone-gland-specific FAR in the silkmoth, Bombyx mori, we have isolated a cDNA clone encoding a protein homologous to a FAR from the desert shrub, Simmondsia chinensis, commonly known as jojoba . The deduced amino acid sequence of this clone predicts a 460-aa protein with a consensus NAD(P)H binding motif within the amino terminus . Northern blot analysis indicated that 2-kb transcripts of this gene were specifically expressed in the pheromone gland at 1 day before adult eclosion . Functional expression of this gene in the yeast Saccharomyces cerevisiae not only confirmed the long-chain FAR activity, but also indicated a distinct substrate specificity . Finally, the transformed yeast cells evoked typical mating behavior in male moths when cultured with the pheromone precursor fatty acid, (E,Z)-10,12-hexadecadienoic acid. J Biol Chem, 2003 Oct 3, 278(40), 39051 - 8 Epub 2003 Jul 18. Coordinate expression of NADPH-dependent flavin reductase, Fre-1, and Hint-related 7meGMP-directed hydrolase, DCS-1; Kwasnicka DA et al.; A novel human cytosolic flavin reductase, Nr1, was recently described that contains FMN, FAD, and NADPH cofactors . Though the targets of the related NADPH-dependent flavoprotein reductases, cytochrome P450 reductase, methionine synthase reductase, and nitric oxide synthase, are known, the cellular function of Nr1 is not clear . To explore expression and regulation of Nr1, we cloned fre-1, the Caenorhabditis elegans ortholog of Nr1, and discovered that it is transcribed as a bicistronic pre-mRNA together with dcs-1, the ortholog of the recently described scavenger mRNA decapping enzyme . We used the novel substrate, 7meGpppBODIPY, to demonstrate that DCS-1 has low micromolar specificity for guanine ribonucleotides with the 7me modification, whereas trimethylated G substrates are poor competitors . Contrary to earlier classification, DCS-1 is not a pyrophosphatase but a distant member of the Hint branch of the histidine triad superfamily of nucleotide hydrolases and transferases . These observations are consistent with the hypothesis that DCS-1 homologs may function in the metabolism of capped oligonucleotides generated following exosome-dependent degradation of short-lived mRNA transcripts . We find that fre-1 and dcs-1 are coordinately expressed through worm development, are induced by heat shock, and have a nearly identical expression profile in human tissues . Furthermore, immunocytochemical analysis of the endogenous proteins in COS cells indicates that both are present in the nucleus and concentrated in a distinct perinuclear structure . Though no connection between these enzymes had been anticipated, our data and data from global expression and protein association studies suggest that the two enzymes jointly participate in responses to DNA damage, heat shock, and other stresses. J Biol Chem, 2003 Oct 3, 278(40), 39037 - 43 Epub 2003 Jul 18. Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation; Rondon AG et al.; THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1 . Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects . To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes . With these assays, we demonstrate that hpr1delta, tho2delta, and mft1delta mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation . The mRNA expression defect of hpr1delta mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease . This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export. Immunity, 2003 Jul, 19(1), 145 - 53 Regulation of the Th2 cytokine locus by a locus control region; Lee GR et al.; The Th2 cytokine genes IL4, IL5, and IL13 are clustered and expressed in a cell lineage-specific manner . We investigated the global locus-specific regulation of these genes using BAC transgenic mice containing the murine Th2 cytokine cluster carrying an IL4 promoter-luciferase reporter . IL4 promoter activity in effector CD4 T cells from these transgenic mice was strong, Th2 specific, and copy number dependent, suggesting the presence of an LCR in the locus . The production of IL4 and IL13, but not IL5, by these cells was also copy number dependent . Deletion analysis defined a 25 kb fragment in the RAD50 gene as the region containing the LCR activity . Expression of the IL4 promoter-luciferase reporter was transactivated by GATA-3 irrespective of position in the locus, suggesting the global nature of this regulation . The LCR itself, however, does not respond directly to GATA-3. Mycoses, 2003 Apr, 46(3-4), 104 - 13 Glycosylphosphatidylinositols synthesized by Trichophyton rubrum in a cell-free system; Pusch U et al.; The opportunistic fungi Trichophyton rubrum and T . mentagrophytes, are responsible for relatively non-inflammatory chronic dermatophytes infections in immunocompromised patients but also in healthy individuals . This chronic infection is associated with immunosuppressive effects of the cell wall components particularly the polysaccharides secreted by these organisms . We have studied glycosylphosphatidylinositol (GPI) anchor biosynthesis in the pathogenic fungus T . rubrum and could demonstrate that T . rubrum is able to synthesize GPI structures . Glycolipids synthesized in a cell-free system prepared from the dermatophyte T . rubrum and labeled with {3H}mannose, and {3H}galactose using GDP-{3H}mannose and UDP-{3H}galactose, respectively, were identified and structurally characterized as GPIs . The evolutionary conserved backbone of T . rubrum GPIs incorporates galactose . Further, all glycolipids lack the acyl group on the inositol which was shown for Saccharomyces cerevisiae and mammalian GPIs . Our data suggest significant differences in the GPI biosynthetic pathway between mammalian and T . rubrum cells that could perhaps be exploited for the development of an antimycotic for Trichophyton infection. Drug Metab Dispos, 2003 Aug, 31(8), 993 - 8 Glucuronidation and excretion of nonylphenol in perfused rat liver; Daidoji T et al.; Nonylphenol, an environmental estrogenic chemical, is reported to have adverse effects on the reproductive organs of animals . In this study, the metabolism of nonylphenol and that of other alkylphenols in the rat liver was investigated using liver perfusion . Alkylphenols (nonylphenol, hexylphenol, butylphenol, and ethylphenol) were glucuronidated by rat liver microsomes . Nonylphenol was found to be conjugated with glucuronic acid by an isoform of UDP-glucuronosyltransferase, UGT2B1, expressed in yeast AH22 cells . However, when nonylphenol was perfused into rat liver in situ, it was difficult for free nonylphenol and conjugated metabolite to be excreted into the bile or vein, and most of the perfused nonylphenol remained free and as a glucuronide conjugate in the liver tissue, even after 1 h of perfusion . After 1 h of perfusion of the other alkylphenols, most of them were excreted into the bile as glucuronides . Ethylphenol, which has the shortest alkyl chain, was excreted rapidly into both the bile and vein; however, the excretion rates of alkylphenols having longer alkyl chains tended to be slow . MRP-2-deficient Eisai hyperbilirubinemic rats could not secrete alkylphenol-glucuronides into the bile, indicating that alkylphenol-glucuronides are transported by MRP-2 to the bile in normal Sprague-Dawley rats . The results indicate that the kinetics of excretion of alkylphenol-glucuronides into the bile or vein depends on the length of alkyl chain and suggest that nonylphenol-glucuronide formed in the liver cannot be transported by MRP-2. Curr Biol, 2003 Jul 15, 13(14), 1227 - 33 Rsp5p is required for ER bound Mga2p120 polyubiquitination and release of the processed/tethered transactivator Mga2p90; Shcherbik N et al.; A number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes . Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid . Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides . In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor . It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins . Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5 Delta cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants . We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER . In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination . Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo . In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor. J Exp Clin Cancer Res, 2003 Jun, 22(2), 201 - 12 Induction of apoptosis in mouse and human carcinoma cell lines by Emblica officinalis polyphenols and its effect on chemical carcinogenesis; Rajeshkumar NV et al.; Plant-derived phenolic compounds manifest many beneficial effects and can potentially inhibit several stages of carcinogenesis . In the present study, we investigated the efficacy of Emblica officinalis (E . officinalis) polyphenol fraction (EOP) on the induction of apoptosis in mouse and human carcinoma cell lineses and its modulatory effect on N- nitrosodiethylamine (NDEA) induced liver tumors in rats . The results indicate that EOP treatment could induce apoptosis in Dalton's Lymphoma Ascites (DLA) and CeHa cell lines At 200 microg/ml dose EOP induced membrane blebbing, chromatin condensation and intenucleosomal breaks as evident from the morphology and DNA ladder pattern obtained in gel electrophoresis . The results also suggested that EOP treatment could decrease the liver tumour development induced by NDEA . Animals administered (oral) with NDEA (0.02%, 2.5 ml/rat, 5 days a week, 20 weeks) developed visible liver tumours by the end of the 20th week and the liver weight raised to 5.2 +/- 1.1 g/ 100 g body weight . Only 11% of the animals treated with EOP (60 mg/kg, oral, 5 days a week for 20 weeks) developed visible liver tumours by this period and the liver weights were reduced to 3.2 +/- 0.7 g/ 100 g body weight . gamma-glutamyl transpeptidase activity was raised to 88.4 +/- 16.2 U/l in serum of NDEA treated group was reduced to 48.4 +/- 14.8 U/l by EOP treatment . Elevated levels of serum alkaline phosphatase (ALP), glutamate pyruvate transaminase (GPT), bilirubin, liver glutathione S-transferase (GST) and glutathione (GSH) in the NDEA administered group were significantly reduced by EOP treatment . The EOP was found to scavenge superoxide and hydroxyl radicals and inhibit lipid peroxidation in vitro . EOP also inhibited DNA topoisomerase I in Saccharomyces cervisiae mutant cell cultures and the activity of cdc25 tyrosine phosphatase. Planta Med, 2003 Jun, 69(6), 547 - 51 Two new antifungal saponins from the Tibetan herbal medicine Clematis tangutica; Du Z et al.; Bioassay-guided fractionation of the ethanol extract of the aerial parts of Clematis tangutica led to the isolation of two new antifungal triterpene saponins . Their structures were determined to be 3- O-alpha- L-arabinopyranosyl hederagenin 28- O-alpha- L-rhamnopyranosyl ester ( 1) and 3- O-beta- D-glucopyranosyl-(1--> 4)-alpha- L-arabinopyranosyl hederagenin 28- O-alpha- L-rhamnopyranosyl ester ( 2) on the basis of spectral data and chemical evidence . Inhibitory activities of the two saponins against seven fungal strains were evaluated . Compounds 1 and 2 showed evident antifungal activity (MIA approximately 2.5 micrograms/disc) against Saccharomyces cerevisiae, similar to the positive control amphotericin B and ordinary activities (MIA approximately 10 micrograms/disc) against Penicillium avellaneum UC-4376, Candida glabrata, Trichosporon beigelii and Pyricularia oryzae . Compound 2 is a better antifungal agent than compound 1 against most of the fungal strains that were tested. Mol Med, 2003 Mar-Apr, 9(3-4), 65 - 76 The molecular mechanism of autophagy; Wang CW et al.; Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions . During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to a degradative organelle, the vacuole in yeast and the lysosome in mammalian cells, for breakdown and recycling . Autophagy is induced under starvation conditions and in mammalian cells is also invoked in response to specific hormones . In yeast, under nutrient-rich conditions, a constitutive biosynthetic pathway, termed the cytoplasm to vacuole targeting (Cvt) pathway, utilizes most of the same molecular machinery and topologically similar vesicles for the delivery of the resident hydrolase aminopeptidase I to the vacuole . Both autophagy and the Cvt pathway have been extensively studied and comprehensively reviewed in the past few years . In this review, we focus on the yeast system, which has provided most of the insight into the molecular mechanism of autophagy and the Cvt pathway, and highlight the most recent additions to our current knowledge of both pathways. Genes Dev, 2003 Jul 15, 17(14), 1789 - 802 Recruitment of Thr 319-phosphorylated Ndd1p to the FHA domain of Fkh2p requires Clb kinase activity: a mechanism for CLB cluster gene activation; Reynolds D et al.; Activation of the CLB gene cluster through the assembly of Mcm1p-Fkh2p complexes at target promoters is essential for mitotic entry and transition through M phase . We show that the activation of this mitotic transcriptional program is dependent on the recruitment of Ndd1p, a coactivator that performs its essential function by acting through Fkh2p . Although an essential gene, NDD1 is dispensable in cells expressing a truncated form of Fkh2p lacking its C terminus . When phosphorylated on T319, Ndd1p is recruited to CLB cluster promoters by association with the forkhead-associated (FHA) domain of Fkh2p . Substitution of T319 for alanine significantly reduces recruitment of Ndd1p, resulting in loss of normal transcriptional regulation, severe impairment of cell growth, and a budding defect reminiscent of cells with a Cdk-Clb kinase deficiency . Finally, we show that phosphorylation of T319 and recruitment of Ndd1p to CLB2 and SWI5 promoters is dependent on Cdc28-Clb kinase activity . These data provide a model describing the activation of G2/M transcription through the phosphorylation of Ndd1p by Cdc28-Clb kinase activity. Genes Dev, 2003 Jul 15, 17(14), 1755 - 67 Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53; Osborn AJ et al.; When DNA replication is stalled, a signal transduction pathway is activated that promotes the stability of stalled forks and resumption of DNA synthesis . In budding yeast, this pathway includes the kinases Mec1 and Rad53 . Here we report that the Mediator protein Mrc1, which is required for normal DNA replication and for activation of Rad53, is present at replication forks . Mrc1 initially binds early-replicating sequences and moves along chromatin with the replication fork . Blocking initiation of DNA replication blocks Mrc1 loading onto origins, providing an explanation for why so many mutants in DNA replication show checkpoint defects . In the presence of replication blocks, we find that Mec1 is recruited to regions of stalled replication, where it encounters and presumably phosphorylates Mrc1 . Mutation of the canonical Mec1 phosphorylation sites on Mrc1 prevents Mrc1 phosphorylation and blocks Rad53 activation, but does not alter Mrc1's role in DNA replication . Our results suggest a model whereby in response to DNA replication interference, the Mec1 kinase is recruited to sites of replication blocks and phosphorylates a component of the DNA replication complex, Mrc1, thereby setting up a solid-state Rad53 activation platform to initiate the checkpoint response. Mol Cell Biol, 2003 Aug, 23(15), 5409 - 20 The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1; Yoon Y et al.; The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p . In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined . In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells . Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission . Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria . Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology . Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1 . These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol . We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency. Mol Cell Biol, 2003 Aug, 23(15), 5388 - 400 Cdc34 self-association is facilitated by ubiquitin thiolester formation and is required for its catalytic activity; Varelas X et al.; Using a coimmunoprecipitation strategy, we showed that the Cdc34 ubiquitin (Ub)-conjugating enzyme from Saccharomyces cerevisiae self-associates in cell lysates, thereby indicating an in vivo interaction . The ability of Cdc34 to interact with itself is not dependent on its association with the ubiquitin ligase Skp1-Cdc53/Cul1-Hrt1-F-box complex . Rather, this interaction depends upon the integrity of the Cdc34-Ub thiolester . Furthermore, several principal determinants within the Cdc34 catalytic domain, including the active-site cysteine, amino acid residues S73 and S97, and its catalytic domain insertion, also play a role in self-association . Mutational studies have shown that these determinants are functionally important in vivo and operate at the levels of both Cdc34-Ub thiolester formation and Cdc34-mediated multi-Ub chain assembly . These determinants are spatially situated in a region that is close to the active site, corresponding closely to the previously identified E2-Ub interface . These observations indicate that the formation of the Cdc34-Ub thiolester is important for Cdc34 self-association and that the interaction of Cdc34-Ub thiolesters is in turn a prerequisite for both multi-Ub chain assembly and Cdc34's essential function(s) . A conclusion from these findings is that the placement of ubiquitin on the Cdc34 surface is a structurally important feature of Cdc34's function. Mol Cell Biol, 2003 Aug, 23(15), 5269 - 81 Human T-lymphotropic virus type 1 oncoprotein tax promotes unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1 and causes chromosomal instability; Liu B et al.; Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia . The HTLV-1 transactivator, Tax, is implicated as the viral oncoprotein . Naive cells expressing Tax for the first time develop severe cell cycle abnormalities that include increased DNA synthesis, mitotic arrest, appearance of convoluted nuclei with decondensed DNA, and formation of multinucleated cells . Here we report that Tax causes a drastic reduction in Pds1p/securin and Clb2p/cyclin B levels in yeast, rodent, and human cells and a loss of cell viability . With a temperature-sensitive mutant of the CDC23 subunit of the anaphase-promoting complex (APC), cdc23(ts); a temperature-sensitive mutant of cdc20; and a cdh1-null mutant, we show that the diminution of Pds1p and Clb2p brought on by Tax is mediated via the Cdc20p-associated anaphase-promoting complex, APC(Cdc20p) . This loss of Pds1p/securin and Clb2p/cyclin B1 occurred before cellular entry into mitosis, caused a G(2)/M cell cycle block, and was accompanied by severe chromosome aneuploidy in both Saccharomyces cerevisiae cells and human diploid fibroblasts . Our results support the notion that Tax aberrantly targets and activates APC(Cdc20p), leading to unscheduled degradation of Pds1p/securin and Clb2p/cyclin B1, a delay or failure in mitotic entry and progression, and faulty chromosome transmission . The chromosomal instability resulting from a Tax-induced deficiency in securin and cyclin B1 provides an explanation for the highly aneuploid nature of adult T-cell leukemia cells. J Biol Chem, 2003 Sep 26, 278(39), 37998 - 8003 Epub 2003 Jul 14. Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein; Scheper W et al.; Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A . E., and van Waes, M . A . (1999) Annu . Rev . Cell Dev . Biol . 15, 799-842) . Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M . O., Deshaies, R . J., Schekman, R., and Kepes, F . (1993) EMBO J . 12, 4083-4093) . Sec61p is a polytopic membrane protein that lines the protein translocation channel . The role of Sss1p is unknown . During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed . In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T . A . (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B . (1999) FEBS Lett . 457, 316-322) . Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation . We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites . Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins. Pharmacol Res, 2003 Sep, 48(3), 237 - 40 Possible involvement of opioid receptors in moclobemide-induced hypothermia in mice; Ginawi OT; Effect of moclobemide, a selective monoamine oxidase-type A enzyme inhibitor, was investigated on the body temperature of male mice . Moclobemide (15-30 mg kg(-1), i.p.) produced significant reductions of body temperature in both normal and yeast-induced hyperthermic male mice . The hypothermic effect of moclobemide was moderate and short-lasting . Moclobemide-induced hypothermia was not antagonized by previous administration of prazosin (10 and 20 mg kg(-1), s.c.), propranolol (5, 10, and 20 mg kg(-1), s.c.), haloperidol (2 and 10 mg kg(-1), s.c.), atropine (10 and 20 mg kg(-1), s.c.), mepyramine (25 and 50 mg kg(-1), s.c.), or methysergide (0.5, 1, and 2 mg kg(-1), s.c.) . Pretreatment with the opioid antagonist naloxone (10 mg kg(-1), s.c.), however, was able to reverse the hypothermic effect of moclobemide (30 mg kg(-1), i.p.) in both normal and yeast-induced hyperthermic mice . The present results indicate a possible role for central opioid receptors in the hypothermic effect of moclobemide . Also, a peripheral component for this effect of moclobemide at the mitochondria of peripheral tissues is suspected . The peripheral tissue mitochondria could be considered a common target for moclobemide and opioids actions on body temperature. FEBS Lett, 2003 Jul 17, 547(1-3), 157 - 61 Characterization of Pellino2, a substrate of IRAK1 and IRAK4; Strelow A et al.; Interleukin-1 (IL-1) receptor-associated kinases (IRAKs) are central components of Toll/IL-1 receptor (TIR) signaling pathways . In an attempt to discover novel signal transducers in TIR signaling, we identified human Pellino2 as an interaction partner of IRAK4 . Pellino2 interacts with kinase-active as well as kinase-inactive IRAK1 and IRAK4 . Furthermore, Pellino2 is one of the first substrates identified for IRAK1 and IRAK4 . Functional studies using overexpression or RNAi knock-down of Pellino2 suggest a role of Pellino2 as a scaffolding protein similar to Pellino1 . However, unlike Pellino1, Pellino2 does not seem to activate a specific transcription factor, but links TIR signaling to basic cellular processes. J Mol Biol, 2003 Jul 25, 330(5), 1165 - 75 Cross-beta order and diversity in nanocrystals of an amyloid-forming peptide; Diaz-Avalos R et al.; The seven-residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms amyloid fibers, colloidal aggregates and highly ordered nanocrystals, provides a model system for characterizing the elusively protean cross-beta conformation . Depending on preparative conditions, orthorhombic and monoclinic crystals with similar lath-shaped morphology have been obtained . Ultra high-resolution (<0.5A spacing) electron diffraction patterns from single nanocrystals show that the peptide chains pack in parallel cross-beta columns with approximately 4.86A axial spacing . Mosaic striations 20-50 nm wide observed by electron microscopy indicate lateral size-limiting crystal growth related to amyloid fiber formation . Frequently obtained orthorhombic forms, with apparent space group symmetry P2(1)2(1)2(1), have cell dimensions ranging from /a/=22.7-21.2A, /b/=39.9-39.3A, /c/=4.89-4.86A for wet to dried states . Electron diffraction data from single nanocrystals, recorded in tilt series of still frames, have been mapped in reciprocal space . However, reliable integrated intensities cannot be obtained from these series, and dynamical electron diffraction effects present problems in data analysis . The diversity of ordered structures formed under similar conditions has made it difficult to obtain reproducible X-ray diffraction data from powder specimens; and overlapping Bragg reflections in the powder patterns preclude separated structure factor measurements for these data . Model protofilaments, consisting of tightly paired, half-staggered beta strands related by a screw axis, can be fit in the crystal lattices, but model refinement will require accurate structure factor measurements . Nearly anhydrous packing of this hydrophilic peptide can account for the insolubility of the crystals, since the activation energy for rehydration may be extremely high . Water-excluding packing of paired cross-beta peptide segments in thin protofilaments may be characteristic of the wide variety of anomalously stable amyloid aggregates. Biochemistry, 2003 Jul 22, 42(28), 8579 - 86 NMR analysis of methyl groups at 100-500 kDa: model systems and Arp2/3 complex; Kreishman-Deitrick M et al.; Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses . Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range . Biochemical methods also must be developed in concert to produce such systems in selectively labeled form . Here, we present (1)H/(13)C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to approximately 560 kDa . Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to approximately 330 ns . We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained (1)H/(13)C-HSQC spectra of this assembly . Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 microM . Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies. Nat Struct Biol, 2003 Aug, 10(8), 614 - 21 The RNA-binding SAM domain of Smaug defines a new family of post-transcriptional regulators; Aviv T et al.; Anteroposterior patterning in Drosophila melanogaster is dependent on the sequence-specific RNA-binding protein Smaug, which binds to and regulates the translation of nanos (nos) mRNA . Here we demonstrate that the sterile-alpha motif (SAM) domain of Smaug functions as an RNA-recognition domain . This represents a new function for the SAM domain family, which is well characterized for mediating protein-protein interactions . Using homology modeling and site-directed mutagenesis, we have localized the RNA-binding surface of the Smaug SAM domain and have elaborated the RNA consensus sequence required for binding . Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain-containing proteins, suggesting that the function of the domain is conserved from yeast to humans . We show here that the SAM domain of Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and that Vts1 induces transcript degradation through a mechanism involving the cytoplasmic deadenylase CCR4 . Together, these results suggest that Smaug and Vts1 define a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism. J Virol, 2003 Aug, 77(15), 8345 - 53 A heterologous coiled coil can substitute for helix I of the Sindbis virus capsid protein; Perera R et al.; Alphavirus core assembly proceeds along an assembly pathway involving a dimeric assembly intermediate . Several regions of the alphavirus capsid protein have been implicated in promoting and stabilizing this dimerization, including a putative heptad repeat sequence named helix I . This sequence, which spans residues 38 to 55 of the Sindbis virus capsid protein, was implicated in stabilizing dimeric contacts initiated through the C-terminal two-thirds of the capsid protein and nucleic acid . The studies presented here demonstrate that helix I can be functionally replaced by the corresponding sequence of a related alphavirus, western equine encephalitis virus, and also by an unrelated sequence from the yeast transcription activator, GCN4, that was previously shown to form a dimeric coiled coil . Replacing helix I with the entire leucine zipper domain of GCN4 (residues 250 to 281) produced a virus with the wild-type phenotype as determined by plaque assay and one-step growth analysis . However, replacement of helix I with a GCN4 sequence that favored trimer formation produced a virus that exhibited approximately 40-fold reduction in virus replication compared to the wild-type Sindbis virus . Changing residues within the Sindbis virus helix I sequence to favor trimer formation also produced a virus with reduced replication . Peptides corresponding to helix I inhibited core-like particle assembly in vitro . On the basis of these studies, it is proposed that helix I favors capsid protein-capsid protein interactions through the formation of dimeric coiled-coil interactions and may stabilize assembly intermediates in the alphavirus nucleocapsid core assembly pathway. Mol Biol Cell, 2003 Jul, 14(7), 3055 - 63 Epub 2003 Apr 17. Oligomerization of a cargo receptor directs protein sorting into COPII-coated transport vesicles; Sato K et al.; Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII) . The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors . Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER . Exit of Emp46p from the ER was saturable and dependent on the expression level of Emp47p . Emp46p binding to Emp47p occurs in the ER through the coiled-coil region in the luminal domains of both Emp47p and Emp46p, and dissociation occurs in the Golgi . Further, this coiled-coil region is also required for Emp47p to form an oligomeric complex of itself in the ER, which is essential for exit of Emp47p from the ER . Our results suggest that Emp47p is a receptor protein for Emp46p that allows for the selective transport of this protein, and this event involves receptor oligomerization. Mol Biol Cell, 2003 Jul, 14(7), 3013 - 26 Epub 2003 Apr 04. Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p; Yuzyuk T et al.; Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway . A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells . Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle . We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity . Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p . spa2delta cells, like ssk2delta cells, were defective for actin recovery from osmotic stress . These spa2delta defects could be suppressed by overexpression of catalytically active Ssk2p . Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells . The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2delta and spa2delta yeast cells . We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p-actin complex and the polarized localization of Ssk2p . Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly. Mol Biol Cell, 2003 Jul, 14(7), 2756 - 67 Epub 2003 Mar 20. Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways; Spear ED et al.; The accumulation of aberrantly folded proteins can lead to cell dysfunction and death . Currently, the mechanisms of toxicity and cellular defenses against their effects remain incompletely understood . In the endoplasmic reticulum (ER), stress caused by misfolded proteins activates the unfolded protein response (UPR) . The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes to maintain cellular homeostasis . We studied the effects of ER stress in budding yeast through expression of the well-characterized misfolded protein, CPY* . By challenging cells within their physiological limits to resist stress, we show that the UPR is required to maintain essential functions including protein translocation, glycosylation, degradation, and transport . Under stress, the ER-associated degradation (ERAD) pathway for misfolded proteins is saturable . To maintain homeostasis, an "overflow" pathway dependent on the UPR transports excess substrate to the vacuole for turnover . The importance of this pathway was revealed through mutant strains compromised in the vesicular trafficking of excess CPY* . Expression of CPY* at levels tolerated by wild-type cells was toxic to these strains despite retaining the ability to activate the UPR. Mol Biol Cell, 2003 Jul, 14(7), 2744 - 55 Rpb4p, a subunit of RNA polymerase II, mediates mRNA export during stress; Farago M et al.; Changes in gene expression represent a major mechanism by which cells respond to stress . We and other investigators have previously shown that the yeast RNA polymerase II subunit Rpb4p is required for transcription under various stress conditions, but not under optimal growth conditions . Here we show that, in addition to its role in transcription, Rpb4p is also required for mRNA export, but only when cells are exposed to stress conditions . The roles of Rpb4p in transcription and in mRNA export can be uncoupled genetically by specific mutations in Rpb4p . Both functions of Rpb4p are required to maintain cell viability during stress . We propose that Rpb4p participates in the cellular responses to stress at the interface of the transcription and the export machineries. Plant Physiol, 2003 Jul, 132(3), 1642 - 51 beta-alanine N-methyltransferase of Limonium latifolium . cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectant beta-alanine betaine; Raman SB et al.; Beta-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae . In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of beta-Ala by the action of a trifunctional S-adenosyl L-methionine (Ado-Met): beta-Ala N-methyltransferase (NMTase) . Peptide sequences from purified beta-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated . The 5' end of the cDNA was cloned using a 5'-rapid amplification of cDNA ends protocol . A 500-bp cDNA was used as a probe to screen a lambda-gt10 L . latifolium leaf cDNA library . Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3'-untranslated regions . The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5'-untranslated region . The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met . The NMTase mRNA was expressed in L . latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for beta-Ala betaine . NMTase mRNA expression was high in young and mature leaves and was enhanced by light . NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter . Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala as methyl acceptors . NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control . The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes . Phylogenetic analyses suggested that beta-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases. Plant Physiol, 2003 Jul, 132(3), 1560 - 76 Abscisic acid and gibberellin differentially regulate expression of genes of the SNF1-related kinase complex in tomato seeds; Bradford KJ et al.; The SNF1/AMP-activated protein kinase subfamily plays central roles in metabolic and transcriptional responses to nutritional or environmental stresses . In yeast (Saccharomyces cerevisiae) and mammals, activating and anchoring subunits associate with and regulate the activity, substrate specificity, and cellular localization of the kinase subunit in response to changing nutrient sources or energy demands, and homologous SNF1-related kinase (SnRK1) proteins are present in plants . We isolated cDNAs corresponding to the kinase (LeSNF1), regulatory (LeSNF4), and localization (LeSIP1 and LeGAL83) subunits of the SnRK1 complex from tomato (Lycopersicon esculentum Mill.) . LeSNF1 and LeSNF4 complemented yeast snf1 and snf4 mutants and physically interacted with each other and with LeSIP1 in a glucose-dependent manner in yeast two-hybrid assays . LeSNF4 mRNA became abundant at maximum dry weight accumulation during seed development and remained high when radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red light, or dormancy, but was low or undetected in seeds that had completed germination or in gibberellin (GA)-deficient seeds stimulated to germinate by GA . In leaves, LeSNF4 was induced in response to ABA or dehydration . In contrast, LeSNF1 and LeGAL83 genes were essentially constitutively expressed in both seeds and leaves regardless of the developmental, hormonal, or environmental conditions . Regulation of LeSNF4 expression by ABA and GA provides a potential link between hormonal and sugar-sensing pathways controlling seed development, dormancy, and germination. Plant Physiol, 2003 Jul, 132(3), 1391 - 404 Interacting transcription factors from the three-amino acid loop extension superclass regulate tuber formation; Chen H et al.; Using the yeast (Saccharomyces cerevisiae) two-hybrid system and a potato (Solanum tuberosum) KNOX protein, designated POTH1, as bait, we have identified seven distinct interacting proteins from a stolon library of potato . All seven cDNAs are members of the BEL1-like family of transcription factors . Among these proteins, there are at least four regions of high sequence conservation including the homeodomain, the proline-tyrosine-proline three-amino acid loop extension, the SKY box, and a 120-amino acid region upstream from the homeodomain . Through deletion analysis, we identified a protein-binding domain present in the carboxy end of the KNOX domain of POTH1 . The protein-binding domain in the BEL1 protein is located in the amino-terminal one-half of the 120-residue conserved region of the BELs . RNA-blot analysis showed differential patterns of RNA accumulation for the BELs in various potato organs . The level of StBEL5 mRNA increased in response to a short-day photoperiod in both leaves and stolons . Similar to sense mutants of POTH1, transgenic lines that overexpressed StBEL5 exhibited enhanced tuber formation even under noninductive conditions . Unlike POTH1 sense lines, however, these BEL lines did not exhibit the extreme leaf and stem morphology characteristic of KNOX overexpressers and displayed a more rapid rate of growth than control plants . Both StBEL5 and POTH1 sense lines exhibited an increase in cytokinin levels in shoot tips . StBEL5 lines also exhibited a decrease in the levels of GA 20-oxidase1 mRNA in stolon tips from long-day plants . Our results demonstrate an interaction between KNOX and BEL1-like transcription factors of potato that may potentially regulate processes of development. Plant Physiol, 2003 Jul, 132(3), 1315 - 21 Arabidopsis D-type cyclin CYCD4;1 is a novel cyclin partner of B2-type cyclin-dependent kinase; Kono A et al.; B-type cyclin-dependent kinases (CDKs) are unique to plants and are assumed to be involved in the control of the G2-to-M phase progression and mitotic events . However, little is known about their cyclin partners . In Arabidopsis, we isolated cDNA encoding the D-type cyclin CYCD4;1 by a yeast (Saccharomyces cerevisiae) two-hybrid screening using CDKB2;1 as bait . In vitro pull-down assay showed that CYCD4;1 bound to CDKB2;1 and CDKA;1 . Protein complexes of CYCD4;1-CDKA;1 and CYCD4;1-CDKB2;1 in insect cells exhibited histone H1-kinase activity . Promoter analysis using the luciferase reporter gene showed that CDKB2;1 was expressed from early G2 to M phase, whereas CYCD4;1 was expressed throughout the cell cycle . In situ hybridization of plant tissues revealed that both CDKB2;1 and CYCD4;1 transcripts accumulated in the shoot apical meristem, leaf primordia, vasculature of leaves, and tapetal cells in anthers . Our results suggest that CDKB2;1 and CYCD4;1 may form an active kinase complex during G2/M phase and control the development of particular tissues. Plant Mol Biol, 2003 May, 52(2), 331 - 41 Molecular characterization of His-Asp phosphorelay signaling factors in maize leaves: implications of the signal divergence by cytokinin-inducible response regulators in the cytosol and the nuclei; Asakura Y et al.; Genes for histidyl-aspartyl (His-Asp) phosphorelay components (His-containing phosphotransfer proteins, HP, and response regulators, RR) were isolated from Zea mays L . to characterize their function in cytokinin signaling . Six type-A RRs (ZmRR1, ZmRR2, ZmRR4-ZmRR7), 3 type-B RRs (ZmRR8-ZmRR10), and 3 HPs (ZmHP1-ZmHP3) were found in leaves . All type-A RR genes expressed in leaves were up-regulated by exogenous cytokinin . Transient expression of fusion products of the signaling modules with green fluorescent protein in epidermal leaf cells suggested cytosolic and nuclear localizations of ZmHPs, whereas type-B ZmRR8 was restricted to the nucleus . Type-A RRs were localized partly to the cytosol (ZmRR1, ZmRR2, and ZmRR3) and partly to the nucleus (ZmRR4, ZmRR5, and ZmRR6) . In the yeast two-hybrid assay, ZmHP1 and ZmHP3 interacted with both cytosolic ZmRR1 and nuclear type-B ZmRRs . In vitro experiments demonstrated that ZmHPs function as a phospho-donor for ZmRRs; turnover rates of the phosphorylated state were tenfold lower in ZmRR8 and ZmRR9 than in ZmRR1 and ZmRR4 . These results suggest that the His-Asp phosphorelay signaling pathway might diverge into a cytosolic and a nuclear branch in leaves of maize, and that the biochemical nature of ZmRRs is different in terms of stability of the phosphorylated status. Pflugers Arch, 2003 Sep, 446(6), 633 - 40 Epub 2003 Jul 10. KCNE2 modulates current amplitudes and activation kinetics of HCN4: influence of KCNE family members on HCN4 currents; Decher N et al.; The HCN4 gene encodes a hyperpolarization-activated cation current contributing to the slow components of the pacemaking currents I(f) in the sinoatrial node and I(h) or I(q) in the thalamus . Heterologous expression studies of individual HCN channels have, however, failed to reproduce fully the diversity of native I(f/h/q) currents, suggesting the presence of modulating auxiliary subunits . Consistent with this is the recent description of KCNE2, which is highly expressed in the sinoatrial node, as a beta-subunit of rapidly activating HCN1 and HCN2 channels . To determine whether KCNE2 can also modulate the slow component of native I(f/h/q) currents, we co-expressed KCNE2 with HCN4 in Xenopus oocytes and in Chinese hamster ovary (CHO) cells and analysed the resulting currents using two-electrode voltage-clamp and patch-clamp techniques, respectively . In both cell types, co-expressed KCNE2 enhanced HCN4-generated current amplitudes, slowed the activation kinetics and shifted the voltage for half-maximal activation of currents to more negative voltages . In contrast, the related family members KCNE1, KCNE3 and KCNE4 did not change current characteristics of HCN4 . Consistent with these electrophysiological results, the carboxy-terminal tail of KCNE2, but not of other KCNE subunits, interacted with the carboxy-terminal tail of HCN4 in yeast two-hybrid assays . KCNE2, by modulating I(f) or I(h) currents, might thus contribute to the electrophysiological diversity of known pacemaking currents in the heart and brain. Nat Cell Biol, 2003 Aug, 5(8), 748 - 53 Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint; Chung E et al.; The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules . When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC) . Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68 . Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X . laevis egg extracts . However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins . This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20 . Our results also have implications for the spindle checkpoint silencing mechanism. Bioinformatics, 2003, 19 Suppl 1, i273 - 82 Genome-wide discovery of transcriptional modules from DNA sequence and gene expression; Segal E et al.; In this paper, we describe an approach for understanding transcriptional regulation from both gene expression and promoter sequence data . We aim to identify transcriptional modules--sets of genes that are co-regulated in a set of experiments, through a common motif profile . Using the EM algorithm, our approach refines both the module assignment and the motif profile so as to best explain the expression data as a function of transcriptional motifs . It also dynamically adds and deletes motifs, as required to provide a genome-wide explanation of the expression data . We evaluate the method on two Saccharomyces cerevisiae gene expression data sets, showing that our approach is better than a standard one at recovering known motifs and at generating biologically coherent modules . We also combine our results with binding localization data to obtain regulatory relationships with known transcription factors, and show that many of the inferred relationships have support in the literature. Bioinformatics, 2003, 19 Suppl 1, i264 - 71 Discovering molecular pathways from protein interaction and gene expression data; Segal E et al.; In this paper, we describe an approach for identifying 'pathways' from gene expression and protein interaction data . Our approach is based on the assumption that many pathways exhibit two properties: their genes exhibit a similar gene expression profile, and the protein products of the genes often interact . Our approach is based on a unified probabilistic model, which is learned from the data using the EM algorithm . We present results on two Saccharomyces cerevisiae gene expression data sets, combined with a binary protein interaction data set . Our results show that our approach is much more successful than other approaches at discovering both coherent functional groups and entire protein complexes. Bioinformatics, 2003, 19 Suppl 1, i183 - 9 Predicting phenotype from patterns of annotation; King OD et al.; MOTIVATION:Predicting the outcome of specific experiments (such as the growth of a particular mutant strain in a particular medium) has the potential to allow researchers to devote resources to experiments with higher expected numbers of 'hits' . RESULTS: We use decision trees to predict phenotypes associated with Saccharomyces cerevisiae genes on the basis of Gene Ontology (GO) functional annotations from the Saccharomyces Genome Database (SGD) and other phenotypic annotations from the Yeast Phenotype Catalog at the Munich Information Center for Protein Sequences (MIPS) . We assess the methodology in three ways: (1) we use cross-validation on the phenotypic annotations listed in MIPS, and show ROC curves indicating the tradeoff between true-positive rate and false-positive rate; (2) we do a literature-search for 100 of the predicted gene-phenotype associations that are not listed in MIPS, and find evidence for 43 of them; (3) we use deletion strains to experimentally assess 61 predicted gene-phenotype associations not listed in MIPS; significantly more of these deletion strains show abnormal growth than would be expected by chance. Exp Gerontol, 2003 Jul, 38(7), 807 - 11 The Ras and Sch9 pathways regulate stress resistance and longevity; Longo VD; Depending on the availability of extracellular nutrients, yeast can enter either high or low metabolism survival phases . We have identified two pathways that regulate longevity and stress resistance in both the low and high metabolism phases . The deletion of SCH9, which encodes for a serine threonine kinase, triples the mean life span and increases resistance to oxidative and thermal stress . Mutations that decrease the activity of the Ras/Cyr1/PKA pathway also extend longevity and increase stress resistance by activating transcription factors Msn2/Msn4 and the mitochondrial antioxidant enzyme superoxide dismutase (Sod2) . Although only one intracellular pathway that includes genes homologous to SCH9 and SOD2 has been identified in worms, our studies in yeast suggest that longevity in higher eukaryotes may also be negatively regulated by the Ras pathway. Thyroid, 2003 May, 13(5), 427 - 35 Leucine at codon 428 in the ninth heptad of thyroid hormone receptor beta1 is necessary for interactions with the transcriptional cofactors and functions regardless of dimer formations; Monden T et al.; Structure/function studies of the thyroid hormone receptor (TR) beta(1) have demonstrated that single amino acid substitutions in either position 428 or 429 in the ligand-binding domain (LBD) can alter heterodimerizations and homodimerizations, respectively . A leucine at 428 is located in a highly conserved region corresponding to the putative ninth heptad repeat of a leucine-zipper-like motif in the LBD of TRbeta(1) . To investigate how the side chain of amino acids at 428 affect receptor characteristics, gel-shift mobility shift assays and yeast two-hybrid assays were analyzed . The neutral status amino acids such as a leucine (wild-type) or a glutamine at 428 preferred heterodimerization with RXR . Furthermore, a positively charged side chain of amino acids at 428 such as an arginine or a lysine, preserved homodimer formation . Irrespective of charge, ninth heptad mutant receptors did not bind the ligand and were not able to interact with either corepressor or coactivating proteins . Limited trypsinization assays revealed no major conformational change in the ninth heptad mutant receptors . Together, these findings suggested that a leucine at 428 was a critical amino acid for both interaction with the thyroid hormone receptor associated proteins and ligand-independent and -dependent functions regardless of dimer formations. Nucleic Acids Res, 2003 Jul 15, 31(14), 4059 - 70 The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity; Huard S et al.; The catalytic subunit of telomerase (TERT) contains conserved reverse transcriptase-like motifs but N- and C-terminal regions unique to telomerases . Despite weak sequence conservation, the C terminus of TERTs from various organisms has been implicated in telomerase-specific functions, including telomerase activity, functional multimerization with other TERT molecules, enzyme processivity and telomere length maintenance . We studied hTERT proteins containing small C-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase activity, hTERT multimerization and processivity . Using sequence alignment of five vertebrate TERTs and Arabidopsis thaliana TERT, we identified blocks of highly conserved amino acids that were required for human telomerase activity and functional hTERT complementation . We adapted the non-PCR-based telomerase elongation assay to characterize telomerase expressed and reconstituted in the in vitro transcription/translation rabbit reticulocyte lysate system . Using this assay, we found that the hTERT C terminus, like the C terminus of Saccharomyces cerevisiae TERT, contributes to successive nucleotide addition within a single 6-base telomeric repeat (type I processivity) . Certain mutations in the hTERT C terminus also reduced the repetitive addition of multiple telomeric repeats (type II processivity) . Our results suggest a functionally conserved role for the TERT C terminus in telomerase enzyme processivity. Nucleic Acids Res, 2003 Jul 15, 31(14), 4041 - 50 Region and amino acid residues required for Rad51C binding in the human Xrcc3 protein; Kurumizaka H et al.; The Xrcc3 protein, which is required for the homologous recombinational repair of damaged DNA, forms a complex with the Rad51C protein in human cells . Mutations in either the Xrcc3 or Rad51C gene cause extreme sensitivity to DNA-damaging agents and generate the genomic instability frequently found in tumors . In the present study, we found that the Xrcc3 segment containing amino acid residues 63-346, Xrcc3(63-346), is the Rad51C-binding region . Biochemical analyses revealed that Xrcc3(63-346) forms a complex with Rad51C, and the Xrcc3(63-346)- Rad51C complex possesses ssDNA and dsDNA binding abilities comparable to those of the full-length Xrcc3-Rad51C complex . Based on the structure of RecA, which is thought to be the ancestor of Xrcc3, six Xrcc3 point mutants were designed . Two-hybrid and biochemical analyses of the Xrcc3 point mutants revealed that Tyr139 and Phe249 are essential amino acid residues for Rad51C binding . Superposition of the Xrcc3 Tyr139 and Phe249 residues on the RecA structure suggested that Tyr139 may function to ensure proper folding and Phe249 may be important to constitute the Rad51C-binding interface in Xrcc3. EMBO J, 2003 Jul 15, 22(14), 3557 - 67 The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome; Imai J et al.; Hsp90 has a diverse array of cellular roles including protein folding, stress response and signal transduction . Herein we report a novel function for Hsp90 in the ATP-dependent assembly of the 26S proteasome . Functional loss of Hsp90 using a temperature-sensitive mutant in yeast caused dissociation of the 26S proteasome . Conversely, these dissociated constituents reassembled in Hsp90-dependent fashion both in vivo and in vitro; the process required ATP-hydrolysis and was suppressed by the Hsp90 inhibitor geldanamycin . We also found genetic interactions between Hsp90 and several proteasomal Rpn (Regulatory particle non-ATPase subunit) genes, emphasizing the importance of Hsp90 to the integrity of the 26S proteasome . Our results indicate that Hsp90 interacts with the 26S proteasome and plays a principal role in the assembly and maintenance of the 26S proteasome. EMBO J, 2003 Jul 15, 22(14), 3524 - 35 The structure of the cell cycle protein Cdc14 reveals a proline-directed protein phosphatase; Gray CH et al.; The Cdc14 family of dual-specificity protein phosphatases (DSPs) is conserved within eukaryotes and functions to down-regulate mitotic Cdk activities, promoting cytokinesis and mitotic exit . We have integrated structural and kinetic analyses to define the molecular mechanism of the dephosphorylation reaction catalysed by Cdc14 . The structure of Cdc14 illustrates a novel arrangement of two domains, each with a DSP-like fold, arranged in tandem . The C-terminal domain contains the conserved PTP motif of the catalytic site, whereas the N-terminal domain, which shares no sequence similarity with other DSPs, contributes to substrate specificity, and lacks catalytic activity . The catalytic site is located at the base of a pronounced surface channel formed by the interface of the two domains, and regions of both domains interact with the phosphopeptide substrate . Specificity for a pSer-Pro motif is mediated by a hydrophobic pocket that is capable of accommodating the apolar Pro(P+1) residue of the peptide . Our structural and kinetic data support a role for Cdc14 in the preferential dephosphorylation of proteins modified by proline-directed kinases. Cell Cycle, 2003 Jul-Aug, 2(4), 384 - 96 FHA domain-mediated DNA checkpoint regulation of Rad53; Schwartz MF et al.; Saccharomyces cerevisiae Rad53 is a protein kinase central to the DNA damage and DNA replication checkpoint signaling pathways . In addition to its catalytic domain, Rad53 contains two forkhead homology-associated (FHA) domains (FHA1 and FHA2), which are phosphopeptide binding domains . The Rad53 FHA domains are proposed to mediate the interaction of Rad53 with both upstream and downstream branches of the DNA checkpoint signaling pathways . Here we show that concurrent mutation of Rad53 FHA1 and FHA2 causes DNA checkpoint defects approaching that of inactivation or loss of RAD53 itself . Both FHA1 and FHA2 are required for the robust activation of Rad53 by the RAD9-dependent DNA damage checkpoint pathway, while an intact FHA1 or FHA2 allows the activation of Rad53 in response to replication block . Mutation of Rad53 FHA1 causes the persistent activation of the RAD9-dependent DNA damage checkpoint pathway in response to replicational stress, suggesting that the RAD53-dependent stabilization of stalled replication forks functions through FHA1 . Rad53 FHA1 is also required for the phosphorylation-dependent association of Rad53 with the chromatin assembly factor Asf1, although Asf1 itself is apparently not required for the prevention of DNA damage in response to replication block. Cell Cycle, 2003 Jul-Aug, 2(4), 316 - 24 Cyclin-dependent kinases and S phase control in mammalian cells; Woo RA et al.; Mammalian DNA replication is an elegantly choreographed process in which multiple components are assembled at the origins to form the prereplication complex . Formation and activation of the prereplication complex requires coordinate actions of G1and S phase cyclin-dependent kinases . Cyclin E-CDK2 and cyclin A-CDK2, together with DBF4-CDC7, phosphorylate several components of the prereplication complex and replication machinery . In this review, we summarize the current understanding of the mechanism of initiation of DNA replication in mammalian cells . The roles of cyclin A/E-CDK2 complexes in driving replication, their relationship with other regulators of S phase, and their role in keeping replication to only once per cell cycle will be discussed . In addition, an important issue is the checks and balances that prevent inappropriate DNA replication, and how a breakdown in these checkpoints can lead to genomic instability and cancer . A critical mediator of these checkpoints, ATM, signals through a comprehensive network of proteins leading to CDK2 inhibition thus preventing DNA synthesis . This will be reviewed in addition to other mechanisms involved in the intra-S phase DNA damage checkpoint. Cell Cycle, 2003 Jul-Aug, 2(4), 307 - 9 Replication origins: why do we need so many? Bielinsky AK. During the G1 phase of the cell cycle, replication origins are prepared to fire, a process that is referred to as origin licensing . It was often pondered what a cell's fate would be if not all of its replication origins were licensed and subsequently activated during S phase . One obvious prediction was that S phase would simply be prolonged . As it turns out, however, the consequences are much more complex . A short G1 phase enforced by premature entry into S phase, or other events that negatively affect origin licensing, will ultimately compromise the cell's ability to complete DNA replication before entering mitosis . As a result, the cell becomes genomically unstable when it attempts to repair unreplicated DNA during anaphase . Thus, the density of active replication origins in the chromosomes of eukaryotic cells determines S phase dynamics and chromosome stability during mitosis. Cell Cycle, 2003 Jul-Aug, 2(4), 303 - 6 It's all in the timing: linking S phase to chromatin structure and chromosome dynamics; Bailis JM et al.; Many aspects of chromosome biology are fundamentally linked to events that occur during the DNA synthesis (S) phase of the cell cycle . The DNA must be duplicated once, and once exactly, each S phase . Local chromatin structure must also be re-assembled each S phase to incorporate newly replicated sister chromatids . The replication fork is the one complex that potentially interacts with every nucleotide of the genome, providing a mechanism to couple chromatin assembly to S phase passage . Importantly, eukaryotic genomes contain regions of structurally distinct chromatin, such as heterochromatin, defined by distinct patterns of histone modification and specific protein associations . Heterochromatin is generally associated with repeated sequence elements near centromeres, telomeres and other sites . Evidence suggests that heterochromatin assembled during S phase supports the association of multiprotein complexes required for many chromosome transactions, including transcriptional silencing, sister-chromatid cohesion, and kinetochore function . These complexes are in turn essential for regulated gene expression, equal chromosome segregation and genomic stability . Intriguingly, recent studies indicate that these processes are linked to S phase by temporal mechanisms as well as by replication-dependent activities. Mol Membr Biol, 2003 Apr-Jun, 20(2), 141 - 54 Membrane dynamics and the biogenesis of lysosomes; Luzio JP et al.; Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways . They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs . Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic . Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration . This process is important in plasma membrane repair . The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae . Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles . Several multi-protein oligomeric complexes required for these processes have been identified . The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes. Mol Membr Biol, 2003 Apr-Jun, 20(2), 117 - 27 Golgi apparatus partitioning during cell division; Rabouille C et al.; This review discusses the mitotic segregation of the Golgi apparatus . The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles . Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm . Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle . This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy . Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion . Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum . This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum . We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis . We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms. Mol Biochem Parasitol, 2003 Jul, 129(2), 191 - 8 The C-terminal domain of the Plasmodium falciparum acyl-CoA synthetases PfACS1 and PfACS3 functions as ligand for ankyrin; Tellez M et al.; Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm establishing novel interactions between host and parasite proteins, particularly at the membrane skeleton that modifies both the structural and functional properties of the red cell . We present evidences that two members of the P . falciparum acyl-CoA synthetase (PfACS) family, responsible for the activation of long-chain fatty acids by thio-esterification with CoA, are transported in vesicle-like structures toward the host erythrocyte cytoplasm where they interact with the cytoskeletal protein ankyrin . Carboxyl-terminal domain (CTD) overlay studies indicated that PfACS1 and PfACS3 bind to the 78-kDa fragment of ankyrin corresponding with its spectrin-binding domain . Co-immunoprecipitation of ankyrin and PfACS1/3 indicates that at least a fraction of these proteins are physically associated in the infected erythrocytes and provide evidence for a novel specific interaction which suggest that such a binding may bring these enzymes closer to the host erythrocyte membrane where exogenous fatty acids are available. Mol Biochem Parasitol, 2003 Jul, 129(2), 127 - 35 Characterization of proteins localized to a subcellular compartment associated with an alternate secretory pathway of the malaria parasite; Cortes GT et al.; Monoclonal antibodies recognizing proteins localized to a unique subcellular compartment within the malaria parasite are described in this report . These monoclonal antibodies recognize Plasmodium falciparum proteins of 68, 45 and 22 kDa proteins which are also conserved in rodent Plasmodium species . Co-localization studies indicate that these proteins are located in a brefeldin A-induced compartment which was previously proposed to be an early step in the export of proteins from the parasite into the infected erythrocyte . COPII coat proteins, Sar1p and Sec31p, and the endoplasmic reticulum-associated chaperone, BiP, all partially co-localize with the 68 and 22 kDa proteins, thus suggesting that this subcellular compartment has some similarities to the endoplasmic reticulum or that this compartment represents a domain of the endoplasmic reticulum . The 68 and 22 kDa proteins are highly soluble in non-ionic detergent and are likely to be located within the lumen of a membrane-bound compartment . These proteins found within this subcellular compartment are present throughout the blood stage from very early rings to segmenters . The results of this study further substantiate the existence of an alternate secretory pathway in the malaria parasite which plays a role in the export of proteins into the host erythrocyte. Biochem Biophys Res Commun, 2003 Jul 18, 307(1), 31 - 40 The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD1; Abramczyk O et al.; The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function . Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes . The key role in regulation of this process is played by specific, second messenger-independent protein kinases . The PK60S kinase was one of the enzymes phosphorylating P-proteins . The enzyme has been purified from yeast and characterised . Pure enzyme has properties similar to those reported for casein kinase type 2 . Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')) . Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt) . The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed. J Am Chem Soc, 2003 Jul 16, 125(28), 8655 - 65 Scavenging with TEMPO* to identify peptide- and protein-based radicals by mass spectrometry: advantages of spin scavenging over spin trapping; Wright PJ et al.; The detection and characterization of radicals in biomolecules are challenging due to their high reactivity and low concentration . Mass spectrometry (MS) provides a tool for the unambiguous identification of protein-based radicals by exploiting their reactivity with suitable reagents . To date, protein-radical detection by MS has been modeled after electron paramagnetic resonance experiments, in which diamagnetic spin traps, such as 3,5-dibromo-4-nitrosobenzene sulfonic acid, convert unstable radicals to more stable spin adducts . Since MS detects mass changes, and not unpaired spins, conversion of radicals to stable diamagnetic adducts is more desirable . The use of 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) in the MS identification of protein-based radicals was explored here to establish whether scavenging via radical combination would give rise to TEMPO adducts that were stable to MS analysis . The horseradish peroxidase/H(2)O(2) reaction was used to generate radicals in derivatives of tyrosine, tryptophan, and phenylalanine as models of protein-based radicals . TEMPO(*) was added as a radical scavenger, and the products were analyzed by electrospray ionization (ESI) MS . Dramatically higher mass-adduct yields were obtained using radical scavenging vs radical trapping, which greatly enhanced the sensitivity of radical detection . The efficiency of TEMPO(*) in protein radical scavenging was examined in horse heart myoglobin and cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae . On H(2)O(2) binding to their ferric hemes, two oxidizing equivalents are transferred to the proteins as an Fe(IV)=O species and a polypeptide-based radical . In addition, CCP has been shown to reduce up to 10 equiv of H(2)O(2) using endogenous donors, thereby generating as many as 20 radicals on its polypeptide . Following myoglobin and CCP incubation with a 10-fold molar excess of H(2)O(2) and TEMPO(*), matrix-assisted laser desorption ionization (MALDI) time-of-flight analysis of the tryptic peptides derived from the proteins revealed 1 and 9 TEMPO adducts of myoglobin and CCP, respectively . Given the high scavenging efficiency of TEMPO(*) and the stability of TEMPO-labeled peptides in ESI and MALDI sources, scavenging by stable nitroxide radicals coupled with MS analysis should provide sensitive and powerful technology for the characterization of protein-based radicals. J Agric Food Chem, 2003 Jul 16, 51(15), 4382 - 8 Protocol for the production of concentrated extracts of food folate for use in human bioavailability studies; McKillop DJ et al.; To provide a tool to study folate bioavailability under controlled conditions, a methodology was developed to produce extracts representative of natural food folates but removed from their matrix and sufficiently concentrated so as to elicit a response in biomarkers of folate status without distorting usual dietary intake patterns . Egg, spinach, and yeast were selected to represent the wide range in extent of folate conjugation found in foods (0, 60, and 100% polyglutamyl folate, respectively) . The protocol, which was based on extracting food folates using only reagents safe for human consumption, was optimized in the laboratory (thermal extraction for 10 min in a 2% ascorbate solution at pH 5) and then adapted for industrial scale production in a food-processing facility . Results showed that the extracts were 2.3-12 times more concentrated in folate compared with their corresponding food sources . Neither the mono- to polyglutamate ratio nor the distribution of the main folate derivatives was altered during processing, making these extracts suitable for use in human bioavailability studies. J Cell Biol, 2003 Jul 7, 162(1), 113 - 24 ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma; Krauss M et al.; Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane . Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180 . Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2 . We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect . These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse. J Cell Biol, 2003 Jul 7, 162(1), 71 - 84 Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains; Ye Y et al.; A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome . Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway . Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain . The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol . The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane . Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1 . We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane. Int J Cancer, 2003 Sep 1, 106(3), 364 - 71 The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines; Arhel NJ et al.; Although the retinoblastoma susceptibility gene RB1 is inactivated in a wide range of human tumours, overexpression in colonic carcinomas has been linked to the antiapoptotic function of the protein . In the current study we show that the Retinoblastoma susceptibility protein (Rb) protein interacts with Bag-1, an apoptotic regulator, in human colonic adenoma- and carcinoma-derived cell lines . Coimmunoprecipitation demonstrated that endogenous Rb and Bag-1 interact in both adenoma- and carcinoma-derived cell lines . The specificity of the interaction was demonstrated by expression of human Papillomavirus E7 oncoprotein, an inhibitor of Rb protein interactions, which disrupted the Rb/Bag-1 complex . We report that Bag-1 is predominantly localised in the nucleus of colorectal adenoma- and carcinoma-derived epithelial cells . Disruption of the Rb/Bag-1 complex through expression of E7 changes the subcellular distribution of Bag-1, decreasing nuclear localised Bag-1 . Our work establishes that the Rb protein interacts with the Bag-1 apoptotic regulator protein, and introduces a novel function for Rb, involving modulation of the subcellular localisation of Bag-1 in human colonic epithelial cells . Sci Aging Knowledge Environ . 2003 Apr 23;2003(16):PE8. Is DNA cut out for a long life? Sinclair D. Much attention has been focused on the DNA repair hypothesis of aging . Studies in mammals that seek to test the validity of this model are complicated by both the functional redundancy and the essential nature of genes involved in the repair process . Compared to mammals, the study of DNA repair and aging in yeast has considerably fewer complicating factors . In this Perspective, I discuss results presented in this month's issue of Aging Cell that address whether the types of DNA damage repaired by the base excision repair pathway cause aging in yeast. J Biol Chem, 2003 Sep 19, 278(38), 36051 - 8 Epub 2003 Jul 04. A novel zinc finger is required for Mcm10 homocomplex assembly; Cook CR et al.; Mcm10 is a DNA replication factor that interacts with multiple subunits of the MCM2-7 hexameric complex . We report here that Mcm10 self-interacts and assembles into large homocomplexes (approximately 800 kDa) . A conserved domain of 210 amino acid residues is sufficient for mediating self-interaction and complex assembly . A novel zinc finger within the conserved domain, CX10CX11CX2H, is essential for the homocomplex formation . Mutant alleles with amino acid substitutions at conserved cysteines and histidine in the zinc finger fail to assemble homocomplexes . A defect in homocomplex assembly correlates with defects in DNA replication and cell growth in the mutants . These observations suggest that homocomplex assembly is essential for Mcm10 function . Multisubunit Mcm10 homocomplexes may provide the structural basis for Mcm10 to interact with multiple subunits of the MCM2-7 hexamer. J Biol Chem, 2003 Sep 12, 278(37), 35660 - 7 Epub 2003 Jul 02. Transcription initiation factor IID-interactive histone chaperone CIA-II implicated in mammalian spermatogenesis; Umehara T et al.; Histones are thought to have specific roles in mammalian spermatogenesis, because several subtypes of histones emerge that are post-translationally modified during spermatogenesis . Though regular assembly of nucleosome is guaranteed by histone chaperones, their involvement in spermatogenesis is yet to be characterized . Here we identified a histone chaperone-related factor, which we designated as CCG1-interacting factor A-II (CIA-II), through interaction with bromodomains of TAFII250/CCG1, which is the largest subunit of human transcription initiation factor IID (TFIID) . We found that human CIA-II (hCIA-II) localizes in HeLa nuclei and is highly expressed in testis and other proliferating cell-containing tissues . Expression of mouse CIA-II (mCIA-II) does not occur in the germ cell-lacking testes of adult WBB6F1-W/Wv mutant mice, indicating its expression in testis to be specific to germ cells . Fractionation of testicular germ cells revealed that mCIA-II transcripts accumulate in pachytene spermatocytes but not in spermatids . In addition, the mCIA-II transcripts in testis were present as early as 4 days after birth and decreased at 56 days after birth . These findings indicate that mCIA-II expression in testis is restricted to premeiotic to meiotic stages during spermatogenesis . Also, we found that hCIA-II interacts with histone H3 in vivo and with histones H3/H4 in vitro and that it facilitates supercoiling of circular DNA when it is incubated with core histones and topoisomerase I in vitro . These data suggest that CIA-II is a histone chaperone and is implicated in the regulation of mammalian spermatogenesis. J Mol Biol, 2003 Jul 11, 330(3), 621 - 37 Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges; Marti DN et al.; Electrostatic interactions play a complex role in stabilizing proteins . Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS) . The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure . pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form . The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)) . Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)) . The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)) . To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline) . Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing . Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability. J Mol Biol, 2003 Jul 11, 330(3), 473 - 84 Unique features in the C-terminal domain provide caltractin with target specificity; Hu H et al.; Caltractin (centrin) is a member of the calmodulin (CaM) superfamily of EF-hand calcium-binding proteins . It is an essential component of the centrosomal structures in a wide range of organisms . Caltractin and calmodulin apparently function in distinct calcium signaling pathways despite substantial sequence similarity . In an effort to understand the structural basis for such differences, the high-resolution three-dimensional solution structure of the complex between the Ca(2+)-activated C-terminal domain of Chlamydomonas reinhardtii caltractin (CRC-C) and a 19 residue peptide fragment comprising the putative cdc31p-binding region of Kar1p (K(19)) has been determined by multi-dimensional heteronuclear NMR spectroscopy . Formation of the complex is calcium-dependent and is stabilized by extensive interactions between CRC-C and three key hydrophobic anchors (Trp10, Leu13 and Leu14) in the peptide as well as favorable electrostatic interactions at the protein-peptide interface . In-depth comparisons have been made to the structure of the complex of Ca(2+)-activated calmodulin and R(20), the CaM-binding domain of smooth muscle myosin light-chain kinase . Although the overall structures of CRC and CaM domains in their respective complexes are very similar, differences in critical regions in the sequences of these proteins and their targets lead to clear differences in the complementarity of their respective binding surfaces . These subtle differences reveal the structural basis for the Ca(2+)-dependent regulation of distinct cellular signaling events by CRC and CaM. Cancer Cell, 2003 Jun, 3(6), 577 - 87 Regulation of HDM2 activity by the ribosomal protein L11; Lohrum MA et al.; The HDM2 protein plays an important role in regulating the stability and function of the p53 tumor suppressor protein . In this report, we show that the ribosomal protein L11 can interact with HDM2 and inhibit HDM2 function, thus leading to the stabilization and activation of p53 . The inhibition of HDM2 activity by L11 shows some similarity to the previously described activity of ARF, and expression of either ARF or L11 can induce a p53 response . Enhancement of the interaction between endogenous L11 and HDM2 following treatment of cells with low levels of actinomycin-D suggests that the HDM2/L11 interaction represents a novel pathway for p53 stabilization in response to perturbations in ribosome biogenesis. Curr Biol, 2003 Jul 1, 13(13), R525 - 7 UCS proteins: managing the myosin motor; Yu Q et al.; Recent studies indicate that myosin molecular motors interact inside cells with proteins containing a conserved 'UCS' domain . This appears to ensure proper folding of myosin heads so that they can perform their ATP-dependent actin-based motor functions. Curr Biol, 2003 Jul 1, 13(13), 1140 - 4 Proteasome disassembly and downregulation is correlated with viability during stationary phase; Bajorek M et al.; During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased . We show that a parallel decrease in proteasome-dependent proteolysis also occurs . The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components . Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry . Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs . By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity . This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation . In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells . Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes. J Nutr, 2003 Jul, 133(7 Suppl), 2485S - 2493S Inhibition of histone deacetylase activity by butyrate; Davie JR; This article reviews the effects of the short-chain fatty acid butyrate on histone deacetylase (HDAC) activity . Sodium butyrate has multiple effects on cultured mammalian cells that include inhibition of proliferation, induction of differentiation and induction or repression of gene expression . The observation that butyrate treatment of cells results in histone hyperacetylation initiated a flurry of activity that led to the discovery that butyrate inhibits HDAC activity . Butyrate has been an essential agent for determining the role of histone acetylation in chromatin structure and function . Interestingly, inhibition of HDAC activity affects the expression of only 2% of mammalian genes . Promoters of butyrate-responsive genes have butyrate response elements, and the action of butyrate is often mediated through Sp1/Sp3 binding sites (e.g., p21(Waf1/Cip1)) . We demonstrated that Sp1 and Sp3 recruit HDAC1 and HDAC2, with the latter being phosphorylated by protein kinase CK2 . A model is proposed in which inhibition of Sp1/Sp3-associated HDAC activity leads to histone hyperacetylation and transcriptional activation of the p21(Waf1/Cip1) gene; p21(Waf1/Cip1) inhibits cyclin-dependent kinase 2 activity and thereby arrests cell cycling . Pending the cell background, the nonproliferating cells may enter differentiation or apoptotic pathways . The potential of butyrate and HDAC inhibitors in the prevention and treatment of cancer is presented. Genome Res, 2003 Jul, 13(7), 1706 - 18 Subsystem identification through dimensionality reduction of large-scale gene expression data; Kim PM et al.; The availability of parallel, high-throughput biological experiments that simultaneously monitor thousands of cellular observables provides an opportunity for investigating cellular behavior in a highly quantitative manner at multiple levels of resolution . One challenge to more fully exploit new experimental advances is the need to develop algorithms to provide an analysis at each of the relevant levels of detail . Here, the data analysis method non-negative matrix factorization (NMF) has been applied to the analysis of gene array experiments . Whereas current algorithms identify relationships on the basis of large-scale similarity between expression patterns, NMF is a recently developed machine learning technique capable of recognizing similarity between subportions of the data corresponding to localized features in expression space . A large data set consisting of 300 genome-wide expression measurements of yeast was used as sample data to illustrate the performance of the new approach . Local features detected are shown to map well to functional cellular subsystems . Functional relationships predicted by the new analysis are compared with those predicted using standard approaches; validation using bioinformatic databases suggests predictions using the new approach may be up to twice as accurate as some conventional approaches. EMBO J, 2003 Jul 1, 22(13), 3337 - 45 The oncogenic RAS2(val19) mutation locks respiration, independently of PKA, in a mode prone to generate ROS; Hlavata L et al.; The RAS2(val19) allele, which renders the cAMP-PKA pathway constitutively active and decreases the replicative life-span of yeast cells, is demonstrated to increase production of reactive oxygen species (ROS) and to elevate oxidative protein damage . Mitochondrial respiration in the mutant is locked in a non-phosphorylating mode prone to generate ROS but this phenotype is not linked to a constitutively active PKA pathway . In contrast, providing RAS2(val19) cells with the mammalian uncoupling protein UCP1 restores phosphorylating respiration and reduces ROS levels, but does not correct for PKA-dependent defects . Thus, the RAS2(val19) allele acts like a double-edged sword with respect to oxidation management: (i) . it diminishes expression of STRE element genes required for oxidative stress defenses in a PKA-dependent fashion, and (ii) . it affects endogenous ROS production and the respiratory state in a PKA-independent way . The effect of the oncogenic RAS allele on the replicative life-span is primarily asserted via the PKA-dependent pathway since Pde2p, but not UCP1, overproduction suppressed premature aging of the RAS2(val19) mutant. Genes Cells, 2003 Jul, 8(7), 603 - 18 {PHI+}, a novel Sup35-prion variant propagated with non-Gln/Asn oligopeptide repeats in the absence of the chaperone protein Hsp104; Crist CG et al.; BACKGROUND: The {PSI+} element of the budding yeast is an aggregated form of the translation release factor Sup35 that is propagated and transmitted cytoplasmically in a manner analogous to that of mammalian prions . The N-terminal of Sup35, necessary for {PSI+}, contains oligopeptide repeats and multiple Gln/Asn residues . RESULTS: We replaced the Gln/Asn-rich prion repeats of Sup35 with non-Gln/Asn repeats from heterologous yeast strains . These non-Gln/Asn repeat Sup35s propagated a novel {PSI+} variant, {PHI+}, that appeared de novo 103 times more frequent than {PSI+} . {PHI+} was stably inherited in a non-Mendelian fashion, but not eliminated upon the inactivation of Hsp104, unlike known {PSI+} elements . In vitro, non-Gln/Asn repeat domains formed amyloid fibres that were shorter and grew more slowly than did Gln/Asn-rich prion domains, while {PHI+} aggregates were smaller than {PSI+} aggregates in vivo . CONCLUSIONS: These findings suggest the existence of an alternative, Hsp104-independent pathway to replicate non-Gln/Asn variant Sup35 prion seeds. Nat Rev Genet, 2003 Jul, 4(7), 520 - 34 Condensin and cohesin: more than chromosome compactor and glue; Hagstrom KA et al.; Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division . Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure . Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes . Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis . Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization. J Pediatr, 2003 Jun, 142(6), 709 - 13 Biochemical effect of intravenous arginine butyrate in X-linked adrenoleukodystrophy; McGovern MM et al.; OBJECTIVES: To determine the biochemical and clinical effects of intravenous arginine butyrate in X-linked Adrenoleukodystrophy (X-ALD) . STUDY DESIGN: Arginine butyrate was intravenously infused over a 4-month period in a patient with the rapid cerebral form of X-ALD . Very long chain fatty acids (VLCFA), complete blood counts, and serum chemistries were monitored, and serial MRI of the brain and clinical neurologic examinations were performed . RESULTS: All blood chemical and hematologic values remained within the normal range for age throughout the therapy . After completion of the first day of infusion, the C 26:0 value fell from 1.01 microg/mL to 0.445 microg/mL, which is below the mean value for an X-ALD heterozygote . Throughout the remainder of the trial, all C26:0 levels fell below the mean -1 SD for X-ALD hemizygotes (mean, 1.18 microg/mL, 1 SD = 0.53), ranging from 0.321 to 0.565 microg/mL . Despite reduction of the plasma VLCFA, the patient continued to deteriorate neurologically . CONCLUSIONS: Intravenous arginine butyrate resulted in a rapid decrease in plasma VLCFA but no effect on the neurologic progression of the disease in this patient . Additional studies are needed to determine minimum effective dosage and interval, what proportion of patients respond, and whether the agent can prevent neurologic degeneration. Plant Cell, 2003 Jul, 15(7), 1578 - 90 The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein alpha-subunit GPA1 and regulates seed germination and early seedling development; Lapik YR et al.; Heterotrimeric G proteins are implicated in diverse signaling processes in plants, but the molecular mechanisms of their function are largely unknown . Finding G protein effectors and regulatory proteins can help in understanding the roles of these signal transduction proteins in plants . A yeast two-hybrid screen was performed to search for proteins that interact with Arabidopsis G protein alpha-subunit (GPA1) . One of the identified GPA1-interacting proteins is the cupin-domain protein AtPirin1 . Pirin is a recently defined protein found because of its ability to interact with a CCAAT box binding transcription factor . The GPA1-AtPirin1 interaction was confirmed in an in vitro binding assay . We characterized two atpirin1 T-DNA insertional mutants and established that they display a set of phenotypes similar to those of gpa1 mutants, including reduced germination levels in the absence of stratification and an abscisic acid-imposed delay in germination and early seedling development . These data indicate that AtPirin1 likely functions immediately downstream of GPA1 in regulating seed germination and early seedling development. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8844 - 9 Epub 2003 Jul 01. The Rieske FeS protein encoded and synthesized within mitochondria complements a deficiency in the nuclear gene; Golik P et al.; The Rieske FeS protein, an essential catalytic subunit of the mitochondrial cytochrome bc1 complex, is encoded in yeast by the nuclear gene RIP1, whose deletion leads to a respiratory-deficient phenotype . By using biolistic transformation, we have relocated the nuclear RIP1 gene into mitochondria . To allow its expression within the organelle and to direct its integration downstream of the cox1 gene, we have fused the 3' end of the Saccharomyces douglasii cox1 gene upstream of the mitochondrial copy of RIP1 (RIP1m) flanked by the Saccharomyces cerevisiae cox1 promoter and terminator regions . We show that RIP1m integrated between the cox1 and atp8 genes is mitotically stable and expressed, and it complements a deletion of the nuclear gene . Immunodetection experiments demonstrate that the mitochondrial genome containing RIP1m is able to produce the Rip1 protein in lower steady-state amounts than the wild type but still sufficient to maintain a functional cytochrome bc1 complex and respiratory competence to a RIP1-deleted strain . Thus, this recombined mitochondrial genome is a fully functional mitochondrial chromosome with an extended gene content . This successful mitochondrial expression of a nuclear gene essential for respiration can be viewed at the evolutionary level as an artificial reversal of evolutionary events. Chem Biol, 2003 Jun, 10(6), 533 - 40 In vivo evolution of an RNA-based transcriptional activator; Buskirk AR et al.; From random RNA libraries expressed in yeast, we evolved RNA-based transcriptional activators that are comparable in potency to the strongest natural protein activation domains . The evolved RNAs activated transcription up to 53-fold higher than a three-hybrid positive control using the Gal4 activation domain and only 2-fold lower than the highly active VP16 activation domain . Using a combination of directed evolution and site-directed mutagenesis, we dissected the functional elements of the evolved transcriptional activators . A surprisingly large fraction of RNAs from our library are capable of activating transcription, suggesting that nucleic acids may be well suited for binding transcriptional machinery elements normally recruited by proteins . In addition, our work demonstrates an RNA evolution-based approach to perturbing natural cellular function that may serve as a general tool for studying selectable or screenable biological processes in living cells. Nucleic Acids Res Suppl, 2001, (1), 91 - 2 Comparative analysis for expressed genes by polymerase chain reaction using module-shuffling primers; Uematsu C et al.; We have developed a method for comparative analysis of gene expression . It is based on competitive PCR amplification with module-shuffling primers, followed by gel electrophoresis in a fluorescent DNA sequencer . In this method, tagged-cDNA restriction fragments derived from different sources were amplified in one tube at the same amplification efficiency . The method can detect different amounts of each expressed gene, up to difference in amounts of 30% . The method was successfully used for comparative analysis of expressed genes in yeast. Planta, 2003 Oct, 217(6), 931 - 50 Epub 2003 Jun 27. A signal peptide secretion screen in Fucus distichus embryos reveals expression of glucanase, EGF domain-containing, and LRR receptor kinase-like polypeptides during asymmetric cell growth; Belanger KD et al.; Zygotes of the brown alga Fucus distichus (L.) Powell develop polarity prior to the first embryonic cell division and retain a pattern of asymmetric growth during early embryogenesis . In order to identify F . distichus polypeptides secreted during asymmetric cell growth, we used a functional assay in Saccharomyces cerevisiae to screen a cDNA library generated from asymmetrically growing Fucus embryos for sequences encoding polypeptides that function as signal peptides for secretion . We isolated and sequenced 222 plasmids containing Fucus cDNAs encoding signal peptide activity . The cDNA inserts from these plasmids were translated in silico into 244 potential polypeptide sequences, 169 of which are predicted to contain signal peptides . BlastP analysis of the Fucus sequences revealed similarity between many Fucus proteins and cell surface proteins that function in development in other eukaryotes, including epidermal growth factor (EGF)-like repeat-containing proteins, plant leucine-rich repeat (LRR)-receptor kinases, and algal beta-1, 3-exoglucanase . However, most of the isolated Fucus polypeptides lack similarity to known proteins . The isolation of cDNAs encoding secreted Fucus proteins provides an important step toward characterizing cell surface proteins important for asymmetric organization and growth in fucoid embryos. EMBO Rep, 2003 Jul, 4(7), 699 - 703 Structural similarity in the absence of sequence homology of the messenger RNA export factors Mtr2 and p15; Fribourg S et al.; The association between Mtr2 and Mex67 is essential for the nuclear export of bulk messenger RNA in yeast . In metazoans, the analogous function is carried out by the TAP-p15 heterodimer . Whereas Mex67 and TAP are highly conserved proteins, their binding partners, Mtr2 and p15, share no sequence similarity, but are nevertheless functionally homologous . Here, we report the 2.8-A resolution crystal structure of Mtr2 in complex with the NTF2-like domain of Mex67 . Mtr2 is a novel member of the NTF2-like family and interacts with Mex67, forming a complex with a similar structural architecture to that of TAP-p15 . Mtr2 fulfils an analogous function to that of human p15 in maintaining the structural integrity of the heterodimer . In addition, Mtr2 presents a long internal loop, which contains residues that affect the export of the large ribosomal subunit. J Biol Chem, 2003 Sep 19, 278(38), 36547 - 55 Epub 2003 Jun 30. Genetic dissection of p23, an Hsp90 cochaperone, reveals a distinct surface involved in estrogen receptor signaling; Oxelmark E et al.; p23 is an Hsp90-associated protein that regulates signal transduction by the estrogen receptor alpha (ER); however, the mechanism through which p23 governs ER function remains enigmatic . To obtain a collection of p23 molecules with distinct effects on ER signaling, we screened in yeast a series of random mutations as well as specific sequence alterations based on the p23 crystal structure and further analyzed these mutations for their effect on p23-Hsp90 association in vitro and in vivo . We found that the ability of the p23 mutants to decrease or increase ER signal transduction correlated with their association with Hsp90 . We also identified a mutation in the C-terminal tail of p23, which displayed a dominant inhibitory effect on ER transcriptional activation and associates more avidly with Hsp90 relative to the wild type p23 . Interestingly, this mutant interacts with Hsp90 in its non-ATP-bound state, whereas the wild type p23 protein interacts exclusively with the ATP-bound form of Hsp90, which may account for its dominant phenotype . In addition, we have uncovered a novel activity of p23 that antagonizes Hsp90 action during times of cell stress . Using molecular modeling and the p23 crystal structure, we found that the p23 mutations affecting ER signaling identified in the screen localized to one face of the molecule, whereas those that had no effect mapped to other parts of the protein . Thus, our structure/function analysis has identified an important regulatory surface on p23 involved in ER signaling and p23 binding to Hsp90. Proteomics, 2003 Jun, 3(6), 847 - 50 Shotgun collision-induced dissociation of peptides using a time of flight mass analyzer; Purvine S et al.; Parallel collision-induced dissociation (CID) of peptides rather than serial, as is customary, results in loss of the obvious parent-fragment ion lineage available from CID on a single ion . We report proof-of-principle results suggesting the feasibility of parallel peptide CID, referred to here as shotgun CID, for protein identification when using the measured mass accuracies available from a time of flight mass analyzer and currently available search routines such as SEQUEST . Additionally, we report that parent-fragment ion lineage may be reconstructed from information encoded in the chromatographic single ion current traces of peptides. J Pharmacol Sci, 2003 Jun, 92(2), 115 - 23 Involvement of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) and Rac1 in the phagocytosis of amyloid-beta(1-42) in rat microglia; Kitamura Y et al.; Alzheimer's disease (AD) is characterized by the accumulation of extracellular amyloid-beta (A beta) fibrils with microglia . Recently, there has been great interest in the microglial phagocytosis of A beta, because the microglial pathway is considered to be one of the A beta clearance pathways in the brain parenchyma . However, the mechanism of microglial phagocytosis of A beta is not fully understood and, thus, was investigated in this study . At one minute after exposure to A beta(1-42) (A beta 42), A beta immunoreactivity was detected at the cell surface of microglia . After 1 h, marked immunoreactivity was observed in the cytosolic vesicles . At 12 h, delayed phagocytosis of fibrillar A beta 42 was also observed with the formation of a large phagocytic cup . The microglial cell shape rapidly changed to an ameboid form during the process of phagocytosis . Although neither neural Wiskott-Aldrich syndrome protein (N-WASP) nor WASP interacting SH3 protein (WISH) immunoreactivity was co-localized with filamentous actin (F-actin) distribution, both WASP family verprolin-homologous protein (WAVE) and Rac1 immunoreactivity was co-localized with F-actin in the lamellipodia of phogocytic microglia . These results suggest that WAVE and Rac1 participate in the phagocytosis of A beta 42 by microglia. Mol Biol Evol, 2003 Oct, 20(10), 1588 - 97 Epub 2003 Jun 27. Positive selection and subfunctionalization of duplicated CCT chaperonin subunits; Fares MA et al.; To reach a functional and energetically stable conformation, many proteins need molecular helpers called chaperonins . Among the group II chaperonins, CCT proteins provide crucial machinery for the stabilization and proper folding of several proteins in the cytosol of eukaryotic cells through interactions that are subunit-specific and geometry-dependent . CCT proteins are made up of eight different subunits, all with similar sequences, positioned in a precise arrangement . Each subunit has been proposed to have a specialized function during the binding and folding of the CCT protein substrate . Here, we demonstrate that functional divergence occurred after several CCT duplication events due to the fixation of amino acid substitutions by positive selection . Sites critical for ATP binding and substrate binding were found to have undergone positive selection and functional divergence predominantly in subunits that bind tubulin but not actin . Furthermore, we show clear functional divergence between CCT subunits that bind the C-terminal domains of actin and tubulin and those that bind the N-terminal domains . Phylogenetic analyses could not resolve the deep relationships between most subunits, except for the groups alpha/beta/eta and delta/epsilon, suggesting several almost simultaneous ancient duplication events . Together, the results support the idea that, in contrast to homo-oligomeric chaperonins such as GroEL, the high divergence level between CCT subunits is the result of positive selection after each duplication event to provide a specialized role for each CCT subunit in the different steps of protein folding. Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8205 - 10 Epub 2003 Jun 27. Optimization of protease-inhibitor interactions by randomizing adventitious contacts; Komiyama T et al.; Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c . Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface . We hypothesized that the second class of "adventitious" contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases . We began with a modified eglin c, Arg-42-Arg-45-eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family . We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin . Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7 . These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin) . One variant, Asp-49-Arg-42-Arg-45-eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells . The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein-protein interactions. Mol Cell Biol, 2003 Jul, 23(14), 5018 - 30 Mitotic exit regulation through distinct domains within the protein kinase Cdc15; Bardin AJ et al.; The mitotic exit network (MEN), a Ras-like signaling cascade, promotes the release of the protein phosphatase Cdc14 from the nucleolus and is essential for cells to exit from mitosis in Saccharomyces cerevisiae . We have characterized the functional domains of one of the MEN components, the protein kinase Cdc15, and investigated the role of these domains in mitotic exit . We show that a region adjacent to Cdc15's kinase domain is required for self-association and for binding to spindle pole bodies and that this domain is essential for CDC15 function . Furthermore, we find that overexpression of CDC15 lacking the C-terminal 224 amino acids results in hyperactivation of MEN and premature release of Cdc14 from the nucleolus, suggesting that this domain within Cdc15 functions to inhibit MEN signaling . Our findings indicate that multiple modes of MEN regulation occur through the protein kinase Cdc15. Mol Cell Biol, 2003 Jul, 23(14), 4826 - 40 Combination of two activating mutations in one HOG1 gene forms hyperactive enzymes that induce growth arrest; Yaakov G et al.; Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis . Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known . These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes . We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation . These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway . Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically . We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties . These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system . This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes . Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176 . Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death . This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect. Mol Cell Biol, 2003 Jul, 23(14), 4814 - 25 Sum1 and Ndt80 proteins compete for binding to middle sporulation element sequences that control meiotic gene expression; Pierce M et al.; A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes . Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE) . Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation . We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs . Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant . A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs . We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound . Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator. Genes Dev, 2003 Jul 15, 17(14), 1768 - 78 Epub 2003 Jun 27. Slx1-Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1-Top3; Fricke WM et al.; The RecQ DNA helicases human BLM and yeast Sgs1 interact with DNA topoisomerase III and are thought to act on stalled replication forks to maintain genome stability . To gain insight into this mechanism, we previously identified SLX1 and SLX4 as genes that are required for viability and for completion of rDNA replication in the absence of SGS1-TOP3 . Here we show that SLX1 and SLX4 encode a heteromeric structure-specific endonuclease . The Slx1-Slx4 nuclease is active on branched DNA substrates, particularly simple-Y, 5'-flap, or replication fork structures . It cleaves the strand bearing the 5' nonhomologous arm at the branch junction and generates ligatable nicked products from 5'-flap or replication fork substrates . Slx1 is the founding member of a family of proteins with a predicted URI nuclease domain and PHD-type zinc finger . This subunit displays weak structure-specific endonuclease activity on its own, is stimulated 500-fold by Slx4, and requires the PHD finger for activity in vitro and in vivo . Both subunits are required in vivo for resistance to DNA damage by methylmethane sulfonate (MMS) . We propose that Sgs1-Top3 acts at the termination of rDNA replication to decatenate stalled forks, and, in its absence, Slx1-Slx4 cleaves these stalled forks. Mol Endocrinol, 2003 Sep, 17(9), 1715 - 25 Epub 2003 Jun 26. Closing the gap: identification of human 3-ketosteroid reductase, the last unknown enzyme of mammalian cholesterol biosynthesis; Marijanovic Z et al.; The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7 . Its ability to synthesize 17beta-estradiol in vitro has been reported previously . However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor . Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium . A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis . Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders . We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway . In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism. Biophys J, 2003 Jul, 85(1), 255 - 66 Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy; Lorenz-Fonfria VA et al.; The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies . When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates . By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process . To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein . From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands . We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features . The functional consequences of this hypothesis are discussed. J Biol Chem, 2003 Sep 12, 278(37), 35421 - 7 Epub 2003 Jun 25. Implications of nectin-like molecule-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 in cell-cell adhesion and transmembrane protein localization in epithelial cells; Shingai T et al.; Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules that play roles in organization of a variety of cell-cell junctions in cooperation with or independently of cadherins . Four nectins have been identified . Five nectin-like molecules, which have domain structures similar to those of nectins, have been identified, and we characterized here nectin-like molecule-2 (Necl-2)/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 . Necl-2 showed Ca2+-independent homophilic cell-cell adhesion activity . It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-1/TSLL1/SynCAM3 and nectin-3 . Necl-2 was widely expressed in rat tissues examined . Necl-2 localized at the basolateral plasma membrane in epithelial cells of the mouse gall bladder, but not at specialized cell-cell junctions, such as tight junctions, adherens junctions, and desmosomes . Nectins bind afadin, whereas Necl-2 did not bind afadin but bound Pals2, a membrane-associated guanylate kinase family member known to bind Lin-7, implicated in the proper localization of the Let-23 protein in Caenorhabditis elegans, the homologue of mammalian epidermal growth factor receptor . These results indicate the unique localization of Necl-2 and its possible involvement in localization of a transmembrane protein(s) through Pals2. Plant Cell Physiol, 2003 Jun, 44(6), 597 - 606 Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas; Kobayashi I et al.; An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene . AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type . By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3 . The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues . This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure . Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake . These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake . Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type. Plant Cell Physiol, 2003 Jun, 44(6), 555 - 64 Arabidopsis TERMINAL FLOWER 2 gene encodes a heterochromatin protein 1 homolog and represses both FLOWERING LOCUS T to regulate flowering time and several floral homeotic genes; Kotake T et al.; Floral transition should be strictly regulated because it is one of the most critical developmental processes in plants . Arabidopsis terminal flower 2 (tfl2) mutants show an early-flowering phenotype that is relatively insensitive to photoperiod, as well as several other pleiotropic phenotypes . We found that the early flowering of tfl2 is caused mainly by ectopic expression of the FLOWERING LOCUS T (FT) gene, a floral pathway integrator . Molecular cloning of TFL2 showed that it encodes a protein with homology to heterochromatin protein 1 (HP1) of animals and Swi6 of fission yeast . TFL2 protein localizes in subnuclear foci and expression of the TFL2 gene complemented yeast swi6(-) mutants . These results suggested that TFL2 might function as an HP1 in Arabidopsis: Gene expression analyses using DNA microarrays, however, did not show an increase in the expression of heterochromatin genes in tfl2 mutants but instead showed the upregulation of the floral homeotic genes APETALA3, PISTILLATA, AGAMOUS and SEPALLATA3 . The pleiotropic phenotype of the tfl2 mutant could reflect the fact that TFL2 represses the expression of multiple genes . Our results demonstrate that despite its homology to HP1, TFL2 is involved in the repression of specific euchromatin genes and not heterochromatin genes in Arabidopsis. Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8571 - 6 Epub 2003 Jun 25. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis; Durfee T et al.; The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens . The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1 . In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development . This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity . However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development . In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants . These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity. Proc Natl Acad Sci U S A, 2003 Jul 8, 100(14), 8424 - 9 Epub 2003 Jun 25. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis; Kato S et al.; Inactivation of the tumor suppressor p53 by missense mutations is the most frequent genetic alteration in human cancers . The common missense mutations in the TP53 gene disrupt the ability of p53 to bind to DNA and consequently to transactivate downstream genes . However, it is still not fully understood how a large number of the remaining mutations affect p53 structure and function . Here, we used a comprehensive site-directed mutagenesis technique and a yeast-based functional assay to construct, express, and evaluate 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and correlated p53 function with structure- and tumor-derived mutations . This high-resolution mutation analysis allows evaluation of previous predictions and hypotheses through interrelation of function, structure and mutation. DNA Repair (Amst), 2003 Jul 16, 2(7), 787 - 94 Rad54, a Jack of all trades in homologous recombination; Tan TL et al.; Homologous recombination mediates the transfer or exchange of genetic information between homologous DNA molecules . It plays important roles in central processes in the cell such as genome duplication and DNA damage repair . Recent experiments reveal the surprising versatility of one of its central actors, the Rad54 protein. Biochem J, 2003 Sep 15, 374(Pt 3), 667 - 75 The transcriptional repressor protein PRH interacts with the proteasome; Bess KL et al.; PRH (proline-rich homeodomain protein)/Hex is important in the control of cell proliferation and differentiation . We have shown previously that PRH contains two domains that can bring about transcriptional repression independently; the PRH homeodomain represses transcription by binding to TATA box sequences, whereas the proline-rich N-terminal domain can repress transcription by interacting with members of the Groucho/TLE (transducin-like enhancer of split) family of co-repressor proteins . The proteasome is a multi-subunit protein complex involved in the processing and degradation of proteins . Some proteasome subunits have been suggested to play a role in the regulation of transcription . In the present study, we show that PRH interacts with the HC8 subunit of the proteasome in the context of both 20 and 26 S proteasomes . Moreover, we show that PRH is associated with the proteasome in haematopoietic cells and that the proline-rich PRH N-terminal domain is responsible for this interaction . Whereas PRH can be cleaved by the proteasome, it does not appear to be degraded rapidly in vitro or in vivo, and the proteolytic activity of the proteasome is not required for transcriptional repression by PRH . However, proteasomal digestion of PRH can liberate truncated PRH proteins that retain the ability to bind to DNA . We discuss these findings in terms of the biological role of PRH in gene regulation and the control of cell proliferation. DNA Seq, 2003 Apr, 14(2), 103 - 7 Isolation of the carbon catabolite repressor (CREA) gene from the plant-pathogenic fungus Cochliobolus carbonum; Tonukari NJ et al.; The CREA gene has been implicated in glucose repression in several fungi . The product of this gene, CreA, binds to the promoter region of several enzymes and down-regulates gene expression . An ortholog of CREA was isolated and characterized from the maize pathogenic fungus, Cochliobolus carbonum . The deduced amino acid sequence of the C . carbonum CREA gene is very similar to the CreA proteins of Aspergillus niger, Gibberella fujikuroi, Sclerotinia sclerotiorum and Trichoderma reesei, as well as the Mig1 protein of Saccharomyces cerevisiae . And like the other fungal proteins, C . carbonum CreA has two zinc finger regions and a nuclear localization signal . Putative CreA binding sequences were also identified in the 5' region of three C . carbonum cell wall degrading enzyme genes suggesting that the protein may play a role in the regulatory process that controls these enzymes expression. DNA Seq, 2003 Apr, 14(2), 79 - 86 Molecular cloning, characterisation and tissue-specific expression of human LAG3, a member of the novel Lag1 protein family; Cai XF et al.; We identified and characterised the cDNA coding for human Lag3, a member of the novel eukaryotic Lag1 protein family which was related to the Saccharomyces cerevisiae longevity-assurance gene LAG1 . The composite nucleotide sequence revealed a coding region of 1140 bp, a 5'-UTR of 210 bp and a 3'-UTR of 900 bp . The deduced sequence of 380 amino acids had six transmembrane regions and the conserved Lag1 motif . Northern blot analysis revealed that the approximately 2.2 kb LAG3Hs transcript was expressed in all 12 human tissues, high in kidney and liver, moderate in brain, heart, skeletal muscle, placenta and lung and low in colon, thymus, spleen, small intestine and peripheral blood leukocyte . Because of its structural similarity to LAG1, it was postulated that LAG3Hs may mediate sphingolipids metabolism. Cancer Sci, 2003 May, 94(5), 421 - 4 Fatty acid-CoA ligase 4 is overexpressed in human hepatocellular carcinoma; Sung YK et al.; Fatty acid-CoA ligase 4 (FACL4) is a central enzyme controlling the unesterified arachidonic acid (AA) level in cells . It has been shown that FACL4 blocks apoptosis and promotes colon carcinogenesis by lowering the cellular level of unesterified AA . Consistent with this, FACL4 is upregulated in colon adenocarcinoma . The status of FACL4 in other tumors including hepatocellular carcinoma (HCC) is not known . Here, we report that FACL4 is overexpressed in human HCC compared with adjacent normal liver tissues . FACL4 mRNA and protein were overexpressed in 5 out of 12 (41.7%) and 3 out of 8 (37.5%) cases of HCC, respectively . Immunohistochemical staining showed strong fine granular intracytoplasmic staining in tumor cells, whereas we observed occasional weak staining in normal liver tissues surrounding the tumors . We found that 14 out of 37 (37.8%) HCC expressed moderate to strong FACL4 immunostaining . Both normal adult and fetal liver tissues showed very weak to no detectable staining, whereas 3 out of 10 (30%) cirrhotic livers expressed weak staining . In addition, we found that 4 out of 8 (50%) human hepatoma cell lines expressed high levels of FACL4 by northern blot analysis . Our results show that FACL4 is a new molecular marker for HCC and suggest that the FACL4 pathway may be involved in liver carcinogenesis. Nucleic Acids Res, 2003 Jul 1, 31(13), 3586 - 8 YMF: A program for discovery of novel transcription factor binding sites by statistical overrepresentation; Sinha S et al.; A fundamental challenge facing biologists is to identify DNA binding sites for unknown regulatory factors, given a collection of genes believed to be coregulated . The program YMF identifies good candidates for such binding sites by searching for statistically overrepresented motifs . More specifically, YMF enumerates all motifs in the search space and is guaranteed to produce those motifs with greatest z-scores . This note describes the YMF web software, available at http://bio.cs.washington.edu/software.html. Nucleic Acids Res, 2003 Jul 1, 31(13), 3572 - 5 SiteSeer: Visualisation and analysis of transcription factor binding sites in nucleotide sequences; Boardman PE et al.; The regulation of gene expression is a fundamental process within every living cell, which allows organisms to manage the precise levels of functional gene products with high sensitivity . It is well established that specific DNA sequences located upstream of the transcriptional start site are important in facilitating the binding of regulatory proteins that control the transcription of the gene . Indeed, microarray-based studies have successfully mined the upstream regions of co-expressed genes and discovered over-represented sequences corresponding to known promoter sites . Here we describe a tool for the visualisation of mapped transcription factor binding sites in the upstream regions of either single or grouped eukaryotic genes, which allows users to examine the positions of known and user-defined sites . SiteSeer allows the user to map different sections of the TRANSFAC and SCPD databases (or a set of user-defined sites) onto nucleotide sequences . Additionally, users may restrict the analysis by expectation values for certain DNA words as well as by known binding sites specific to a given organism . We believe this tool will prove particularly valuable for biologists who wish to examine sets of co-expressed or functionally-related genes and those who wish to visualise the positions of promoter sequences and generate displays for publications. Nucleic Acids Res, 2003 Jul 1, 31(13), 3491 - 6 Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays; Nielsen HB et al.; Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters . The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer with an overview of these parameters . We present here a flexible tool named OligoWiz for designing oligonucleotides for multiple purposes . OligoWiz presents a set of parameter scores in a graphical interface to facilitate an overview for the user . Additional custom parameter scores can easily be added to the program to extend the default parameters: homology, DeltaTm, low-complexity, position and GATC-only . Furthermore we present an analysis of the limitations in designing oligonucleotide sets that can detect transcripts from multiple organisms . OligoWiz is available at www.cbs.dtu.dk/services/OligoWiz/. Nucleic Acids Res, 2003 Jul 1, 31(13), 3487 - 90 REDUCE: An online tool for inferring cis-regulatory elements and transcriptional module activities from microarray data; Roven C et al.; REDUCE is a motif-based regression method for microarray analysis . The only required inputs are (i) a single genome-wide set of absolute or relative mRNA abundances and (ii) the DNA sequence of the regulatory region associated with each gene that is probed . Currently supported organisms are yeast, worm and fly; it is an open question whether in its current incarnation our approach can be used for mouse or human . REDUCE uses unbiased statistics to identify oligonucleotide motifs whose occurrence in the regulatory region of a gene correlates with the level of mRNA expression . Regression analysis is used to infer the activity of the transcriptional module associated with each motif . REDUCE is available online at This web site provides functionality for the upload and management of microarray data . REDUCE analysis results can be viewed and downloaded, and optionally be shared with other users or made publicly accessible. J Biol Chem, 2003 Sep 12, 278(37), 34943 - 51 Epub 2003 Jun 23. The roles of intersubunit interactions in exosome stability; Estevez AM et al.; In eukaryotes, at least 10 proteins associate in a 3'-5' exonuclease complex, the exosome, which is involved in the processing of many RNA species . A recent model for the exosome placed six RNase PH-related components in a hexameric ring core structure, with three S1 domain proteins associated with the ring surface . So far, however, this model lacks experimental support . Using a combination of RNA interference, complex affinity purification, and yeast two-hybrid approaches, we show here that the RNase PH homologues are important for maintenance of complex integrity . In contrast, the S1 domain proteins are not required for complex stability, although they are required for exosome function . Our results are partially consistent with the proposed model of the exosome, but indicate a different arrangement of the RNase PH proteins. Antimicrob Agents Chemother, 2003 Jul, 47(7), 2273 - 82 Molecular basis for fungal selectivity of novel antimitotic compounds; Lila T et al.; Compounds that selectively disrupt fungal mitosis have proven to be effective in controlling agricultural pests, but no specific mitotic inhibitor is available for the treatment of systemic mycoses in mammalian hosts . In an effort to identify novel mitotic inhibitors, we used a cell-based screening strategy that exploited the hypersensitivity of a yeast alpha-tubulin mutant strain to growth inhibition by antimitotic agents . The compounds identified inhibited yeast nuclear division and included one structural class of compounds shown to be fungus specific . MC-305904 and structural analogs inhibited fungal cell mitosis and inhibited the in vitro polymerization of fungal tubulin but did not block mammalian cell microtubule function or mammalian tubulin polymerization . Extensive analysis of yeast mutations that specifically alter sensitivity to MC-305904 structural analogs suggested that compounds in the series bind to a site on fungal beta-tubulin near amino acid 198 . Features of the proposed binding site explain the observed fungal tubulin specificity of the series and are consistent with structure-activity relationships among a library of related compounds. Mol Cell, 2003 Jun, 11(6), 1599 - 607 Histones are first hyperacetylated and then lose contact with the activated PHO5 promoter; Reinke H et al.; We have analyzed the histone modification status of the PHO5 promoter from yeast by the ChIP technology and have focused on changes occurring upon activation . Using various acetylation-specific antibodies, we found a dramatic loss of the acetylation signal upon induction of the promoter . This turned out to be due, however, to the progressive loss of histones altogether . The fully remodeled promoter appears to be devoid of histones as judged by ChIP analyses . Local histone hyperacetylation does indeed occur, however, prior to remodeling . This can explain the delay in chromatin remodeling in the absence of histone acetyltransferase activity of the SAGA complex that was previously documented for the PHO5 promoter . Our findings shed new light on the nucleosomal structure of fully remodeled chromatin . At the same time, they point out the need for novel controls when the ChIP technique is used to study histone modifications in the context of chromatin remodeling in vivo. Mol Cell, 2003 Jun, 11(6), 1587 - 98 Nucleosomes unfold completely at a transcriptionally active promoter; Boeger H et al.; It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation . Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes . We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae . Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation. Mol Cell, 2003 Jun, 11(6), 1435 - 44 Context of multiubiquitin chain attachment influences the rate of Sic1 degradation; Petroski MD et al.; The ubiquitin-dependent targeting of proteins to the proteasome is an essential mechanism for regulating eukaryotic protein stability . Here we define the minimal signal for the degradation of the S phase CDK inhibitor Sic1 . Of 20 lysines scattered throughout Sic1, 6 N-terminal lysines serve as major ubiquitination sites . Sic1 lacking these lysines (K0N) is stable in vivo, but readdition of any one restores turnover . Nevertheless, ubiquitin chains attached at different N-terminal lysines specify degradation in vitro at markedly different rates . Moreover, although K0N can be ubiquitinated by SCF(Cdc4)/Cdc34 in vitro in the absence (but not in the presence) of S-CDK, it is degraded slowly . Our results reveal that a single multiubiquitin chain can sustain a physiological turnover rate, but that chain position plays an unexpectedly significant role in the rate of proteasomal proteolysis. Mol Cell, 2003 Jun, 11(6), 1421 - 3 A new link for a linker histone; Conconi A et al.; Classically, the functions of linker histones (histones H1 and variants) have been related mainly to chromatin organization and the ensuing consequences on transcription . Remarkably, yeast histone H1 may not comply, as it appears to regulate homologous recombination specifically. Biochemistry, 2003 Jul 1, 42(25), 7855 - 62 Functional analysis of ISC1 by site-directed mutagenesis; Okamoto Y et al.; We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p {Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y . A . (2000) J . Biol . Chem . 275, 39793-39798} . The present study was conducted to determine specific domains in Isc1p required for catalysis . Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1 . Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues . Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins . In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain" . G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis . D163A and K168A lost activity completely . Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity . The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p . Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding . Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain . This study provides novel insight into a new domain found in Isc1p and related enzymes. Nat Genet, 2003 Jul, 34(3), 330 - 6 Downregulation of FUSE-binding protein and c-myc by tRNA synthetase cofactor p38 is required for lung cell differentiation; Kim MJ et al.; p38 is associated with a macromolecular tRNA synthetase complex . It has an essential role as a scaffold for the complex, and genetic disruption of p38 in mice causes neonatal lethality . Here we investigated the molecular mechanisms underlying lethality of p38-mutant mice . p38-deficient mice showed defects in lung differentiation and respiratory distress syndrome . p38 was found to interact with FUSE-binding protein (FBP), a transcriptional activator of c-myc . Binding of p38 stimulated ubiquitination and degradation of FBP, leading to downregulation of c-myc, which is required for differentiation of functional alveolar type II cells . Transforming growth factor-beta (TGF-beta) induced p38 expression and promoted its translocation to nuclei for the regulation of FBP and c-myc . Thus, this work identified a new activity of p38 as a mediator of TGF-beta signaling and its functional importance in the control of c-myc during lung differentiation. J Vet Sci, 2002 Dec, 3(4), 273 - 7 Requirement of metabolic activation for estrogenic activity of Pueraria mirifica; Lee YS et al.; A wide range of chemicals derived from plant and human-made xenobiotics are reported to have hormonal activities . The present study was performed to examine the estrogenic effect of Kwao Keur, Pueraria mirifica (PM), that has been used as a rejuvenating folk medicine in Thailand, using recombinant yeast, MCF-7 cell proliferation and HepG2 cell transient transfection assay . In recombinant yeast assay, 0.025, 0.25, 2.5, 25, 2.5 x 10(2), 2.5 x 10(3), 2.5 x 10(4) ng/ml concentrations of PM did not show any estrogenic activities, while 10(-9) of 17 beta-estradiol (positive control) showed high estrogenic activity . Estrogenic activities were induced at 2.5 ng/ml to 25 microg/ml concentrations of PM in a dose-dependent manner on MCF-7 cells and the estrogenic effect of PM was blocked by tamoxifen treatment, a well-known anti-estrogen . PM also showed estrogenic effect on human hepatoma cell line, HepG2 cells, containing estrogen receptor and luciferase reporter gene . Taken together, PM in itself may have no estrogenicity in yeast system, but it has estrogenicity in MCF-7 & HepG2 cells that have human metabolic enzymes . The results indicated that PM may require metabolic activation for estrogenic activity. Genome Res, 2003 Jun, 13(6B), 1416 - 29 Identification and analysis of chromodomain-containing proteins encoded in the mouse transcriptome; Tajul-Arifin K et al.; The chromodomain is 40-50 amino acids in length and is conserved in a wide range of chromatic and regulatory proteins involved in chromatin remodeling . Chromodomain-containing proteins can be classified into families based on their broader characteristics, in particular the presence of other types of domains, and which correlate with different subclasses of the chromodomains themselves . Hidden Markov model (HMM)-generated profiles of different subclasses of chromodomains were used here to identify sequences encoding chromodomain-containing proteins in the mouse transcriptome and genome . A total of 36 different loci encoding proteins containing chromodomains, including 17 novel loci, were identified . Six of these loci (including three apparent pseudogenes, a novel HP1 ortholog, and two novel Msl-3 transcription factor-like proteins) are not present in the human genome, whereas the human genome contains four loci (two CDY orthologs and two apparent CDY pseudogenes) that are not present in mouse . A number of these loci exhibit alternative splicing to produce different isoforms, including 43 novel variants, some of which lack the chromodomain . The likely functions of these proteins are discussed in relation to the known functions of other chromodomain-containing proteins within the same family. Genome Res, 2003 Jun, 13(6B), 1389 - 94 The comparative proteomics of ubiquitination in mouse; Semple CA; RIKEN GER Group; GSL Members; Ubiquitination is a common posttranslational modification in eukaryotic cells, influencing many fundamental cellular processes . Defects in ubiquitination and the processes it mediates are involved in many human disease states . The ubiquitination of a substrate involves four classes of enzymes:a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin protein ligase (E3), and a de-ubiquitinating enzyme (DUB) . A substantial number of E1s (four), E2s (13), E3s (97), and DUBs (six) that were previously unknown in the mouse are included in the FANTOM2 Representative Transcript and Protein Set (RTPS) . Many of the genes encoding these proteins will constitute promising candidates for involvement in disease . In addition, the RTPS provides the basis for the most comprehensive survey of ubiquitination-associated proteins across eukaryotes undertaken to date . Comparisons of these proteins across human and other organisms suggest that eukaryotic evolution has been associated with an increase in the number and diversity of E3s (possessing either zinc-finger RING, F-box, or HECT domains) and DUBs (containing the ubiquitin thiolesterase family 2 domain) . These increases in numbers are too large to be accounted for by the presence of fragmentary proteins in the data sets examined . Much of this innovation appears to have been associated with the emergence of multicellular organisms, and subsequently of vertebrates, increasing the opportunity for complex regulation of ubiquitination-mediated cellular and developmental processes. Genome Res, 2003 Jun, 13(6B), 1335 - 44 Mouse proteome analysis; Kanapin A et al.; A general overview of the protein sequence set for the mouse transcriptome produced during the FANTOM2 sequencing project is presented here . We applied different algorithms to characterize protein sequences derived from a nonredundant representative protein set (RPS) and a variant protein set (VPS) of the mouse transcriptome . The functional characterization and assignment of Gene Ontology terms was done by analysis of the proteome using InterPro . The Superfamily database analyses gave a detailed structural classification according to SCOP and provide additional evidence for the functional characterization of the proteome data . The MDS database analysis revealed new domains which are not presented in existing protein domain databases . Thus the transcriptome gives us a unique source of data for the detection of new functional groups . The data obtained for the RPS and VPS sets facilitated the comparison of different patterns of protein expression . A comparison of other existing mouse and human protein sequence sets (e.g., the International Protein Index) demonstrates the common patterns in mammalian proteomes . The analysis of the membrane organization within the transcriptome of multiple eukaryotes provides valuable statistics about the distribution of secretory and transmembrane proteins Adv Colloid Interface Sci, 2003 Jul 1, 104, 159 - 73 Field-induced disturbance of the double layer electro-neutrality and non-linear electrophoresis; Shilov V et al.; The influence of applied electric field on the field-induced variation of the electrolyte concentration (concentration polarization) disturbs the electro-neutrality of the system, represented by a dispersed particle and its electric double layer in electrolyte solution . The manifestation of this electro-neutrality disturbance in the non-linear electrophoresis was considered in the framework of a procedure of successive approximations in powers of the applied field strength . Analytic expressions describing the component of the electrophoretic velocity proportional to the cubic power of the applied field strength (cubic electrophoresis) were obtained for arbitrary values of the surface conductivity (of Dukhin number) . The model restrictions are spherical non-conducting particle with homogeneous surface and thin double layer. J Mol Biol, 2003 Jun 27, 330(1), 129 - 35 Conformational flexibility of pyruvate dehydrogenase complexes: a computational analysis by quantized elastic deformational model; Kong Y et al.; Pyruvate dehydrogenase complex (PDC) is one of the largest multienzyme complexes known and consists of a dodecahedral E2 core to which other components are attached . We report the results of applying a new computational method, quantized elastic deformational model, to simulating the conformational fluctuations of the truncated E2 core, using low-resolution electron cryomicroscopy density maps . The motional features are well reproduced; especially, the symmetric breathing mode revealed in simulation is nearly identical with what was observed experimentally . Structural details of the motions of the trimeric building blocks, which are critical to facilitating the global expansion and contraction of the complex, were revealed . Using the low-resolution maps from electron cryomicroscopy reconstructions, the simulations showed a picture of the motional mechanism of the PDC core, which is an example without precedent of thermally activated global dynamics . Moreover, the current results support an earlier suggestion that, at low resolution and without the use of amino acid sequence and atomic coordinates, it is possible for computer simulations to provide an accurate description of protein dynamics. Neuron, 2003 Jun 19, 38(6), 871 - 85 Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system; Watts RJ et al.; Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases . However, little is known about the cellular and molecular mechanisms of axon pruning . We use the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms . Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning . In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning . Our findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury. Isr J Psychiatry Relat Sci, 2003, 40(1), 57 - 66 Attention deficit and hyperactivity disorder: review of genetic association studies; Galili-Weisstub E et al.; Attention Deficit and Hyperactivity Disorder (ADHD) is a common idiopathic childhood neurodevelopmental disorder, exacting a significant clinical and public health toll . It impairs schooling and social adaptation, resulting in high rates of depression, conduct disorder, school dropouts, and substance abuse, and necessitating exposure of many children to prolonged courses of stimulant psychotropic medication . Although the biological basis of ADHD is unknown, it has been shown to possess considerable heritability . Candidate gene association studies proved to be a productive strategy leading to replicated association findings of genetic loci contributing to susceptibility to ADHD . Based on the mechanism of action of stimulant drugs effective in the alleviation of ADHD symptoms, current association studies have focused mainly on dopaminergic genes . Promising exploratory findings have also been reported for genes affecting other neurotransmitter systems . The current article reviews the rationale, methodology, and main findings in the field, and outlines future directions . Locating the actual genes mediating ADHD susceptibility will have far reaching implications for understanding the pathophysiology of ADHD as well as for understanding mechanisms of therapeutic drug action and genetic determinants of response. Biol Chem, 2003 May, 384(5), 805 - 9 Contribution of the absolutely conserved B8Gly to the foldability of insulin; Guo ZY et al.; B8Gly is absolutely conserved in insulin from different species, and in other members of the insulin superfamily the corresponding position is always occupied by a Gly residue . However, the reasons for its conservation are still unclear; probably many factors contribute to this phenomenon . In our previous work, B8Gly was replaced by an Ala residue, which suggested that biological activity is one of the factors contributing to its conservation . In order to identify more factors contributing to this positional conservation, the secretion efficiency, structural stability, disulfide stability, and in vitro refolding of single-chain insulin (PIP) and a mutant with B8Gly replaced by Ala, were investigated . Compared with wild-type PIP, the B8Ala replacement decreased the secretion efficiency, structural stability, disulfide stability, and in vitro refolding efficiency of the PIP sequence . These results suggest that B8Gly is important to the secretion, folding, and stability of the insulin sequence. Biol Chem, 2003 May, 384(5), 791 - 803 Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms; Hundt C et al.; Recently, crystallization of the prion protein in a dimeric form was reported . Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric . The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP . The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains . Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system . In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions . Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases. Genes Dev, 2003 Jun 15, 17(12), 1463 - 8 Aberrant histone acetylation, altered transcription, and retinal degeneration in a Drosophila model of polyglutamine disease are rescued by CREB-binding protein; Taylor JP et al.; Sequestration of the transcriptional coactivator CREB-binding protein (CBP), a histone acetyltransferase, has been implicated in the pathogenesis of polyglutamine expansion neurodegenerative disease . We used a Drosophila model to demonstrate that polyglutamine-induced neurodegeneration is accompanied by a defect in histone acetylation and a substantial alteration in the transcription profile . Furthermore, we demonstrate complete functional and morphological rescue by up-regulation of endogenous Drosophila CBP (dCBP) . Rescue of the degenerative phenotype is associated with eradication of polyglutamine aggregates, recovery of histone acetylation, and normalization of the transcription profile . These findings suggest that histone acetylation is an early target of polyglutamine toxicity and indicate that transcriptional dysregulation is an important part of the pathogenesis of polyglutamine-induced neurodegeneration. J Biol Chem, 2003 Sep 19, 278(38), 35869 - 77 Epub 2003 Jun 18. The caspase-like sites of proteasomes, their substrate specificity, new inhibitors and substrates, and allosteric interactions with the trypsin-like sites; Kisselev AF et al.; Proteasomes are the primary sites for protein degradation in mammalian cells . Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites . Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites . We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites . Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates . Highly selective inhibitors of the caspase-like sites were also generated . They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide . Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites . Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity . Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site. J Biol Chem, 2003 Sep 19, 278(38), 35979 - 87 Epub 2003 Jun 17. M phase-specific phosphorylation of BRCA2 by Polo-like kinase 1 correlates with the dissociation of the BRCA2-P/CAF complex; Lin HR et al.; BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression . Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51 . In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF) . To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined . We found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase . This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents . Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain "phosphorylation consensus motifs" for Polo-like kinase 1 (Plk1) . Phosphorylation of BR-N1 with Plk1 recapitulated the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells . Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolished Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1 . Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation . Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression. J Biol Chem, 2003 Sep 5, 278(36), 34253 - 8 Epub 2003 Jun 18. Regulation of transforming growth factor-beta signaling by protein inhibitor of activated STAT, PIASy through Smad3; Imoto S et al.; Smads proteins play a key role in the intracellular signaling of the transforming growth factor (TGF)-beta family of growth factors, which exhibits a diverse set of cellular responses, including cell proliferation and differentiation . In particular, Smad7 acts as an antagonist of TGF-beta signaling, which could determine the intensity or duration of its signaling cascade . In this study we identified a protein inhibitor of activated STAT (signal transducers and activators of transcription), PIASy, as a novel interaction partner of Smad7 by yeast two-hybrid screening using the MH2 domain of Smad7 as bait . The association of Smad7 and PIASy was confirmed using co-expressed tagged proteins in 293T cells . Moreover, we found that other Smads including Smad3 also associated with PIASy through its MH2 domain, and PIASy suppressed TGF-beta-mediated activation of Smad3 . PIASy also stimulated the sumoylation of Smad3 in vivo . Furthermore, endogenous PIASy expression was induced by TGF-beta in Hep3B cells . These findings provide the first evidence that a PIAS family protein, PIASy, associates with Smads and involves the regulation of TGF-beta signaling using the negative feedback loop. J Proteome Res, 2003 May-Jun, 2(3), 303 - 11 Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins; Pedersen SK et al.; Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects . In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span . By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins . More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level. J Proteome Res, 2003 May-Jun, 2(3), 249 - 54 Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies; Chen J et al.; Capillary isoelectric focusing (CIEF) involves the use of the entire capillary filled with a mixture containing protein/peptide analytes and carrier ampholytes . Thus, the preparative capabilities of CIEF are inherently greater than most capillary-based electrokinetic separation techniques . To further increase sample loading and, therefore, the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from a solution reservoir, is demonstrated using a low p/ protein calibration kit and tryptic peptides from Saccharomyces cerevisiae . The proteins/peptides continuously migrate into the capillary and encounter a pH gradient established by carrier ampholytes originally present in the capillary for focusing and separation . Dynamic introduction and focusing in CIEF can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength . Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility . Depending on the mobilities of yeast peptides, the loading capacity of each peptide is measured to be around 8 to 45-fold of that obtained in conventional CIEF . By comparing with the concentrations of dilute yeast peptides originally present in the reservoir, an overall concentration factor of 1400-7700 together with excellent separation resolution is achieved using dynamic introduction and focusing . This concentration effect is further illustrated by detecting 10 pg/microL of bradykinin peptide spiked in yeast protein digest using only ultraviolet absorption. Oncogene, 2003 Jun 19, 22(25), 3943 - 51 Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells; Kim TY et al.; DLC-1 (deleted in liver cancer) gene is frequently deleted in hepatocellular carcinoma . However, little is known about the genetic status and the expression of this gene in gastric cancer . In this study, Northern and Southern analysis showed that seven of nine human gastric cancer cell lines did not express DLC-1 mRNA, but contained the DLC-1 gene . To identify the mechanism of the loss of DLC-1 mRNA expression in these cell lines, we investigated the methylation status of DLC-1 gene by using methylation-specific PCR (MSP) and Southern blot, and found that five of seven DLC-1 nonexpressing gastric cancer cell lines were methylated in the DLC-1 CpG island . Treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) induced DLC-1 mRNA expression in the gastric cancer cell lines that have the methylated alleles . Studies using SNU-601 cell line with methylated DLC-1 alleles revealed that nearly all CpG sites within DLC-1 CpG island were methylated, and that the in vitro methylation of the DLC-1 promoter region is enough to repress DLC-1 mRNA expression, regardless of the presence of transcription factors capable of inducing this gene . In all, 29 of 97 (30%) primary gastric cancers were also shown to be methylated, demonstrating that methylation of the DLC-1 CpG island is not uncommon in gastric cancer . In addition, we demonstrated that DLC-1 mRNA expression was induced, and an increase in the level of acetylated H3 and H4 was detected by the treatment with trichostatin A (TSA) in two DLC-1 nonexpressing cell lines that have the unmethylated alleles . Taken together, the results of our study suggest that the transcriptional silencing of DLC-1, by epigenetic mechanism, may be involved in gastric carcinogenesis. Oncogene, 2003 Jun 19, 22(25), 3853 - 8 Inhibition of histone deacetylase activity increases chromosomal instability by the aberrant regulation of mitotic checkpoint activation; Shin HJ et al.; Histone modification through acetylation and deacetylation is a key process in transcription, DNA replication, and chromosome segregation . During mitosis, histones are highly acetylated and chromatin is condensed . Here, we investigate the mechanistic involvement of histone deacetylase (HDAC) activity in the regulation of mitotic checkpoint activation . Inhibition of HDAC activity was found to cause the improper kinetochore localization of the mitotic checkpoint proteins, and to prolong mitotic arrest, and thus to lead to chromosomal instability due to aberrant exit from the mitotic cell cycle arrest . In addition, treatment with HDAC inhibitor attenuated the activations of p38 and ERK kinases, and increased the expression levels of cIAP-1, suggesting that the observed increased adaptation and chromosomal instability induced by inhibiting HDAC activity might be directly connected with the activations of cell survival and/or antiapoptotic signals . Moreover, the treatment of cells with mitotic defects with HDAC inhibitor increased their susceptibility to chromosomal instability . These results support the notion that HDAC activity plays an important role in the regulation of mitotic checkpoint activation, and thus the aberrant control of HDAC activity contributes to chromosomal instability. Oncogene, 2003 Jun 19, 22(25), 3833 - 41 Interaction of the HPV E7 proteins with the pCAF acetyltransferase; Avvakumov N et al.; Most cervical carcinomas express the E6 and E7 proteins of a high-risk human papillomavirus (HPV) . These proteins affect growth control by interfering with the functions of cell regulatory proteins, promoting oncogenic transformation . A key target of E7 is the tumor suppressor protein pRb, which directly interacts with E7 . However, binding to additional cellular regulatory proteins is clearly required for oncogenesis, as mutants of E7 have been identified that bind to pRb, yet fail to transform efficiently . Here we demonstrate the interaction of the HPV 6, 16 and 18 E7 proteins with the pCAF acetyltransferase, which has been reported to function as a coactivator for a variety of transcription factors including p53 . Mutation of a highly conserved leucine residue within the zinc finger region of HPV 16 E7 disrupts binding to pCAF and also impairs transformation and transcriptional activation . HPV 16 E7 interacts with the acetyltransferase domain of pCAF, and reduces its acetyltransferase activity in vitro . Our analysis of the interaction between the pCAF acetyltransferase and E7 provides new insight into the mechanisms by which the E7 oncoproteins can alter cellular gene expression and growth. J Biol Chem, 2003 Sep 5, 278(36), 34568 - 81 Epub 2003 Jun 17. Interaction codes within the family of mammalian Phox and Bem1p domain-containing proteins; Lamark T et al.; The Phox and Bem1p (PB1) domain constitutes a recently recognized protein-protein interaction domain found in the atypical protein kinase C (aPKC) isoenzymes, lambda/iota- and zeta PKC; members of mitogen-activated protein kinase (MAPK) modules like MEK5, MEKK2, and MEKK3; and in several scaffold proteins involved in cellular signaling . Among the last group, p62 and Par6 (partitioning-defective 6) are involved in coupling the aPKCs to signaling pathways involved in cell survival, growth control, and cell polarity . By mutation analyses and molecular modeling, we have identified critical residues at the interaction surfaces of the PB1 domains of aPKCs and p62 . A basic charge cluster interacts with an acidic loop and helix both in p62 oligomerization and in the aPKC-p62 interaction . Subsequently, we determined the abilities of mammalian PB1 domain proteins to form heteromeric and homomeric complexes mediated by this domain . We report several novel interactions within this family . An interaction between the cell polarity scaffold protein Par6 and MEK5 was found . Furthermore, p62 interacts both with MEK5 and NBR1 in addition to the aPKCs . Evidence for involvement of p62 in MEK5-ERK5 signaling is presented. J Biol Chem, 2003 Aug 22, 278(34), 32390 - 6 Epub 2003 Jun 16. A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1; Wang W et al.; Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway . Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1 . The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure . Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27 . Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1 . Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination . A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction . Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27. J Biol Chem, 2003 Sep 5, 278(36), 33887 - 95 Epub 2003 Jun 17. The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways; Rodriguez-Hernandez CJ et al.; The immunosuppressant Tacrolimus (FK506) has increased the survival rates of organ transplantation . FK506 exerts its immunosuppressive effect by inhibition of the protein phosphatase calcineurin in activated T-cells . Unfortunately, FK506 therapy is associated with undesired non-therapeutic effects involving targets other than calcineurin . To identify these targets we have addressed FK506 cellular toxicity in budding yeast . We show that FK506 increased cell sensitivity upon osmotic challenge independently of calcineurin and the FK506-binding proteins Fpr1p, -2p, -3p, and -4p . FK506 also induced strong amino acid starvation and activation of the general control (GCN) pathway . Tryptophan prototrophy or excess tryptophan overcame FK506 toxicity, showing that tryptophan deprivation mediated this effect . Mutation of the GCN3 and -4 genes partially alleviated FK506 toxicity, suggesting that activation of the GCN pathway by FK506 was also involved in osmotic tolerance . FK506 enhanced osmotic stress-dependent Hog1p kinase phosphorylation that was not accompanied by induction of a Hog1p-dependent reporter . Interestingly, deletion of the GCN2 gene suppressed FK506-dependent Hog1p hyperphosphorylation and restored Hog1p-dependent reporter activity . Conversely, deletion of the HOG1 gene impaired FK506-dependent activation of Gcn2p kinase and translation of a GCN4-LacZ reporter, highlighting functional cross-talk between the Gcn2p and Hog1p protein kinases . Taken together, these data demonstrate that both FK506-induced amino acid starvation and activation of the GCN pathway contribute to cell sensitivity to osmotic stress and reveal a positive regulatory loop between the Hog1p and Gcn2p pathways . Given the conserved nature of Gcn2p and Hog1p pathways, this mechanism of FK506 toxicity could be relevant to the non-therapeutic effects of FK506 therapy. Yi Chuan Xue Bao, 2003 Mar, 30(3), 283 - 8 Isolation and characterization of a carboxylic transport protein JEN1 and its promoter from Metarhizium anisopliae; Fang WG et al.; Based on the flanking sequence of T-DNA of a T-DNA insertion mutant of Beauveria bassiana, T12, the full length cDNA of carboxylic transport protein, designated MaJen1, was cloned from Metarhizium anisopliae . MaJen1 is 1,695 bp long and contained a 1,524 bp ORF which predicted a protein of 508 amino acid . The amino acid sequence of the gene showed 69% and 31% identity to the carboxylic transport protein of Neurospore crassa and Saccharomyces cerevisiae, respectively . The genome sequence, GMaJen1, was amplified by PCR, indicating that there were two introns in GMaJen1 . Southern analysis indicated that GMaJen1 was present as a singl copy in Metarhizium anisopliae . The result of RT-PCR showed that expression of MaJen1 was induced by the cuticle of cockroach and repressed by glucose . A 1,626 bp upstream sequence of GMaJen1 was amplified by YADE method, which contained several putative binding domains of glucose repressor. J Cell Physiol, 2003 Aug, 196(2), 251 - 7 The Sp1 transcription factor does not directly interact with the HIV-1 Tat protein; Loregian A et al.; The role of Sp1 in regulating the trans-activating activity of the human immunodeficiency virus type 1 (HIV-1) Tat protein has not yet been clearly defined . In fact, studies on the physical and functional interaction between Sp1 and Tat have yielded contradictory results . Here we investigated whether a physical interaction between Sp1 and Tat indeed occurs, exploiting both biochemical and genetic techniques that allow detection of direct protein-protein interactions . Studies performed with the yeast two-hybrid system indicate that Sp1 does not directly interact with the HIV-1 Tat protein . Control experiments demonstrated that both proteins are functionally expressed in the yeast cells . In vitro binding assays further confirmed that Sp1 does not physically bind Tat . These data suggest that in vivo Tat and Sp1 most likely take part of a multicomponent complex and thus encourage the search of the molecule(s) which mediate Tat-Sp1 interaction . Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7812 - 7 Epub 2003 Jun 16. Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells; Szondy Z et al.; Tissue transglutaminase (TGase2) is a protein-crosslinking enzyme known to be associated with the in vivo apoptosis program . Here we report that apoptosis could be induced in TGase2-/- mice; however, the clearance of apoptotic cells was defective during the involution of thymus elicited by dexamethasone, anti-CD3 antibody, or gamma-irradiation, and in the liver after induced hyperplasia . The lack of TGase2 prevented the production of active transforming growth factor-beta1 in macrophages exposed to apoptotic cells, which is required for the up-regulation of TGase2 in the thymus in vivo, for accelerating deletion of CD4+CD8+ cells and for efficient phagocytosis of apoptotic bodies . The deficiency is associated with the development of splenomegaly, autoantibodies, and immune complex glomerulonephritis in TGase2-/- mice . These findings have broad implications not only for diseases linked to inflammation and autoimmunity but also for understanding the interrelationship between the apoptosis and phagocytosis process. RNA, 2003 Jul, 9(7), 821 - 38 A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA; Marmier-Gourrier N et al.; The 15.5-kD protein and its yeast homolog Snu13p bind U4 snRNA, U3 snoRNA, and the C/D box snoRNAs . In U4 snRNA, they associate with a helix-bulge-helix (K-turn) structure . U3 snoRNA contains two conserved pairs of boxes, C'/D and B/C, which were both expected to bind the 15.5-kD/Snu13 protein . Only binding to the B/C motif was experimentally demonstrated . Here, by chemical probing of in vitro reconstituted RNA/protein complexes, we demonstrate the independent binding of the 15.5-kD/Snu13 protein to each of the two motifs . Due to a highly reduced stem I (1 bp), the K-turn structure is not formed in the naked B/C motif . However, gel-shift experiments revealed a higher affinity of Snu13p for the B/C motif, compared to the C'/D motif . A phylogenetic analysis of U3 snoRNA, coupled with an analysis of Snu13p affinity for variant yeast C'/D and B/C motifs, and a study of the functionality of a truncated yeast U3 snoRNA carrying base substitutions in the C'/D and B/C motifs, revealed that conservation of the identities of residues 2 and 3 in the B/C K-turn is more important for Snu13p binding and U3 snoRNA function, than conservation of the identities of corresponding residues in the C'/D K-turn . This suggests that binding of Snu13p to K-turns with a very short helix I imposes sequence constraints in the bulge . Altogether, the data demonstrate the strong importance of the binding of the 15.5-kD/Snu13 protein to the C'/D and B/C motifs for both U3 snoRNP assembly and activity. J Biol Chem, 2003 Aug 29, 278(35), 33351 - 63 Epub 2003 Jun 16. Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2; Bolger GB et al.; The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay . The interaction was confirmed in biochemical pull-down analyses . The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52 . XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies . Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-{3-(cyclopentyloxy)-4-methoxyphenyl}-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells . XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm . Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram . Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain . The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram . Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity . Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2 . We suggest that XAP2 functionally interacts with PDE4A5 in cells. Curr Opin Pharmacol, 2003 Jun, 3(3), 300 - 8 Role of transcription factors in skeletal muscle and the potential for pharmacological manipulation; Martin PT; Our understanding of the role of transcription factors in skeletal muscle vastly exceeds our ability to manipulate this class of proteins for therapeutic benefit . Transcription factors responsible for controlling the fate, growth, migration, proliferation, differentiation and regeneration of muscle cells have been identified, and additional factors involved in these processes continue to be discovered . These factors are often involved in multiple steps in muscle differentiation and can have redundant activities . As such, a detailed understanding of their intermolecular interactions and the gene programs they control is essential to the rational design of therapeutics . Mutations in transcription factors cause a number of muscle disorders . Moreover, the manipulation of transcriptional signals holds the promise of treating muscle diseases by exploiting the ability of muscle cells to regenerate after injury . Finally, several proteins have recently been shown to inhibit muscular dystrophy in mouse models . Because some of these proteins are enriched at the neuromuscular synapse, the manipulation of factors governing synaptic transcription is a promising new approach to the treatment of muscular dystrophy. Cell, 2003 Jun 13, 113(6), 777 - 87 FY is an RNA 3' end-processing factor that interacts with FCA to control the Arabidopsis floral transition; Simpson GG et al.; The nuclear RNA binding protein, FCA, promotes Arabidopsis reproductive development . FCA contains a WW protein interaction domain that is essential for FCA function . We have identified FY as a protein partner for this domain . FY belongs to a highly conserved group of eukaryotic proteins represented in Saccharomyces cerevisiae by the RNA 3' end-processing factor, Pfs2p . FY regulates RNA 3' end processing in Arabidopsis as evidenced through its role in FCA regulation . FCA expression is autoregulated through the use of different polyadenylation sites within the FCA pre-mRNA, and the FCA/FY interaction is required for efficient selection of the promoter-proximal polyadenylation site . The FCA/FY interaction is also required for the downregulation of the floral repressor FLC . We propose that FCA controls 3' end formation of specific transcripts and that in higher eukaryotes, proteins homologous to FY may have evolved as sites of association for regulators of RNA 3' end processing. Biochem J, 2003 Sep 1, 374(Pt 2), 497 - 503 Association of human kinesin superfamily protein member 4 with BRCA2-associated factor 35; Lee YM et al.; A large portion of human kinesin superfamily protein member 4 (KIF4) is associated with the nuclear matrix during the interphase, while a small portion is found in the cytoplasm . During mitosis, it is associated with chromosomes throughout the entire process . In the present study, we identified a protein that interacts with KIF4 using a yeast two-hybrid system, co-immunoprecipitation and co-fractionation . This protein is BRCA2-associated factor 35 (BRAF35) containing a non-specific DNA binding high-mobility-group domain and a kinesin-like coiled-coil domain . It appeared that the interaction between the two proteins occurs through their respective alpha-helical coiled-coil domains . The co-fractionation experiment revealed that KIF4 and BRAF35 were present in a complex of approx . 540 kDa . The composition and biological significance of this complex should be studied further. Nat Biotechnol, 2003 Jul, 21(7), 813 - 7 Epub 2003 Jun 15. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry; Wenk MR et al.; Phosphoinositides (phosphorylated derivatives of phosphatidylinositol, PI) are versatile intracellular signaling lipids whose occurrence in low concentrations complicates direct mass measurements . Here we present a sensitive method to detect, identify and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP(2)) with different fatty acid compositions (phosphoinositide profiles) in total lipid extracts by electrospray ionization mass spectrometry (ESI-MS) . Using this method, we detected elevated concentrations of PIP2 in human fibroblasts from patients with Lowe syndrome, a genetic disorder that affects phosphoinositide metabolism . Saccharomyces cerevisiae cells deficient in enzymes involved in PIP metabolism--Sac1p, a phosphoinositide phosphatase, and Vps34p and Pik1p, a PI 3-kinase and PI 4-kinase, respectively--showed not only different PIP concentrations but also differential changes in PIP profiles indicating metabolic and/or subcellular pooling . Mass spectrometric analysis of phosphoinositides offers unique advantages over existing approaches and may represent a powerful diagnostic tool for human diseases that involve defective phosphoinositide metabolism. Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7743 - 8 Epub 2003 Jun 13. Targeted cytosine methylation for in vivo detection of protein-DNA interactions; Carvin CD et al.; We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin . M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing . Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites . Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure . In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation . Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus . Thus, TAGM enables rapid detection of DNA-protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level . Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.
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