S. cerevisiae

Biochemistry, 2002 Feb 5, 41(5), 1512 - 9
A single glycine mutation in the equilibrative nucleoside transporter gene, hENT1, alters nucleoside transport activity and sensitivity to nitrobenzylthioinosine; SenGupta DJ et al.; The human equilibrative nucleoside transporter, hENT1, which is sensitive to inhibition by nitrobenzylthioinosine (NBMPR), is expressed in a wide variety of tissues . hENT1 is involved in the uptake of natural nucleosides, including regulation of the physiological effects of extracellular adenosine, and transports nucleoside drugs used in the treatment of cancer and viral diseases . Structure-function studies have revealed that transmembrane domains (TMD) 3 through 6 of hENT1 may be involved in binding of nucleosides . We have hypothesized that amino acid residues within TMD 3-6, which are conserved across equilibrative transporter sequences from several species, may have a critical role in the binding and transport of nucleosides . Therefore, we explored the role of point mutations of two conserved glycine residues, at positions 179 and 184 located in transmembrane domain 5 (TMD 5), using a GFP-tagged hENT1 in a yeast nucleoside transporter assay system . Mutations of glycine 179 to leucine, cysteine, or valine abolished transporter activity without affecting the targeting of the transporter to the plasma membrane, whereas more conservative mutations such as glycine to alanine or serine preserved both targeting to the plasma membrane and transport activity . Similar point mutations at glycine 184 resulted in poor targeting of hENT1 to the plasma membrane and little or no detectable functional activity . Uridine transport by G179A mutant was significantly lower (p < 0.05) and less sensitive (p < 0.05) to inhibition by NBMPR when compared to the wild-type transporter (IC(50) 7.7 +/- 0.8 nM versus 46 +/- 14.6 nM) . Based on these data, we conclude that when hENT1 is expressed in yeast, glycine 179 is critical not only to the ability of hENT1 to transport uridine but also as a determinant of hENT1 sensitivity to NBMPR . In contrast, glycine 184 is likely important in targeting the transporter to the plasma membrane . This is the first identification and characterization of a critical amino acid residue of hENT1 that is important in both nucleoside transporter function and sensitivity to inhibition by NBMPR.

Biochemistry, 2002 Feb 5, 41(5), 1451 - 6
Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP; Jacobsen MD et al.; To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain . Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding . The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation . The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP {K(1/2)(ATP) 12 nM} . Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol . Mutations of Cys(549) did not affect ATP binding . In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).

Cell Mol Life Sci, 2001 Dec, 58(14), 2108 - 16
Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation; Matuoka K et al.; Aging appears to be an irreversible process . Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity . Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system . The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects . Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action . On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered . Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT.

Nat Cell Biol, 2002 Feb, 4(2), 134 - 9
Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48; Jarosch E et al.; Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requires the dislocation of substrates from the ER into the cytosol . It has been speculated that a functional ubiquitin proteasome pathway is not only essential for proteolysis, but also for the preceding export step . Here, we show that short ubiquitin chains synthesized on proteolytic substrates are not sufficient to complete dislocation; the size of the chain seems to be a critical determinant . Moreover, our results suggest that the AAA proteins of the 26S proteasome are not directly involved in substrate export . Instead, a related AAA complex Cdc48, is required for ER-associated protein degradation upstream of the proteasome.

Nucleic Acids Res, 2001 Dec 15, 29(24), 5216 - 25
The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity; Hayman ML et al.; RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with approximately 30% of total gRNAs in Trypanosoma brucei mitochondria . To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners . By these methods, RBP16 does not appear to stably interact with the core editing machinery . However, fractionation of mitochondrial extracts on MBP-RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns . We describe here our analysis of one RBP16-associated protein, p22 . The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p . Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32 . We confirmed the p22-RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16 . p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA . To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays . These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16 . Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16.

J Biol Chem, 2002 Apr 12, 277(15), 12632 - 41 Epub 2002 Jan 25.
Conserved enzymatic production and biological effect of O-acetyl-ADP-ribose by silent information regulator 2-like NAD+-dependent deacetylases; Borra MT et al.; Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging . Yeast Sir2 and several homologues have been shown to be NAD(+)-dependent histone/protein deacetylases . Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite . We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human . Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose . By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres . This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects . In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.

J Mol Biol, 2002 Jan 25, 315(4), 809 - 18
Protein-protein interactions of hCsl4p with other human exosome subunits; Raijmakers R et al.; The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA . We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome . By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p . Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo . These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p .

Anal Biochem, 2002 Feb 1, 301(1), 35 - 48
Characterization of monomeric and dimeric forms of recombinant Sml1p-histag protein by electrospray mass spectrometry; Uchiki T et al.; Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)3, a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair . In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p) . Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway . Neither the structure of Sml1p nor its complex to Rnr1p is well known . In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry . Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS-PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal-ion binding to the protein species, which may alter the function and binding of the protein partners.

Mol Genet Genomics, 2001 Dec, 266(4), 657 - 63 Epub 2001 Oct 06.
The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants; Thumm M et al.; A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers . The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy . The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants . These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.

Nucleic Acids Res, 2002 Feb 1, 30(3), 732 - 9
Suppression of genetic defects within the RAD6 pathway by srs2 is specific for error-free post-replication repair but not for damage-induced mutagenesis; Broomfield S et al.; srs2 was isolated during a screen for mutants that could suppress the UV-sensitive phenotype of rad6 and rad18 cells . Genetic analyses led to a proposal that Srs2 acts to prevent the channeling of DNA replication-blocking lesions into homologous recombination . The phenotypes associated with srs2 indicate that the Srs2 protein acts to process lesions through RAD6-mediated post-replication repair (PRR) rather than recombination repair . The RAD6 pathway has been divided into three rather independent subpathways: two error-free (represented by RAD5 and POL30) and one error-prone (represented by REV3) . In order to determine on which subpathways Srs2 acts, we performed comprehensive epistasis analyses; the experimental results indicate that the srs2 mutation completely suppresses both error-free PRR branches . Combined with UV-induced mutagenesis assays, we conclude that the Polzeta-mediated error-prone pathway is functional in the absence of Srs2; hence, Srs2 is not required for mutagenesis . Furthermore, we demonstrate that the helicase activity of Srs2 is probably required for the phenotypic suppression of error-free PRR defects . Taken together, our observations link error-free PRR to homologous recombination through the helicase activity of Srs2.

Nucleic Acids Res, 2002 Feb 1, 30(3), 667 - 74
Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction; Huang J et al.; The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells . Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay . To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair . We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts . Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence . Factor activity was inhibited by anti-Mre11 antibody . The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified . Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.

Mol Cell Biol, 2002 Feb, 22(4), 1253 - 65
Functional multimerization of human telomerase requires an RNA interaction domain in the N terminus of the catalytic subunit; Moriarty TJ et al.; Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase {hTERT}) containing reverse transcriptase (RT)-like motifs . The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms . We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization . Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity . We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif . Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70% . Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity . Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

Mol Cell Biol, 2002 Feb, 22(4), 1246 - 52
Convergence of TOR-nitrogen and Snf1-glucose signaling pathways onto Gln3; Bertram PG et al.; Carbon and nitrogen are two basic nutrient sources for cellular organisms . They supply precursors for energy metabolism and metabolic biosynthesis . In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively . Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes . Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein . In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor . Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses.

Mol Cell Biol, 2002 Feb, 22(4), 1233 - 45
Developmental defects and male sterility in mice lacking the ubiquitin-like DNA repair gene mHR23B; Ng JM et al.; mHR23B encodes one of the two mammalian homologs of Saccharomyces cerevisiae RAD23, a ubiquitin-like fusion protein involved in nucleotide excision repair (NER) . Part of mHR23B is complexed with the XPC protein, and this heterodimer functions as the main damage detector and initiator of global genome NER . While XPC defects exist in humans and mice, mutations for mHR23A and mHR23B are not known . Here, we present a mouse model for mHR23B . Unlike XPC-deficient cells, mHR23B(-/-) mouse embryonic fibroblasts are not UV sensitive and retain the repair characteristics of wild-type cells . In agreement with the results of in vitro repair studies, this indicates that mHR23A can functionally replace mHR23B in NER . Unexpectedly, mHR23B(-/-) mice show impaired embryonic development and a high rate (90%) of intrauterine or neonatal death . Surviving animals display a variety of abnormalities, including retarded growth, facial dysmorphology, and male sterility . Such abnormalities are not observed in XPC and other NER-deficient mouse mutants and point to a separate function of mHR23B in development . This function may involve regulation of protein stability via the ubiquitin/proteasome pathway and is not or only in part compensated for by mHR23A.

Mol Cell Biol, 2002 Feb, 22(4), 1116 - 25
A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine; McCulloch V et al.; A critical step toward understanding mitochondrial genetics and its impact on human disease is to identify and characterize the full complement of nucleus-encoded factors required for mitochondrial gene expression and mitochondrial DNA (mtDNA) replication . Two factors required for transcription initiation from a human mitochondrial promoter are h-mtRNA polymerase and the DNA binding transcription factor, h-mtTFA . However, based on studies in model systems, the existence of a second human mitochondrial transcription factor has been postulated . Here we report the isolation of a cDNA encoding h-mtTFB, the human homolog of Saccharomyces cerevisiae mitochondrial transcription factor B (sc-mtTFB) and the first metazoan member of this class of transcription factors to which a gene has been assigned . Recombinant h-mtTFB is capable of binding mtDNA in a non-sequence-specific fashion and activates transcription from the human mitochondrial light-strand promoter in the presence of h-mtTFA in vitro . Remarkably, h-mtTFB and its fungal homologs are related in primary sequence to a superfamily of N6 adenine RNA methyltransferases . This observation, coupled with the ability of recombinant h-mtTFB to bind S-adenosylmethionine in vitro, suggests that a structural, and perhaps functional, relationship exists between this class of transcription factors and this family of RNA modification enzymes and that h-mtTFB may perform dual functions during mitochondrial gene expression.

Int Immunol, 2002 Feb, 14(2), 189 - 200
Cis elements for transporter associated with antigen-processing-2 transcription: two new promoters and an essential role of the IFN response factor binding element in IFN-gamma-mediated activation of the transcription initiator; Guo Y et al.; Expression of cell surface MHC class I:peptide complex requires coordinated expression of multiple genes such as MHC class I heavy chain, beta(2)-microglobulin (beta(2)m), transporters associated with antigen-processing (TAP)-1 and TAP-2, and proteosomal components low-molecular weight polypeptide (LMP)-2 and LMP-7 . All of these genes are expressed at defined and distinct levels in normal tissues, and are inducible by IFN-gamma . While the cis elements involved in transcription of the MHC class I heavy chain, beta(2)m, TAP-1 and LMP-2 have been analyzed extensively, those for TAP-2 and LMP-7 have not been well studied . Here we systematically analyzed the cis elements for TAP-2 transcription . We found at least two independent elements that are sufficient to activate transcription of a reporter gene . One (hereby called TAP-2 P1) is located 5' to the TAP-2 exon 1, while the other (hereby called TAP-2 P2) is a transcription initiator residing in intron 1 . Analysis of the 5' sequence of TAP-2 mRNA indicates that both promoters are active . Moreover, while the TAP-2 promoter region contains cis elements that can mediate TAP-2 induction by IFN-gamma, such as gamma-activation site and IFN response factor binding element (IRFE), only the IRFE is required for IFN-gamma induction of TAP-2 promoter in vitro . The IRFE appears to work as an enhancer for the initiator (P2) . Together with another promoter recently identified by others, TAP-2 therefore has three independent promoters that can be differentially regulated.

J Nat Prod, 2002 Jan, 65(1), 65 - 8
A new triterpene saponin from Pittosporum viridiflorum from the Madagascar rainforest; Seo Y et al.; A novel triterpenoid saponin, pittoviridoside (1), which possesses an unusual 2,3,4-trisubstituted glycosidic linkage, has been isolated from Pittosporum viridiflorum using the engineered yeast strains 1138, 1140, 1353, and Sc-7 for bioactivity-guided fractionation . The structure of this compound was determined to be 3-O-{beta-D-glucopyranosyl(1-->2)}-{alpha-D-arabinopyranosyl(1-->3)},{alpha-l-arabinofuranosyl(1-->4)-beta-D-glucuronopyranosyl-21-angeloyl-22-senecioylolean-12-en-3beta,15alpha,16alpha,21beta,22alpha,28-hexol by spectral, chemical, and GC analyses . This compound showed weak cytotoxicity against the A2780 human ovarian cancer cell line.

Nature, 2002 Jan 24, 415(6870), 447 - 50
Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein; Helenius J et al.; N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway . The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER . The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate . This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion . The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins . The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells . The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive . Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

J Cell Biol, 2002 Jan 21, 156(2), 315 - 26 Epub 2002 Jan 21.
Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p; Gladfelter AS et al.; At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence . Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring . The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP) . Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs . In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization . Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly . However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type . Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p . These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent "switch" to turn on effectors or as an EF-Tu-like "assembly factor" using the GTPase cycle to assemble a macromolecular structure.

Curr Genet, 2001 Dec, 40(4), 234 - 42
Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function; Wu X et al.; Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II . Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5 . Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD . Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation . In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI . We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1 . Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect . Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD . The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

Sci STKE . 2002 Jan 22;2002(116):PE4.
A new gun in town: the U box is a ubiquitin ligase domain; Patterson C; Ubiquitin ligases determine protein stability in a highly regulated manner by coordinating the addition of polyubiquitin chains to proteins that are then targeted to the proteasome for degradation . Ubiquitin ligases have generally been separated into two groups--those containing HECT domains and those with RING finger domains . Recently, a third group of ubiquitin ligases has emerged: those containing a U-box domain . Patterson discusses what is known about the few U-box-containing proteins that have been characterized, although the general properties of U-box proteins that distinguish them from other ubiquitin ligases are still a matter of speculation.

J Biol Chem, 2002 Apr 5, 277(14), 11691 - 5 Epub 2002 Jan 22.
Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23; Rao H et al.; Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates . The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation . The major function of Ub is to regulate the in vivo half-life of its substrates . Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it . Molecular mechanisms of the postubiquitylation processes are poorly understood . Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA) . Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation . We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis . Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome . Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.

J Biol Chem, 2002 Mar 29, 277(13), 10852 - 60 Epub 2002 Jan 22.
Adenosine monophosphoramidase activity of Hint and Hnt1 supports function of Kin28, Ccl1, and Tfb3; Bieganowski P et al.; The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases . Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily . Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1) . Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures . Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28 . The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers . Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.

J Biol Chem, 2002 Mar 29, 277(13), 10753 - 5 Epub 2002 Jan 22.
COMPASS, a histone H3 (Lysine 4) methyltransferase required for telomeric silencing of gene expression; Krogan NJ et al.; The trithorax (Trx) family of proteins is required for maintaining a specific pattern of gene expression in some organisms . Recently we reported the isolation and characterization of COMPASS, a multiprotein complex that includes the Trx-related protein Set1 of the yeast Saccharomyces cerevisiae . Here we report that COMPASS catalyzes methylation of the fourth lysine of histone H3 in vitro . Set1 and several other components of COMPASS are also required for histone H3 methylation in vivo and for transcriptional silencing of a gene located near a chromosome telomere.

Genetics, 2002 Jan, 160(1), 137 - 48
Meiotic deletion at the BUF1 locus of the fungus Magnaporthe grisea is controlled by interaction with the homologous chromosome; Farman ML; The Magnaporthe grisea BUF1 gene suffers high-frequency mutation in certain genetic crosses, resulting in buff-colored progeny . Analysis of 16 buf1 mutants arising from a cross with a mutation frequency of 25% revealed that, in every case, the BUF1 gene was deleted . The deletions occurred in only one of the parental chromosomes and were due to intrachromosomal recombination . Tetrad analysis revealed that deletions occurred in 44% of meioses and usually affected both chromatids of the mutable chromosome . This suggests that they happen before the premeiotic round of DNA synthesis . However, they were also almost entirely restricted to heteroallelic crosses . This, together with the discovery of numerous repetitive elements that were present only in the mutable BUF1 locus, suggests that the deletion process is sensitive to pairing interactions between homologous chromosomes, such that only unpaired loci are subject to deletion . Given that karyogamy is not supposed to occur until after premeiotic DNA replication in Pyrenomycetous fungi such as M . grisea, this latter observation would place the time of deletion during, or after, DNA synthesis . These conflicting results suggest that karyogamy might actually precede DNA replication in Pyrenomycetous fungi or that parts of the genome remain unreplicated until after karyogamy and subsequent chromosome pairing have taken place.

Mol Cell, 2002 Jan, 9(1), 8 - 9
The spliceosome: no assembly required?
Nilsen TW.
In this issue of Molecular Cell, Stevens et al . purify a large particle from yeast extracts that contains all five of the U snRNPs required for pre-mRNA splicing . The existence of this "penta-snRNP" suggests the provocative possibility that spliceosome assembly does not depend upon a pre-mRNA substrate.

Eur J Pharm Sci, 2002 Feb, 15(1), 39 - 47
Compromised integrity of excised porcine intestinal epithelium obtained from the abattoir affects the outcome of in vitro particle uptake studies; Pietzonka P et al.; Excised porcine intestinal tissue obtained from the local abattoir was studied for its suitability to examine the uptake and transport of poly(lactic-co-glycolic acid) (PLGA) nanoparticles in Peyer's (PP) and non-Peyer's patch (NPP) tissue in vitro . Incubation of such tissue with fluorescent PLGA and polystyrene particles revealed negligible uptake into the intercellular space with no noticeable difference between PP and NPP tissue . Similarly, yeast cells, which were used as a positive control for selective uptake into PP tissue, were found in the subepithelial area of both PP and NPP tissue . Therefore we examined the morphological integrity of the tissue for the duration of the experiments . For this purpose, excised intestinal tissue from the abattoir transported to the laboratory was examined for morphological changes by light microscopy and compared to intestinal tissue from freshly slaughtered piglets . Already after 25 min postmortem, we observed lysis and defoliation of the epithelial cell layer followed by a complete loss of villus architecture and, consequently, resulting in a complete loss of the integrity of the intestinal tissue . This may explain the limited and non-selective particle uptake when using excised intestinal tissue from the abattoir . It is suggested to avoid small intestine obtained from the abattoir and to use tissue from freshly sacrificed animals within a few minutes postmortem . Experiments should then be performed under adequate oxygenation of the excised intestinal tissue.

Nat Cell Biol, 2002 Feb, 4(2), 140 - 7
IFN-Stimulated transcription through a TBP-free acetyltransferase complex escapes viral shutoff; Paulson M et al.; Type I interferon (IFN) stimulates transcription through a heteromeric transcription factor that contains tyrosine-phosphorylated STAT2 . We show that STAT2 recruits histone acetyltransferases (HAT) through its transactivation domain, resulting in localized transient acetylation of histones . GCN5, but not p300/CBP or PCAF, is required for STAT2 function . However, GCN5 function is impaired by the transcriptional antagonist, adenovirus E1A oncoprotein . The TFIID component TAF(II)130 potentiates STAT2 function, but TAF(II)28 or the HAT activity of TAF(II)250 do not, and transcriptional induction can proceed independently of the TATA-binding protein, TBP . Moreover, IFN-stimulated transcription was resistant to poliovirus-targeted degradation by TBP, and continued despite host-cell transcriptional shutoff during poliovirus infection . We conclude that a non-classical transcriptional mechanism combats an anticellular action of poliovirus, through a TBP-free TAF-containing complex and GCN5.

Mol Biol Evol, 2002 Feb, 19(2), 189 - 200
Evolution of eukaryotic translation elongation and termination factors: variations of evolutionary rate and genetic code deviations; Moreira D et al.; Translation is carried out by the ribosome and several associated protein factors through three consecutive steps: initiation, elongation, and termination . Termination remains the least understood of them, partly because of the nonuniversality of the factors involved . To get some insights on the evolution of eukaryotic translation termination, we have compared the phylogeny of the release factors eRF1 and eRF3 to that of the elongation factors EF-1alpha and EF-2, with special focus on ciliates . Our results show that these four translation proteins have experienced different modes of evolution . This is especially evident for the EF-1alpha, EF-2, and eRF1 ciliate sequences . Ciliates appear as monophyletic in the EF-2 phylogenetic tree but not in the EF-1alpha and eRF1 phylogenetic trees . This seems to be mainly because of phylogeny reconstruction artifacts (the long-branch attraction) produced by the acceleration of evolutionary rate of ciliate EF-1alpha and eRF1 sequences . Interaction with the highly divergent actin found in ciliates, or on the contrary, loss of interaction, could explain the acceleration of the evolutionary rate of the EF-1alpha sequences . In the case of ciliate eRF1 sequences, their unusually high evolutionary rate may be related to the deviations in the genetic code usage found in diverse ciliates . These deviations involve a relaxation (or even abolition) of the recognition of one or two stop codons by eRF1 . To achieve this, structural changes in eRF1 are needed, and this may affect its evolutionary rate . Eukaryotic translation seems to have followed a mosaic evolution, with its different elements governed by different selective pressures . However, a correlation analysis shows that, beneath the disagreement shown by the different translation proteins, their concerted evolution can still be made apparent when they are compared with other proteins that are not involved in translation.

Mol Biol Evol, 2002 Feb, 19(2), 179 - 88
Low nucleotide diversity at the pal1 locus in the widely distributed Pinus sylvestris; Dvornyk V et al.; Nucleotide polymorphism in Scots pine (Pinus sylvestris) was studied in the gene encoding phenylalanine ammonia-lyase (Pal, EC 4.3.1.5) . Scots pine, like many other pine species, has a large current population size . The observed levels of inbreeding depression suggest that Scots pine may have a high mutation rate to deleterious alleles . Many Scots pine markers such as isozymes, RFLPs, and microsatellites are highly variable . These observations suggest that the levels of nucleotide variation should be higher than those in other plant species . A 2,045-bp fragment of the pal1 locus was sequenced from five megagametophytes each from a different individual from each of four populations, from northern and southern Finland, central Russia, and northern Spain . There were 12 segregating sites in the locus . The synonymous site overall nucleotide diversity was only 0.0049 . In order to compare pal1 with other pine genes, sequence was obtained from two alleles of 11 other loci (total length 4,606 bp) . For these, the synonymous nucleotide diversity was 0.0056 . These estimates are lower than those from other plants . This is most likely because of a low mutation rate, as estimated from between-pine species synonymous site divergence . In other respects, Scots pine has the characteristics of a species with a large effective population . There was no linkage disequilibrium even between closely linked sites . This resulted in high haplotype diversity (14 different haplotypes among 20 sequences) . This could also give rise to high per locus diversity at the protein level . Divergence between populations in the main range was low, whereas an isolated Spanish population had slightly lower diversity and higher divergence than the remaining populations.

J Cell Sci, 2002 Jan 1, 115(Pt 1), 131 - 40
Roles of the N- and C-termini of GLUT4 in endocytosis; Al-Hasani H et al.; In insulin target cells, the predominantly expressed glucose transporter isoform GLUT4 recycles between distinct intracellular compartments and the plasma membrane . To characterize putative targeting signals within GLUT4 in a physiologically relevant cell type, we have analyzed the trafficking of hemagglutinin (HA)-epitope-tagged GLUT4 mutants in transiently transfected primary rat adipose cells . Mutation of the C-terminal dileucine motif (LL489/90) did not affect the cell-surface expression of HA-GLUT4 . However, mutation of the N-terminal phenylalanine-based targeting sequence (F5) resulted in substantial increases, whereas deletion of 37 or 28 of the 44 C-terminal residues led to substantial decreases in cell-surface HA-GLUT4 in both the basal and insulin-stimulated states . Studies with wortmannin and coexpression of a dominant-negative dynamin GTPase mutant indicate that these effects appear to be primarily due to decreases and increases, respectively, in the rate of endocytosis . Yeast two-hybrid analyses revealed that the N-terminal phenylalanine-based targeting signal in GLUT4 constitutes a binding site for medium chain adaptins mu1, mu2, and mu3A, implicating a role of this motif in the targeting of GLUT4 to clathrin-coated vesicles.

J Biol Chem, 2002 Apr 12, 277(15), 13016 - 28 Epub 2002 Jan 18.
Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase; Menguy T et al.; We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process . In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment . We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase . Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum . All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion . Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C . (2001) J . Biol . Chem . 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.

Reprod Fertil Dev, 2001, 13(4), 221 - 9
Protein coregulators that mediate estrogen receptor function; Ratajczak T; The recent discovery of estrogen receptor beta as a biological partner with estrogen receptor alpha in mediating the estrogen response has come at precisely the same time as intensive research is revealing the role played by downstream coregulator proteins in linking nuclear hormone receptor activity to general transcription machinery involved in gene transcriptional activation . In what is a rapidly evolving area of research, findings to date have led to a proposed model of hormonal action, in which a receptor activated by estrogen or cell-membrane-derived phosphorylation-dependent signaling pathways promotes recruitment of selected members of the multifunctional steroid receptor coactivator family and the cointegrators, p300/CBP and P/CAF . The intrinsic histone acetylase activity mediated by these coactivator and cointegrator proteins, alters chromatin structure giving rise to increased transcriptional efficiency . On the other hand, antiestrogen-bound receptors favour the assembly of receptor-corepressor complexes containing the sequence-related corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoid and thyroid hormone receptors), localizing histone deacetylase activity to the promoter and leading to transcriptional repression . The model predicts that a change in the balance between corepressor and coactivator expression in favour of coactivators, might result in antiestrogen resistance . Together with available crystal structure data for estrogen- and antiestrogen-bound receptors, these studies have provided valuable insights into events that occur subsequent to receptor interaction with specific DNA sequences and have helped define the molecular basis of estrogen and antiestrogen activity.

Biochem Cell Biol, 2001, 79(6), 681 - 92
Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery; McMaster CR; The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained . Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells . Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles . Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum . This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events.

J Biol Chem, 2002 Mar 29, 277(13), 11401 - 9 Epub 2002 Jan 17.
Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway; Yoo JS et al.; The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown . Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S . I., Bannykh, G . I., Fish, K . N., Moyer, B . D., Riordan, J . R., and Balch, W . E . (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway . We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested . Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi . Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation . We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.

Genes Dev, 2002 Jan 15, 16(2), 183 - 97
The mitotic spindle is required for loading of the DASH complex onto the kinetochore; Li Y et al.; A role for the mitotic spindle in the maturation of the kinetochore has not been defined previously . Here we describe the isolation of a novel and conserved essential gene, ASK1, from Saccharomyces cerevisiae involved in this process . ask1 mutants display either G(2)/M arrest or segregation of DNA masses without the separation of sister chromatids, resulting in massive nondisjunction and broken spindles . Ask1 localizes along mitotic spindles and to kinetochores, and cross-links to centromeric DNA . Microtubules are required for Ask1 binding to kinetochores, and are partially required to maintain its association . We found Ask1 is part of a multisubunit complex, DASH, that contains approximately 10 components, including several proteins essential for mitosis including Dam1, Duo1, Spc34, Spc19, and Hsk1 . The Ipl1 kinase controls the phosphorylation of Dam1 in the DASH complex and may regulate its function . We propose that DASH is a microtubule-binding complex that is transferred to the kinetochore prior to mitosis, thereby defining a new step in kinetochore maturation.

Zhonghua Yi Xue Za Zhi, 2000 Jun, 80(6), 456 - 60
{Function of one novel gene identified by SSH PCR differentially expressed in HBX transfected HepG2 cells}; Liu J et al.; OBJECTIVE: To clone full length differentially expressed genes which are related with HBxAg . METHODS: HepG2-cells were infected with prepared recombinant retroviruses encoding the X antigen . The differences in gene expression between HepG2 x and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR . In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes . The full length cDNA clone of the gene was obtained by 5' and 3' rapid amplification of cDNA ends(race) PCR . HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting (FACS) analysis for DNA content . HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice . RESULTS: The expression of multiple genes were turned on (8) or off (2) in HepG2X compared to HepG2CAT cells . One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG(2) cells . The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids . Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice . Cells transfected with pcDNA3-HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg . C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma . CONCLUSIONS: HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth . Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation.

IUBMB Life, 2001 Sep-Nov, 52(3-5), 101 - 12
Protein import into mitochondria; Paschen SA et al.; Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol . Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria . The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins . The TIM23 complex mediates import of matrix-targeted preproteins with N-terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex . The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane . This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa . In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome . This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration.

J Biol Chem, 2002 Mar 22, 277(12), 9741 - 8 Epub 2002 Jan 16.
Studies on the mode of Ku interaction with DNA; Arosio D et al.; The Ku heterodimer plays a central role in non-homologous end-joining . The binding of recombinant Ku to DNA has been investigated by dynamic light scattering, double-filter binding, fluorescence spectroscopy, and band shift assays . The hydrodynamic radius of Ku in solution is 5.2 nm and does not change when a 25-bp double-strand DNA (dsDNA) fragment (D25) is added, indicating that only one Ku molecule binds to a 25-bp fragment . The dissociation constant (k(d)) for the binding to D25 is 3.8 +/- 0.9 nm . If both ends of the substrate are closed with hairpin loops, Ku is still able to bind with little change in the k(d) . The k(d) is not affected by ATP, Mg(2+), or ionic strength . However, the addition of bovine serum albumin decreases the k(d) by 2-fold . DNA substrates of 50 bp can bind two Ku molecules, whereas three molecules are bound to a 75-bp substrate . Data analysis with the Hill equation yields a value of the Hill coefficient (n) close to 1, and the k(d) values for the binding of Ku to both ends of these substrates are the same . Thus, we demonstrate that there is no cooperative interaction among the Ku heterodimers binding longer substrates.

J Biol Chem, 2002 Apr 19, 277(16), 13848 - 55 Epub 2002 Jan 16.
Transient inhibition of translation initiation by osmotic stress; Uesono Y et al.; Cells respond and adapt to changes in the environment . In this study, we examined the effect of environmental stresses on protein synthesis in the yeast Saccharomyces cerevisiae . We found that osmotic stress causes irreversible inhibition of methionine uptake, transient inhibition of uracil uptake, transient stimulation of glucose uptake, transient repression of ribosomal protein (RP) genes such as CYH2 and RPS27, and the transient inhibition of translation initiation . Rapid inhibition of translation initiation by osmotic stress requires a novel pathway, different from the amino acid-sensing pathway, the glucose-sensing pathway, and the TOR pathway . The Hog1 MAP kinase pathway is not involved in the inhibition of either methionine uptake or translation initiation but is required for the adaptation of translation initiation after inhibition and the repression of RP genes by osmotic stress . These results suggest that the transient inhibition of translation initiation occurs as a result of a combination of both acute inhibition of translation and the long-term activation of translation by the Hog1 pathway.

Hum Mutat, 2002 Feb, 19(2), 173 - 7
A robust method for detecting CHK2/RAD53 mutations in genomic DNA; Sodha N et al.; While screening for germline CHK2 mutations in cancer cases by heteroduplex CSGE, we observed that additional PCR fragments were generated from the 3' end region of the gene that includes exons 11-14 . Direct sequencing of these fragments suggested that homologous loci (possibly pseudogenes) were concomitantly being amplified . Searches of public sequence databases showed that a number of areas of the genome show a high degree of homology to exons 10-14 of the CHK2 gene . The presence of these homologous regions means that standard screening methods for detecting mutations in CHK2, based on PCR of genomic DNA, are prone to error . To circumvent this problem, we have developed a strategy, based on long-range PCR, to screen the functional copy of CHK2 . Using this approach it is possible to carry out a comprehensive mutational analysis of CHK2 from genomic DNA .

Funct Integr Genomics, 2001 Mar, 1(4), 269 - 78
Dynamic models of gene expression and classification; Dewey TG et al.; Powerful new methods, like expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic or pathogenic challenges . This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell . Here we present a general approach to developing dynamic models for analyzing time series of whole genome expression . In this approach, a self-consistent calculation is performed that involves both linear and non-linear response terms for interrelating gene expression levels . This calculation uses singular value decomposition (SVD) not as a statistical tool but as a means of inverting noisy and near-singular matrices . The linear transition matrix that is determined from this calculation can be used to calculate the underlying network reflected in the data . This suggests a direct method of classifying genes according to their place in the resulting network . In addition to providing a means to model such a large multivariate system this approach can be used to reduce the dimensionality of the problem in a rational and consistent way, and suppress the strong noise amplification effects often encountered with expression profile data . Non-linear and higher-order Markov behavior of the network are also determined in this self-consistent method . In data sets from yeast, we calculate the Markov matrix and the gene classes based on the linear-Markov network . These results compare favorably with previously used methods like cluster analysis . Our dynamic method appears to give a broad and general framework for data analysis and modeling of gene expression arrays.

Funct Integr Genomics, 2000 Nov, 1(3), 174 - 85
Constraint structure analysis of gene expression; Rifkin SA et al.; A microarray experiment gives a snapshot of the state of an organism in terms of the relative abundances of its mRNA transcripts, locating the organism at a point in a high dimensional state space where each axis represents the relative expression level of a single gene . Multiple experiments generate a cloud of points in this gene expression space . We present a geometric approach to analyzing the covariational properties of such a cloud and use a dataset from Saccharomyces cerevisiae as an illustration . In particular, we use singular value decomposition to identify significant linear sub-structures in the data and analyze the contributions of both individual genes and functional classes of genes to these major directions of variation . Analyzing the publicly available yeast expression data, we show that under all experimental conditions the variation in expression is limited to a small number of linear dimensions . Projections of individual gene axes onto the significant dimensions can order the contribution of individual genes to variation in expression within an experiment . We show that no particular groups of genes characterize particular experimental conditions . Instead, the particular structure of the coordinated expression of the entire genome characterizes a particular experiment.

Funct Integr Genomics, 2000 May, 1(1), 70 - 5
An integrated web interface for large-scale characterization of sequence data; Cheung KH et al.; Large-scale genome projects require the analysis of large amounts of raw data . This analysis often involves the application of a chain of biology-based programs . Many of these programs are difficult to operate because they are non-integrated, command-line driven, and platform-dependent . The problem is compounded when the number of data files involved is large, making navigation and status-tracking difficult . To demonstrate how this problem can be addressed, we have created a platform-independent Web front end that integrates a set of programs used in a genomic project analyzing gene function by transposon mutagenesis in Saccharomyces cerevisiae . In particular, these programs help define a large number of transposon insertion events within the yeast genome, identifying both the precise site of transposon insertion as well as potential open reading frames disrupted by this insertion event . Our Web interface facilitates this analysis by performing the following tasks . Firstly, it allows each of the analysis programs to be launched against multiple directories of data files . Secondly, it allows the user to view, download, and upload files generated by the programs . Thirdly, it indicates which sets of data directories have been processed by each program . Although designed specifically to aid in this project, our interface exemplifies a general approach by which independent software programs may be integrated into an efficient protocol for large-scale genomic data processing.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 832 - 7 Epub 2002 Jan 15.
Ku86 is essential in human somatic cells; Li G et al.; Ku86 plays a key role in nonhomologous end joining in mammals . Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNA-dependent protein kinase complex, is nevertheless compatible with viability . In contrast, no human patient has been described with mutations in either Ku86 or Ku70 . This has led to the hypotheses that either these genes are performing an additional essential role(s) and/or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans . To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus . Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation . Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time . These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis . These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 727 - 32 Epub 2002 Jan 15.
Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment; Muchowski PJ et al.; Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein . The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein . However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear . Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1) . Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies . Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form . Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1 . Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.

J Cell Sci, 2001 Dec, 114(Pt 24), 4629 - 35
Interaction of the endoplasmic reticulum alpha 1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system; Massaad MJ et al.; The alpha1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum . The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p . A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi alpha1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Deltapep4 cells and in the vacuoles of rer1/Deltapep4 by indirect immunofluorescence . The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo . Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of beta-galactosidase activity . Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction . A weak interaction was observed between Alg5p and Rer1p . These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact . Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

J Natl Cancer Inst, 2002 Jan 16, 94(2), 88 - 94
Cell-based assays for identification of novel double-strand break-inducing agents; Dunstan HM et al.; BACKGROUND:We are developing cell-based assays to identify anticancer agents that are selectively toxic to cells with defined mutations . As a test, we used a three-stage strategy to screen compounds from the National Cancer Institute's repository for agents that are selectively toxic to double-strand break repair-deficient yeast cells . METHODS:Compounds identified in the screen were further analyzed by use of yeast and vertebrate cell-based and in vitroassays to distinguish between topoisomerase I and II poisons . RESULTS:Of the more than 85 000 compounds screened, 126 were selectively toxic to yeast deficient in DNA double-strand break repair . Eighty-seven of these 126 compounds were structurally related to known topoisomerase poisons, and 39 were not . Twenty-eight of the 39 were characterized, and we present data for eight of the compounds . Among these eight compounds, we identified two novel topoisomerase II poisons (NSC 327929 and NSC 638432) that were equipotent to etoposide in biochemical tests and in cells, five (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with topoisomerase I-dependent toxicity in yeast that induced DNA damage and toxicity in mammalian cells, and one (NSC 610898) that directly bound to DNA and induced strand breaks . CONCLUSIONS:Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds.

Bioconjug Chem, 2002 Jan-Feb, 13(1), 136 - 42
Neoglycoconjugates of mannan with bovine serum albumin and their interaction with lectin concanavalin A; Mislovicova D et al.; Neoglycoconjugates were prepared from mannan isolated from yeast Saccharomyces cerevisiae and activated by periodate oxidation to create aldehyde groups . Various degrees of oxidation introduced 11-28 aldehyde groups per mannan molecule and simultaneously resulted in a molar mass decrease from 46 to 44.5-31 kDa . The activated mannans were subsequently conjugated with bovine serum albumin forming neoglycoconjugates . Some parameters of these mannan-bovine serum albumin conjugates were characterized: saccharide content 25-30% w/w, molar mass within the range 169-246 kDa, and polydispersion (M(w)/M(n)) from 2.8 to 3.6 . The interaction of these conjugates with lectin concanavalin A was studied using three different methods: (i) quantitative precipitation in solution; (ii) sorption to concanavalin A immobilized on bead cellulose; and (iii) kinetic measurement of the interaction by surface plasmon resonance . Quantitative precipitation assay showed only negligible differences in the precipitation course of original mannan and the corresponding mannan-bovine serum albumin conjugates . Both the sorption method (equilibrium method) and the surface plasmon resonance measurement (kinetic method) demonstrates that the values of dissociation constant K(D) of all synthetic neoglycoconjugates were within the range 10(-7) - 10(-8) mol x L(-1) (close to K(D) = 10(-8) mol x L(-1) determined by the sorption method for the original mannan) . In conclusion, characterization of synthetic neoglycoconjugates confirmed that the method used for their preparation retained the ability of mannan moiety to interact with concanavalin A.

Bioconjug Chem, 2002 Jan-Feb, 13(1), 97 - 103
An efficient binding chemistry for glass polynucleotide microarrays; Lee PH et al.; A variety of methods have been described for making synthetic polynucleotide microarrays . These include in situ synthesis directly on the array surface, for example, by photolithography or ink-jet printing technologies, and the application of presynthesized polynucleotides that are derivatized with various nucleophiles or electrophiles . In the latter case, a variety of surface chemistries have been developed, and several are available commercially . These chemistries must be compatible with nanoliter-scale volumes of polynucleotide reagents, which contact the array over a small portion of their surface . We reasoned that a three-dimensional polymer coating could potentially offer greater surface contact and higher binding efficiency . Here we describe a polyethylenimine-based coating chemistry that provides exceptional binding and hybridization characteristics . In our preferred process, size-fractionated polyethylenimine polymers are cross-linked onto an aminopropylsilanated glass surface in the presence of cyanuric chloride . The resulting three-dimensional coating binds polynucleotides through a mixture of covalent and noncovalent interactions as evidenced by comparisons between 5'-aminoalkyl modified and unmodified polynucleotides . Binding and hybridization comparisons are presented including analogous two-dimensional electrophilic and electrostatic chemistries.

Biosci Biotechnol Biochem, 2001 Nov, 65(11), 2465 - 71
Substrate recognition mechanism of carboxypeptidase Y; Nakase H et al.; To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)n Ala-OH (n = 1 to 6), Fmoc-(Glu)n Ala-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured . Km for Fmoc-peptides significantly decreased as peptide length increased from n = 1 to n = 5 with only slight changes in kcat . Km for Fmoc-(Glu)(5,6)Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull . Chem . Soc . Jpn., 73, 2587-2590) . These results show that the enzyme has six subsites (S1' and S1-S5) . Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted . For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate . It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1'-S1' interaction, so the amidase activity prevailed for Fmoc-(Glu)(3,5)Ala-NH2 . These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.

Genome Inform Ser Workshop Genome Inform, 2001, 12, 123 - 34
The potential use of SUISEKI as a protein interaction discovery tool; Blaschke C et al.; Relevant information about protein interactions is stored in textual sources . This sources are commonly used not only as archives of what is already known but also as information for generating new knowledge, particularly to pose hypothesis about new possible interactions that can be inferred from the existing ones . This task is the more creative part of scientific work in experimental systems . We present a large-scale analysis for the prediction of new interactions based on the interaction network for the ones already known and detected automatically in the literature . During the last few years it has became clear that part of the information about protein interactions could be extracted with automatic tools, even if these tools are still far from perfect and key problems such as detection of protein names are not completely solved . We have developed a integrated automatic approach, called SUISEKI (System for Information Extraction on Interactions), able to extract protein interactions from collections of Medline abstracts . Previous experiments with the system have shown that it is able to extract almost 70% of the interactions present in relatively large text corpus, with an accuracy of approximately 80% (for the best defined interactions) that makes the system usable in real scenarios, both at the level of extraction of protein names and at the level of extracting interaction between them . With the analysis of the interaction map of Saccharomyces cerevisiae we show that interactions published in the years 2000/2001 frequently correspond to proteins or genes that were already very close in the interaction network deduced from the literature published before these years and that they are often connected to the same proteins . That is, discoveries are commonly done among highly connected entities . Some biologically relevant examples illustrate how interactions described in the year 2000 could have been proposed as reasonable working hypothesis with the information previously available in the automatically extracted network of interactions.

Genome Inform Ser Workshop Genome Inform, 2001, 12, 24 - 33
Minimum spanning trees for gene expression data clustering; Xu Y et al.; This paper describes a new framework for microarray gene-expression data clustering . The foundation of this framework is a minimum spanning tree (MST) representation of a set of multi-dimensional gene expression data . A key property of this representation is that each cluster of the expression data corresponds to one subtree of the MST, which rigorously converts a multi-dimensional clustering problem to a tree partitioning problem . We have demonstrated that though the inter-data relationship is greatly simplified in the MST representation, no essential information is lost for the purpose of clustering . Two key advantages in representing a set of multi-dimensional data as an MST are: (1) the simple structure of a tree facilitates efficient implementations of rigorous clustering algorithms, which otherwise are highly computationally challenging; and (2) as an MST-based clustering does not depend on detailed geometric shape of a cluster, it can overcome many of the problems faced by classical clustering algorithms . Based on the MST representation, we have developed a number of rigorous and efficient clustering algorithms, including two with guaranteed global optimality . We have implemented these algorithms as a computer software EXCAVATOR . To demonstrate its effectiveness, we have tested it on two data sets, i.e., expression data from yeast Saccharomyces cerevisiae, and Arabidopsis expression data in response to chitin elicitation.

J Biol Chem, 2002 Mar 29, 277(13), 11026 - 33 Epub 2002 Jan 14.
Normal peroxisome development from vesicles induced by truncated Hansenula polymorpha Pex3p; Faber KN et al.; We show that the synthesis of the N-terminal 50 amino acids of Pex3p (Pex3p(1-50)) in Hansenula polymorpha pex3 cells is associated with the formation of vesicular membrane structures . Biochemical and ultrastructural findings suggest that the nuclear membrane is the donor membrane compartment of these vesicles . These structures also contain Pex14p and can develop into functional peroxisomes after subsequent reintroduction of the full-length Pex3p protein . We discuss the significance of this finding in relation to peroxisome reintroduction, e.g . in case peroxisomes are lost due to failure in inheritance.

Curr Opin Genet Dev, 2002 Feb, 12(1), 73 - 9
SWI/SNF chromatin remodeling and cancer; Klochendler-Yeivin A et al.; The SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure . Depending on the context, it can be involved in either transcriptional activation or repression . Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in human and mice . Results from biochemical and transfection studies suggest also that SWI/SNF participates either in the inhibition or activation of several oncogenes and tumor suppressor genes and/or control their transcriptional activity . These activities provide molecular insight into the mechanism underlying SWI/SNF function in tumor suppression.

Curr Biol, 2002 Jan 8, 12(1), R18 - 20
Membrane traffic: exocyst III--makes a family; Short B et al.; Although many factors have been implicated in the docking steps that precede vesicle fusion with a target membrane, few similarities have been found between them . New evidence suggests that at least some of these factors form related multimeric complexes that may help to explain the mechanism of vesicle docking.

Yeast, 2002 Jan 30, 19(2), 99 - 114
Phylogenetic footprinting reveals multiple regulatory elements involved in control of the meiotic recombination gene, REC102; Jiao K et al.; REC102 is a meiosis-specific early exchange gene absolutely required for meiotic recombination in Saccharomyces cerevisiae . Sequence analysis of REC102 indicates that there are multiple potential regulatory elements in its promoter region, and a possible regulatory element in the coding region . This suggests that the regulation of REC102 may be complex and may include elements not yet reported in other meiotic genes . To identify potential cis-regulatory elements, phylogenetic footprinting analysis was used . REC102 homologues were cloned from other two Saccharomyces spp . and sequence comparison among the three species defined evolutionarily conserved elements . Deletion analysis demonstrated that the early meiotic gene regulatory element URS1 was necessary but not sufficient for proper regulation of REC102 . Upstream elements, including the binding sites for Gcr1p, Yap1p, Rap1p and several novel conserved sequences, are also required for the normal regulation of REC102 as well as a Rap1p binding site located in the coding region . The data in this paper support the use of phylogenetic comparisions as a method for determining important sequences in complex promoters .

Nat Cell Biol, 2002 Feb, 4(2), 117 - 23
A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies; Reggiori F et al.; Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole . Sorting of some, although not all, proteins into these structures is mediated by ubiquitination . We have identified a transmembrane ubiquitin ligase, Tul1, that is resident in the Golgi apparatus and is required for the ubiquitination of proteins with polar TMDs, including vacuolar proteins such as carboxypeptidase S . We suggest that Tul1 provides quality control, identifying misfolded membrane proteins and marking them for transport to endosomes and degradation in the vacuole.

Plant Physiol, 2002 Jan, 128(1), 165 - 72
Effects of phosphorylation on phosphoenolpyruvate carboxykinase from the C4 plant Guinea grass; Walker RP et al.; In the C4 plant Guinea grass (Panicum maximum), phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in darkened leaves and dephosphorylated in illuminated leaves . To determine whether the properties of phosphorylated and non-phosphorylated PEPCK were different, PEPCK was purified to homogeneity from both illuminated and darkened leaves . The final step of the purification procedure, gel filtration chromatography, further separated phosphorylated and non-phosphorylated forms . In the presence of a high ratio of ATP to ADP, the non-phosphorylated enzyme had a higher affinity for its substrates, oxaloacetate and phosphoenolpyruvate . The activity of the non-phosphorylated form was up to 6-fold higher when measured at low substrate concentrations . Comparison of proteoloytically cleaved PEPCK from Guinea grass, which lacked its N-terminal extension, from yeast (Saccharomyces cerevisiae), which does not possess an N-terminal extension, and from the C4 plant Urochloa panicoides, which possesses an N-terminal extension but is not subject to phosphorylation, revealed similar properties to the non-phosphorylated full-length form from Guinea grass . Assay of PEPCK activity in crude extracts of Guinea grass leaves, showed a large difference between illuminated and darkened leaves when measured in a selective assay (a low concentration of phosphoenolpyruvate and a high ratio of ATP to ADP), but there was no difference under assay conditions used to estimate maximum activity . Immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed no difference in the abundance of PEPCK protein in illuminated and darkened leaves . There were no light/dark differences in activity detected in maize (Zea mays) leaves, in which PEPCK is not subject to phosphorylation.

J Biol Chem, 2002 Mar 22, 277(12), 9929 - 35 Epub 2002 Jan 11.
Mutation in the glucose-6-phosphate dehydrogenase gene leads to inactivation of Ku DNA end binding during oxidative stress; Ayene IS et al.; Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the oxidative pentose phosphate cycle, regulates the NADPH/NADP(+) ratio in eukaryotic cells . G6PD deficiency is one of the most common mutations in humans and is known to cause health problems for hundreds of millions worldwide . Although it is known that decreased G6PD functionality can result in increased susceptibility to oxidative stress, the molecular targets of this stress are not known . Using a Chinese hamster ovary G6PD-null mutant, we previously demonstrated that exposure to a thiol-specific oxidant, hydroxyethyldisulfide, caused enhanced radiation sensitivity and an inability to repair DNA double strand breaks . We now demonstrate a molecular mechanism for these observations: the direct inhibition of DNA end binding activity of the Ku heterodimer, a DNA repair protein, by oxidation of its cysteine residues . Inhibition of Ku DNA end binding was found to be reversible by treatment of the nuclear extract with dithiothreitol, suggesting that the homeostatic regulation of reduced cysteine residues in Ku is a critical function of G6PD and the oxidative pentose cycle . In summary, we have discovered a new layer of DNA damage repair, that of the functional maintenance of repair proteins themselves . In view of the rapidly escalating number of roles ascribed to Ku, these results may have widespread ramifications.

J Biol Chem, 2002 Apr 5, 277(14), 12190 - 9 Epub 2002 Jan 10.
Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein; Lindsay AJ et al.; Rab4 and Rab11 are small GTPases belonging to the Ras superfamily . They both function as regulators along the receptor recycling pathway . We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11 . We name this protein Rab coupling protein (RCP) . RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested . It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor . Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment . Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays . RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

Am J Pathol, 2002 Jan, 160(1), 247 - 54
Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma; Basolo F et al.; The tall-cell variant (TCV) of papillary thyroid carcinoma (PTC), characterized by tall cells bearing an oxyphilic cytoplasm, is more clinically aggressive than conventional PTC . RET tyrosine kinase rearrangements, which represent the most frequent genetic alteration in PTC, lead to the recombination of RET with heterologous genes to generate chimeric RET/PTC oncogenes . RET/PTC1 and RET/PTC3 are the most prevalent variants . We have found RET rearrangements in 35.8% of TCV (14 of 39 cases) . Whereas the prevalences of RET/PTC1 and RET/PTC3 were almost equal in classic and follicular PTC, all of the TCV-positive cases expressed the RET/PTC3 rearrangement . These findings prompted us to compare RET/PTC3 and RET/PTC1 in an in vitro thyroid model system . We have expressed the two oncogenes in PC Cl 3 rat thyroid epithelial cells and found that RET/PTC3 is endowed with a strikingly more potent mitogenic effect than RET/PTC1 . Mechanistically, this difference correlated with an increased signaling activity of RET/PTC3 . In conclusion, we postulate that the correlation between the RET/PTC rearrangement type and the aggressiveness of human PTC is related to the efficiency with which the oncogene subtype delivers mitogenic signals to thyroid cells.

J Theor Biol, 2002 Jan 7, 214(1), 85 - 97
Analysis of the kinetic and equilibrium binding of Ku protein to DNA; Taghva A et al.; The loading of Ku onto a DNA end in a double-strand DNA break is thought to be one of the first steps in the non-homologous DNA end joining (NHEJ) pathway, giving it an essential role in the maintenance of genomic integrity . The binding of Ku to DNA is complicated since DNA can accommodate multiple Ku subunits, which can translocate on the DNA strand . Furthermore, Ku may exhibit cooperativity in the loading process . Therefore, simple one- to-one kinetic models are unable to adequately simulate the process . However, through the use of computer simulation and curve-fitting, we are able to provide a comprehensive mechanistic model and rate constants that closely approximate experimental data for DNA molecules that bind one, two, and three Ku molecules under both kinetic and equilibrium conditions . The model obtains a best fit with Ku having a roughly seven-fold preference to bind to DNA ends rather than internal positions and is consistent with Ku having a strong preference of which face of the protein loads onto the DNA end .

Plant Mol Biol, 2001 Dec, 47(6), 771 - 83
Identification of a S-ribonuclease-binding protein in Petunia hybrida; Sims TL et al.; To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein . These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease . Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific . Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain . Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions . Together, these results suggest that PhSBPI may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.

Mol Cell Biol, 2002 Feb, 22(3), 835 - 48
Role of the Sin3-histone deacetylase complex in growth regulation by the candidate tumor suppressor p33(ING1); Kuzmichev A et al.; Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2 . Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure . We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18 . SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD . SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes . We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein . Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex . The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30 . The N-terminal domain of p33 is present in several uncharacterized human proteins . We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.

Mol Cell Biol, 2002 Feb, 22(3), 737 - 49
Direct activation of mitogen-activated protein kinase kinase kinase MEKK1 by the Ste20p homologue GCK and the adapter protein TRAF2; Chadee DN et al.; Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues . The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood . Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) . These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive . Here we show that endogenous GCK and MEKK1 associate in vivo . In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1 . The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not . Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation . Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo . These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.

Mol Cell Biol, 2002 Feb, 22(3), 693 - 703
Histone-dependent association of Tup1-Ssn6 with repressed genes in vivo; Davie JK et al.; The Tup1-Ssn6 complex regulates diverse classes of genes in Saccharomyces cerevisiae and serves as a model for corepressor functions in many organisms . Tup1-Ssn6 does not directly bind DNA but is brought to target genes through interactions with sequence-specific DNA binding factors . Full repression by Tup1-Ssn6 appears to require interactions with both the histone tails and components of the general transcription machinery, although the relative contribution of these two pathways is not clear . Here, we map Tup1 locations on two classes of Tup1-Ssn6-regulated genes in vivo via chromatin immunoprecipitations . Distinct profiles of Tup1 are observed on a cell-specific genes and DNA damage-inducible genes, suggesting that alternate repressive architectures may be created on different classes of repressed genes . In both cases, decreases in acetylation of histone H3 colocalize with Tup1 . Strikingly, although loss of the Srb10 mediator protein had no effect on Tup1 localization, both histone tail mutations and histone deacetylase mutations crippled the association of Tup1 with target loci . Together with previous findings that Tup1-Ssn6 physically associates with histone deacetylase activities, these results indicate that the repressor complex alters histone modification states to facilitate interactions with histones and that these interactions are required to maintain a stable repressive state.

Eur J Biochem, 2002 Jan, 269(1), 128 - 38
Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose; Segawa H et al.; A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT) . When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells . DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms . Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal . When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal . One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product . However, this mutation was suppressed by an Ile51 to Thr second-site mutation . Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 333 - 8
Amino acid runs in eukaryotic proteomes and disease associations; Karlin S et al.; We present a comparative proteome analysis of the five complete eukaryotic genomes (human, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Arabidopsis thaliana), focusing on individual and multiple amino acid runs, charge and hydrophobic runs . We found that human proteins with multiple long runs are often associated with diseases; these include long glutamine runs that induce neurological disorders, various cancers, categories of leukemias (mostly involving chromosomal translocations), and an abundance of Ca(2 +) and K(+) channel proteins . Many human proteins with multiple runs function in development and/or transcription regulation and are Drosophila homeotic homologs . A large number of these proteins are expressed in the nervous system . More than 80% of Drosophila proteins with multiple runs seem to function in transcription regulation . The most frequent amino acid runs in Drosophila sequences occur for glutamine, alanine, and serine, whereas human sequences highlight glutamate, proline, and leucine . The most frequent runs in yeast are of serine, glutamine, and acidic residues . Compared with the other eukaryotic proteomes, amino acid runs are significantly more abundant in the fly . This finding might be interpreted in terms of innate differences in DNA-replication processes, repair mechanisms, DNA-modification systems, and mutational biases . There are striking differences in amino acid runs for glutamine, asparagine, and leucine among the five proteomes.

EMBO J, 2002 Jan 15, 21(1-2), 181 - 93
Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation; Janke C et al.; We show here that Ask1p, Dad2p, Spc19p and Spc34p are subunits of the budding yeast Duo1p-Dam1p- Dad1p complex, which associate with kinetochores and localize along metaphase and anaphase spindles . Analysis of spc34-3 cells revealed three novel functions of the Duo1-Dam1p-Dad1p subunit Spc34p . First, SPC34 is required to establish biorientation of sister kinetochores . Secondly, SPC34 is essential to maintain biorientation . Thirdly, SPC34 is necessary to maintain an anaphase spindle independently of chromosome segregation . Moreover, we show that in spc34-3 cells, sister centromeres preferentially associate with the pre-existing, old spindle pole body (SPB) . A similar preferential attachment of sister centromeres to the old SPB occurs in cells depleted of the cohesin Scc1p, a protein with a known role in facilitating biorientation . Thus, the two SPBs are not equally active in early S phase . We suggest that not only in spc34-3 and Deltascc1 cells but also in wild-type cells, sister centromeres bind after replication preferentially to microtubules organized by the old SPB . Monopolar attached sister centromeres are resolved to bipolar attachment in wild-type cells but persist in spc34-3 cells.

EMBO J, 2002 Jan 15, 21(1-2), 175 - 80
A role for RAD54B in homologous recombination in human cells; Miyagawa K et al.; In human somatic cells, homologous recombination is a rare event . To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells . Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells . Here, we report that RAD54B plays a critical role in targeted integration in human cells . Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency . Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells . Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54 . In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54 . Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.

EMBO J, 2002 Jan 15, 21(1-2), 12 - 21
Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants; Hattendorf DA et al.; AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes . AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis . Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis . Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state . In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1 . Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2 . In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function.

Cancer Res, 2002 Jan 1, 62(1), 67 - 74
Deregulation of polyamine biosynthesis alters intrinsic histone acetyltransferase and deacetylase activities in murine skin and tumors; Hobbs CA et al.; The essential requirement for polyamines for normal cell growth and differentiation may be partly attributed to their influence on gene expression, a process regulated by the acetylation state of nucleosomal histones . We used transgenic mice to examine the effects of constitutive expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on histone acetylation in epithelial cells in skin . As compared with the skin of normal littermate mice, both intrinsic histone acetyltransferase (HAT) and deacetylase activities are elevated in ODC transgenic skin . Skin tumors that form spontaneously in ODC/Ras double transgenic mice exhibit exceptionally high HAT activity having a distinct specificity for Lys-12 in the tail domain of histone H4, which may have implications for gene transcription . However, acetylation of histones by HAT enzymes was impeded in cultured ODC transgenic keratinocytes, and there were only modest changes in levels of acetylated histones in the skin of ODC transgenic mice . Treatment with the ODC enzyme inhibitor alpha-difluoromethylornithine, which results in regression of ODC/Ras tumors, reverses the effects on HAT and deacetylase enzyme function, implicating polyamine biosynthesis in the regulation of histone acetylation . Polyamines do not directly stimulate the enzymatic activity of either p300 or p300/CREB-binding protein (CBP)-associated factor, members of two distinct classes of HAT enzymes, implying that the elevated CBP/p300-associated HAT activity detected in ODC transgenic skin is attributable to indirect influence of polyamines . These results suggest that multiple mechanisms exist by which endogenous polyamines influence chromatin in mammals . Furthermore, they suggest that the elevated polyamine levels inherent in many solid tumors alter chromatin structure, likely affecting gene expression and promoting the neoplastic process.

Dev Cell, 2002 Jan, 2(1), 9 - 19
Model organisms as a guide to mammalian aging; Tissenbaum HA et al.; Recent studies on aging in model systems such as yeast and roundworms have revealed conserved regulation of the process in response to nutrient availability and specific genes that appear to mediate this regulation . Here we review these findings with a focus on the nematode Caenorhabditis elegans and the budding yeast Saccharomyces cerevisiae and highlight general features of the regulation of aging that may have implications for mammals.

Oncogene, 2001 Dec 6, 20(56), 8116 - 24
Crosstalk between Myc and activating transcription factor 2 (ATF2): Myc prolongs the half-life and induces phosphorylation of ATF2; Miethe J et al.; Myc is a key regulator of cell growth, differentiation and apoptosis, and affects cell fate decisions by activating as well as by inhibiting the expression of cellular genes . Myc is a member of the basic region-helix-loop-helix-leucine zipper (b-HLH-Zip) class of transcription factors, which heterodimerizes with the Max protein and recognizes a consensus Myc binding motif . Stimulation of gene expression by Myc is thought to be mediated by direct binding of Myc-Max heterodimers to specific target genes . So far, only a few genes have been identified as direct binding targets of Myc, raising the possibility that Myc affects gene expression also by indirect mechanisms . In this work we present evidence that v-Myc encoded by the avian retrovirus MC29 stimulates activating transcription factor 2 (ATF2)-dependent transcription . Analysis of the effect of Myc on ATF2 shows that v-Myc prolongs the half-life of ATF2 and induces the phosphorylation of N-terminal sites of ATF2 (Thr-69 and Thr-71) which have previously been identified as the target sites of stress-activated protein kinases and implicated in the regulation of ATF2 activity . Taken together, our results suggest that v-Myc can affect gene expression indirectly by modulating the activity of ATF2.

Eur J Hum Genet, 2001 Nov, 9(11), 823 - 8
Analysis of TSC2 stop codon variants found in tuberous sclerosis patients; Goedbloed MA et al.; Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations to the TSC1 and TSC2 tumour suppressor genes . We detected two sequence changes involving the TSC2 stop codon and investigated the effects of these changes on the expression of tuberin, the TSC2 gene product, and on the binding between tuberin and the TSC1 gene product, hamartin . While elongation of the tuberin open reading frame by 17 amino acids did not interfere with tuberin-hamartin binding, a longer extension prevented this interaction . Our data illustrate how functional protein assays can assist in the verification and characterisation of disease-causing mutations.

Nat Cell Biol, 2001 Dec, 3(12), 1086 - 91
Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly; Hetzer M et al.; Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized . NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion . Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly . Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion . Subsequent NE growth involves a p97-p47 complex . This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

J Cell Biol, 2002 Jan 7, 156(1), 35 - 9 Epub 2002 Jan 07.
Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length; Schott DH et al.; Myosins are molecular motors that exert force against actin filaments . One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells . However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs . Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis . The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene . Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

J Biol Chem, 2002 Mar 15, 277(11), 9342 - 50 Epub 2002 Jan 07.
Inhibition of transforming growth factor beta signaling and Smad-dependent activation of transcription by the Latent Membrane Protein 1 of Epstein-Barr virus; Prokova V et al.; Inhibition of transforming growth factor beta (TGFbeta) signaling by the Epstein-Barr virus Latent Membrane Protein 1 (LMP1) may account, at least in part, for the oncogenic activity of LMP1 . We found that LMP1 is a potent inhibitor of TGFbeta signaling and Smad-dependent activation of transcription in 293 epithelial cells and COS-7 fibroblasts . LMP1 strongly inhibited the uninduced and the Smad-inducible activity of the promoters of the human p21/WAF1/Cip1 gene and the mouse Smad7 gene . Inhibition of TGFbeta signaling and Smad-dependent activation of transcription by LMP1 was greatly reduced by deletion of both C-terminal activating regions 1 and 2 of LMP1 as well as by overexpression of a non-degradable form of IkappaB . In contrast, specific inhibitors of p38 kinase or MEK kinase did not reverse the inhibitory activity of LMP1 . TGFbeta signaling was enhanced by overexpression of dominant negative forms of the LMP1 effectors TRAF2, NIK, and IKKbeta and was abolished by overexpression of p65/RelA or a p50/p65 fusion protein . Deletion of the transactivation domain of p65 abolished its inhibitory activity . Immunoblotting and immunofluorescence microscopy indicated that suppression of TGFbeta signaling and Smad transcriptional activity by LMP1 was not due to Smad degradation or cytoplasmic retention suggesting that LMP1 affects the nuclear function of Smad proteins . Our data are consistent with an essential role of NF-kappaB activation by LMP1 in the inhibition of TGFbeta signaling and Smad-mediated transcriptional responses.

Gastroenterology, 2002 Jan, 122(1), 211 - 9
Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system; Trojan J et al.; BACKGROUND & AIMS: Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC) . Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult . To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae . The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system . METHODS: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1 Delta 9/10) were cloned . On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay . RESULTS: Transfection was successful for all hMLH1 constructs . As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts . The V185G, G244D, and Delta 9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent . CONCLUSIONS: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system.

Biochemistry, 2002 Jan 15, 41(2), 579 - 87
Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response; McGonigle S et al.; Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family . Intracellular proteins that associate with these receptors are likely to play an important role in signaling . 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI . We now describe the identification of S . mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay . S . mansoni eIF2 alpha also interacts with human TGF beta receptors . Strongest association was demonstrated with kinase inactive receptors, particularly the type II TGF beta receptor (T beta RII) . Both T beta RI and T beta RII phosphorylate eIF2 alpha in vitro, at sites other than the previously described eIF2 alpha phosphorylation sites . EIF2 alpha also modulates signaling by TGF beta receptors; however, in contrast to 14-3-3 epsilon, eIF2 alpha overexpression inhibits the TGF beta-driven response . These data suggest a novel function for eIF2 alpha in the TGF beta signaling pathway . In addition, we have demonstrated an independent interaction between eIF2 alpha and 14-3-3 epsilon . Coexpression of 14-3-3 epsilon with eIF2 alpha leads to the abrogation of the inhibitory effect of eIF2 alpha on TGF beta-mediated signaling . The interaction of these two regulatory proteins with each other and with the TGF beta receptors and their relative expression levels are likely to be important in fine-tuning the regulation of TGF beta signal transduction.

RNA, 2001 Dec, 7(12), 1717 - 27
The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes; Coller JM et al.; A major pathway of mRNA turnover in eukaryotic cells initiates with deadenylation, leading to mRNA decapping and subsequent 5' to 3' exonuclease digestion . We show that a highly conserved member of the DEAD box family of helicases, Dhh1p, stimulates mRNA decapping in yeast . In dhh1delta mutants, mRNAs accumulate as deadenylated, capped species . Dhh1p's effects on decapping only occur on normal messages as nonsense-mediated decay still occurs in dhh1delta mutants . The role of Dhh1p in decapping appears to be direct, as Dhh1p physically interacts with several proteins involved in mRNA decapping including the decapping enzyme Dcp1p, as well as Lsm1p and Pat1p/Mrt1p, which function to enhance the decapping rate . Additional observations suggest Dhh1p functions to coordinate distinct steps in mRNA function and decay . Dhh1p also associates with Pop2p, a subunit of the mRNA deadenylase . In addition, genetic phenotypes suggest that Dhh1p also has a second biological function . Interestingly, Dhh1p homologs in others species function in maternal mRNA storage . This provides a novel link between the mechanisms of decapping and maternal mRNA translational repression.

Nature, 2002 Jan 3, 415(6867), 96 - 9
Superoxide activates mitochondrial uncoupling proteins; Echtay KS et al.; Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane . The functions of its homologues, UCP2 and UCP3, in other tissues are debated . UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic . Mild uncoupling would, however, decrease the mitochondrial production of reactive oxygen species, which are important mediators of oxidative damage . Here we show that superoxide increases mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3 . Superoxide-induced uncoupling requires fatty acids and is inhibited by purine nucleotides . It correlates with the tissue expression of UCPs, appears in mitochondria from yeast expressing UCP1, and is absent in skeletal muscle mitochondria from UCP3 knockout mice . Our findings indicate that the interaction of superoxide with UCPs may be a mechanism for decreasing the concentrations of reactive oxygen species inside mitochondria.

Nature, 2001 Dec 20-27, 414(6866), 929 - 33
Stimulatory effect of splicing factors on transcriptional elongation; Fong YW et al.; Transcription and pre-mRNA splicing are tightly coupled gene expression events in eukaryotic cells . An interaction between the carboxy-terminal domain of the largest subunit of RNA polymerase (Pol) II and components of the splicing machinery is postulated to mediate this coupling . Here, we show that splicing factors function directly to promote transcriptional elongation, demonstrating that transcription is more intimately coupled to splicing than previously thought . The spliceosomal U small nuclear ribonucleoproteins (snRNPs) interact with human transcription elongation factor TAT-SF1 (refs 6,7,8,9) and strongly stimulate polymerase elongation when directed to an intron-free human immunodeficiency virus-1 (HIV-1) template . This effect is likely to be mediated through the binding of TAT-SF1 to elongation factor P-TEFb, a proposed component of the transcription elongation complex . Inclusion of splicing signals in the nascent transcript further stimulates transcription, supporting the notion that the recruitment of U snRNPs near the elongating polymerase is important for transcription . Because the TAT-SF1-U snRNP complex also stimulates splicing in vitro, it may serve as a dual-function factor to couple transcription and splicing and to facilitate their reciprocal activation.

Nature, 2001 Dec 20-27, 414(6866), 924 - 8
Selectivity of chromatin-remodelling cofactors for ligand-activated transcription; Lemon B et al.; An array of regulatory protein and multi-subunit cofactors has been identified that directs eukaryotic gene transcription . However, establishing the specific functions of various related cofactors has been difficult owing to the limitations inherent in assaying transcription in animals and cells indirectly . Here we describe, using an integrated chromatin-dependent reconstituted transcription reaction, the purification and identification of a multi-subunit cofactor (PBAF) that is necessary for ligand-dependent transactivation by nuclear hormone receptors . A highly related cofactor, human SWI/SNF, and the ISWI-containing chromatin-remodelling complex ACF both fail to potentiate transcription . We also show that transcriptional activation mediated by nuclear hormone receptors requires TATA-binding protein (TBP)-associated factors (TAFs) as well as the multi-subunit cofactors ARC/CRSP . These studies demonstrate functional selectivity amongst highly related complexes involved in gene regulation and help define a more complete set of factors and cofactors required to activate transcription.

J Biol Chem, 2002 Mar 29, 277(13), 10893 - 902 Epub 2002 Jan 04.
Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome; Dawson S et al.; A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein . Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle . Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin . Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability . Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4 . The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.

J Biol Chem, 2002 Mar 22, 277(12), 10220 - 5 Epub 2002 Jan 04.
The Rpb9 subunit of RNA polymerase II binds transcription factor TFIIE and interferes with the SAGA and elongator histone acetyltransferases; Van Mullem V et al.; Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains . rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs . Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect . Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a beta-addition motif with the "jaw" of the largest subunit (Rpb1) . Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable from rpb9 single mutants . In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors . In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE . The interacting fragment, comprising amino acids 62-164 of Tfa1, belongs to a conserved zinc motif . Tfa1 is immunoprecipitated by RNA polymerase II . This co-purification is strongly reduced in rpb9-Delta, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.

Genome Res, 2002 Jan, 12(1), 203 - 14
Associating genes with gene ontology codes using a maximum entropy analysis of biomedical literature; Raychaudhuri S et al.; Functional characterizations of thousands of gene products from many species are described in the published literature . These discussions are extremely valuable for characterizing the functions not only of these gene products, but also of their homologs in other organisms . The Gene Ontology (GO) is an effort to create a controlled terminology for labeling gene functions in a more precise, reliable, computer-readable manner . Currently, the best annotations of gene function with the GO are performed by highly trained biologists who read the literature and select appropriate codes . In this study, we explored the possibility that statistical natural language processing techniques can be used to assign GO codes . We compared three document classification methods (maximum entropy modeling, naive Bayes classification, and nearest-neighbor classification) to the problem of associating a set of GO codes (for biological process) to literature abstracts and thus to the genes associated with the abstracts . We showed that maximum entropy modeling outperforms the other methods and achieves an accuracy of 72% when ascertaining the function discussed within an abstract . The maximum entropy method provides confidence measures that correlate well with performance . We conclude that statistical methods may be used to assign GO codes and may be useful for the difficult task of reassignment as terminology standards evolve over time.

Genome Res, 2002 Jan, 12(1), 145 - 52
Gene expression analysis with universal n-mer arrays; van Dam RM et al.; Gene expression profiling is one of the many applications that have benefited from the massively parallel nucleic acid detection capability of DNA microarrays . Current expression arrays, however, are expensive and inflexible . They are custom-designed for each organism and they do not offer the possibility of incorporating updated genomic information without production of a new chip . One possible solution is the development of a universal chip, consisting of all 4n possible DNA sequences of length n . Studying different organisms or new genes would simply require modifications to the hybridization pattern analysis software . The key problem is to find a value of n that is large enough to afford sufficient specificity, yet is small enough for practical fabrication and readout . We developed an analytical model, supported by computer-assisted calculation with yeast and mouse transcript data, to argue that it is both practical and useful to fabricate n-mer arrays with 10 < or = n < or = 16.

Genetics, 2001 Dec, 159(4), 1861 - 5
Meiotic alterations in CAG repeat tracts; Schweitzer JK et al.; We have investigated meiotic changes in CAG repeat tracts embedded in a yeast chromosome . Repeat tracts undergo either conversion events between homologs or expansion and contraction events that appear to be confined to a single chromatid . We did not find evidence for conversion of tract interruptions or excess exchange of flanking markers.

Fertil Steril, 2002 Jan, 77(1), 162 - 6
Chlamydial heat shock protein 60--specific T cells in inflamed salpingeal tissue; Kinnunen A et al.; OBJECTIVE: To evaluate the role of chlamydial heat shock protein 60 (CHSP60)-specific T-lymphocytes in tubal factor infertility . DESIGN: Case series of patients with tubal factor infertility . SETTING: Infertility Clinic, Department of Obstetrics and Gynecology, Helsinki University Central Hospital and Laboratory of Cell-Mediated Immunity, National Public Health Institute, Oulu, Finland . PATIENT(S): Five patients with tubal factor infertility who underwent elective salpingectomy because of hydrosalpinges . INTERVENTION: Collection of salpingeal tissue specimens for in vitro culture of T-lymphocytes . MAIN OUTCOME MEASURE(S): Cloning of Chlamydia trachomatis and CHSP60-specific T-lymphocyte lines derived from inflamed salpingeal tissue . Cytokine production analysis of the established T-lymphocyte clones . RESULT(S): Seventy-seven (34%) of the 229 T-lymphocyte clones recognized C . trachomatis and C . pneumoniae elementary bodies as target antigens . One-third of these Chlamydia genus-specific T-lymphocyte clones further recognized CHSP60 as the target antigen . Most of the CHSP60-specific T-lymphocyte clones produced predominantly IL-10 . CONCLUSION(S): CHSP60 may be an important T-lymphocyte antigen involved in the immunopathogenesis of tubal damage associated with chronic C . trachomatis infection.

Mol Cell, 2001 Dec, 8(6), 1383 - 90
Increased accumulation of hybrid V(D)J joins in cells expressing truncated versus full-length RAGs; Sekiguchi JA et al.; RAG1 and RAG2 (RAGs) initiate V(D)J recombination by introducing breaks between two coding segments and flanking recombination signals (RSs) . Nonhomologous end-joining (NHEJ) proteins then join the coding segments and join the RSs . In wild-type cells, both full-length and truncated ("core") RAGs lead to accumulation of "hybrid" V(D)J joins, in which an RS is appended to a different coding sequence . We now show that while hybrid joins do not accumulate in NHEJ-deficient cells that express full-length RAGs, they do accumulate in NHEJ-deficient cells that express the core RAGS; like those catalyzed by core RAGs in vitro, however, they are sealed on just one DNA strand . These results suggest a potential role for the non-core regions in repressing potentially harmful transposition events.

Mol Cell, 2001 Dec, 8(6), 1363 - 73
The nucle(ol)ar Tif6p and Efl1p are required for a late cytoplasmic step of ribosome synthesis; Senger B et al.; Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects . Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit . In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm . In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes . We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.

Mol Cell, 2001 Dec, 8(6), 1243 - 54
Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases; Barlev NA et al.; Cellular DNA damage causes stabilization and activation of the tumor suppressor and transcription factor p53, in part by promoting multiple covalent modifications of the p53 protein, including acetylation . We investigated the importance of acetylation in p53 function and the mechanism by which acetylation influences p53 activity . Acetylation site substitutions reduced p53-dependent transcriptional induction and G1 cell cycle arrest . Chromatin immunoprecipitation analysis of the endogenous p21 promoter showed increased association of p53, coactivators (CBP and TRRAP), and acetylated histones following cell irradiation . Results with acetylation-defective p53 demonstrate that the critical function of acetylation is not to increase the DNA binding affinity of p53 but rather to promote coactivator recruitment and histone acetylation . Therefore, we propose that an acetylation cascade consisting of p53 acetylation-dependent recruitment of coactivators/HATs is crucial for p53 function.

Mol Cell, 2001 Dec, 8(6), 1231 - 41
Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction; Thomson S et al.; Induction of c-fos and c-jun is associated with phosphoacetylation of histone H3 and acetylation of histone H4 . Upon induction, a large population of nucleosomes becomes highly acetylated on histones H3 and H4, whereas a much smaller population of comparable nucleosomes at similar positions along the gene becomes phosphoacetylated . Inhibiting histone H3 phosphorylation with kinase inhibitors does not measurably alter the enhanced acetylation of these nucleosomes . Finally, whereas H3 phosphorylation is a MAP kinase-mediated inducible event, we found acetylation to be continuously turning over by the targeted action of HATs and HDACs in the absence of any stimulation or gene transcription . These studies suggest that phosphorylation and acetylation are independently and dynamically regulated at these genes and reveal the complexity of multiple histone modifications at immediate-early gene chromatin.

Cell, 2001 Dec 28, 107(7), 893 - 903
Complementary signaling pathways regulate the unfolded protein response and are required for C . elegans development; Shen X et al.; The unfolded protein response (UPR) is a transcriptional and translational intracellular signaling pathway activated by the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) . We have used C . elegans as a genetic model system to dissect UPR signaling in a multicellular organism . C . elegans requires ire-1-mediated splicing of xbp-1 mRNA for UPR gene transcription and survival upon ER stress . In addition, ire-1/xbp-1 acts with pek-1, a protein kinase that mediates translation attenuation, in complementary pathways that are essential for worm development and survival . We propose that UPR transcriptional activation by ire-1 as well as translational attenuation by pek-1 maintain ER homeostasis . The results demonstrate that the UPR and ER homeostasis are essential for metazoan development.

Cell, 2001 Dec 28, 107(7), 881 - 91
XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor; Yoshida H et al.; In yeast, the transmembrane protein kinase/endoribonuclease Ire1p activated by endoplasmic reticulum stress cleaves HAC1 mRNA, leading to production of the transcription factor Hac1p that activates the unfolded protein response (UPR) . In mammals, no Hac1p counterpart has yet been discovered despite the presence of Ire1p homologs in the endoplasmic reticulum . Instead, the transcription factor ATF6 specific to the mammalian UPR is regulated by intramembrane proteolysis . Here, we identified the transcription factor XBP1, a target of ATF6, as a mammalian substrate of such an unconventional mRNA splicing system and showed that only the spliced form of XBP1 can activate the UPR efficiently . Our results reveal features of the UPR conserved during evolution and clarify the relationship between IRE1- and ATF6-dependent pathways.

Cell, 2001 Dec 28, 107(7), 827 - 30
The unfolding tale of the unfolded protein response; Ma Y et al.; Surface and secreted proteins are synthesized in the endoplasmic reticulum where they must fold and assemble before being transported . Changes in the ER that interfere with their proper maturation initiate the unfolded protein response pathway . New studies have filled in a missing link between the yeast and mammalian pathways.

Cell, 2001 Dec 28, 107(7), 819 - 22
Multisite phosphorylation and the countdown to S phase; Deshaies RJ et al.; Remarkably, SCF(Cdc4) ubiquitin ligase binds and ubiquitinates Sic1 decorated with six, but not five, phosphates . This numerical wizardry suggests how analog inputs can be rectified to digital outputs . Unraveling the counting mechanism promises to generate new insights into the architecture of protein nanoprocessors.

Int J Radiat Biol, 2002 Feb, 78(2), 75 - 88
Characterization of CDKN1A (p21) binding to sites of heavy-ion-induced damage: colocalization with proteins involved in DNA repair; Jakob B et al.; PURPOSE: To determine an association of locally accumulated CDKN1A and DNA repair proteins at the sites of heavy-ion traversals . MATERIALS AND METHODS: CDKN1A, PCNA, DNA-PK, hMre11 and Rad50 were investigated for their subnuclear localization after irradiation with heavy-ions using immunocytochemical staining and confocal laser-scanning microscopy . Human fibroblasts (normal diploid or XPA, ATM- or NBS1-deficient lines and HPV16 E6-transfected cells) were used . RESULTS: CDKN1A formed nuclear foci in G0/G1 normal human fibroblasts at the sites of particle traversal . Foci were persistent over hours and vanished after treatment with DNase-I . Formation of foci also occurred in NBS1- or ATM-deficient lines and in cells functionally abrogated for TP53 . In normal fibroblasts, CDKN1A foci colocalized with particle-induced foci of the hMre11 and Rad50 proteins . However, only CDKN1A relocalization was observed in irradiated NBS1 cells . PCNA foci temporarily colocalizing with CDKN1A were also detected in normal fibroblasts after exposure to heavy-ions . In contrast, no radiation-induced subnuclear relocalization was found for DNA-PK . CONCLUSIONS: CDKN1A foci arise rapidly at sites of localized DNA damage induced by heavy-ions and are associated with the chromatin . Evidence is provided that localization of CDKN1A to foci is not dependent on functional TP53 and occurs independently of the formation of the hMre11/Rad50/NBS1 complex . The data support a yet unknown role of CDKN1A in sensing or early processing of radiation-induced DNA lesions.

J Cell Biol, 2002 Jan 7, 156(1), 53 - 64 Epub 2002 Jan 03.
Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins; Brownawell AM et al.; We have identified a novel human karyopherin (Kap) beta family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p . Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments . We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction . Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA . We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap . In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3 . Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5 . We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

J Biol Chem, 2002 Mar 15, 277(11), 9375 - 81 Epub 2002 Jan 02.
Conformational regulation of SNARE assembly and disassembly in vivo; Munson M et al.; SNAP receptor (SNARE) proteins function in intracellular trafficking by forming complexes that bridge vesicle and target membranes prior to fusion . Biochemical studies indicate that the entry of certain SNARE proteins into complexes is inhibited by intramolecular interactions that generate a closed conformation . For example, an essential N-terminal regulatory domain of the yeast plasma membrane SNARE Sso1p sequesters the C-terminal SNARE motif and prevents it from binding to its assembly partners Sec9p and Sncp . Here, we introduce mutations into Sso1p that cause it to remain constitutively open . These open mutants can functionally substitute for wild-type Sso1p protein in vivo, demonstrating that inhibition of SNARE assembly is not the essential function of the N-terminal regulatory domain . Furthermore, the open mutants suppress sec9--4, a mutation that causes a severe defect in SNARE assembly . Elevated levels of SNARE complexes are observed in cells expressing the open mutants . In the presence of sufficient Sec9p, these complexes accumulate to levels that cause severe growth defects . Similarly, overexpression of the open mutants in yeast carrying mutations in the SNARE disassembly machinery impairs growth . Our findings indicate that elevated levels of SNARE complexes can be toxic and that these levels are normally controlled by the SNARE disassembly machinery, by the limited availability of Sec9p, and by the closed conformation of Sso1p.

J Am Soc Mass Spectrom, 2002 Jan, 13(1), 59 - 71
Tandem mass spectrometry of protein-protein complexes: cytochrome c-cytochrome b5; Mauk MR et al.; An improved method to interpret triple quadrupole MS/MS experiments of complexes of large ions is presented and applied to a study of the complex formed by the proteins cytochrome c and cytochrome b5 . Modeling of the activation and dissociation process shows that most of the reaction occurs near the collision cell exit where ions have the highest internal energies . Experiments at different collision cell pressures or with different collision gases (Ne, Ar, Kr) are interpreted with a previously proposed collision model (Chen et al., Rapid Commun . Mass Spectrom . 1998, 12, 1003-1010) to calculate the internal energy added to ions to cause dissociation . Small but systematic differences under different experimental conditions are attributed to different times available for reaction . A method to correct for this is presented . Ne, Ar, and Kr are found to have similar energy transfer efficiencies . Complexes of cytochrome c and cytochrome b5 are detected in ESI mass spectra but with abundances less than expected from the solution equilibrium . Dissociation of the cytochrome c-cytochrome b5 complexes with charge k gives as the most abundant fragments, cytochrome b5(+3) and cytochrome c+(k-3) . Adding charges to the complex destabilizes it . A series of cytochrome c variants with Lys residues thought to be involved in solution binding replaced by Ala showed no differences in the energy required to induce dissociation of the gas phase complex . The implications for the binding of the gas phase ions are inconclusive.

J Hum Genet, 2001, 46(12), 730 - 2
Single-nucleotide polymorphisms of the proprotein convertase subtilisin/ kexin type 5 (PCSK5) gene; Cao H et al.; The proprotein convertase . subtilisin/kexin type 5, or PCSK5, mediates post-translational endoproteolytic processing for several integrin alpha subunits . We identified two silent single-nucleotide polymorphisms (SNPs) in PCSK5, which were found to vary in frequency across ethnic groups . The identification of these amplification primers and SNPs provides tools to investigate PCSK5 for association with inflammatory or vascular phenotypes.

Pigment Cell Res, 2001 Dec, 14(6), 475 - 84
'VIT1', a novel gene associated with vitiligo; Le Poole IC et al.; To define genes associated with the pigmentary disorder vitiligo, gene expression was compared in non-lesional melanocytes cultured from three vitiligo patients and from three control melanocyte cultures by differential display . A basic local alignment search tool search did not reveal homology of six differentially expressed cDNA fragments to previously identified expressed sequence tags; thus, one was used to screen a melanocyte cDNA library . The underlying VIT1 gene maps to chromosome 2p16 . The 3' portion of the VIT1 message is complementary to the 3' end of hMSH6 mRNA, enabling the formation of RNA-RNA hybrids, which may interfere with G/T mismatch repair function . Moreover, the aligned cDNA sequence revealed an open reading frame identical to a hypothetical protein expressed in brain, with a similarity to Drosophila calmodulin, and containing a zinc-finger motif partially identical to N-recognin . Expression of ORF mRNA was confirmed for multiple skin cell types, suggesting its importance for skin physiology.

Anal Chem, 2001 Dec 1, 73(23), 5683 - 90
An automated multidimensional protein identification technology for shotgun proteomics; Wolters DA et al.; We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry . The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column . We detail the improvements over a system described by Link et al . (Link, A . J.; Eng, J.; Schieltz, D . M.; Carmack, E.; Mize, G . J.; Morris, D . R.; Garvik, B . M.; Yates, J . R., III . Nat . Biotechnol . 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides . Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis . In addition, we describe the chromatographic benchmarks of MudPIT . MudPIT was reproducible within 0.5% between two analyses . Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated . By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 128 - 33 Epub 2002 Jan 02.
Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA; Bassi GS et al.; Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclear-encoded protein, Mrs1 . Both proteins bind independently to the bI3 RNA . The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA . The active six-subunit complex has a molecular mass of 420 kDa, splices with a k(cat) of 0.3 min(-1), and binds the guanosine nucleophile with an affinity comparable to other group I introns . The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNA-binding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity . Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding . Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions.

J Biol Chem, 2002 Mar 8, 277(10), 7989 - 95 Epub 2001 Dec 31.
Role of the Ada2 and Ada3 transcriptional coactivators in histone acetylation; Balasubramanian R et al.; Previous studies have shown that the transcriptional coactivator protein Gcn5 functions as a catalytic histone acetyltransferase (HAT) . In this work, we examine the roles of the Ada2 and Ada3 coactivator proteins that are functionally linked to Gcn5 . We show that yeast Ada2, Ada3, and Gcn5 form a catalytic core of the ADA and Spt-Ada-Gcn5-acetyltransferase HAT complexes, which is necessary and sufficient in vitro for nucleosomal HAT activity and lysine specificity of the intact HAT complexes . We also demonstrate that Ada3 is necessary for Gcn5-dependent nucleosomal HAT activity in yeast extracts . Our results suggest that Ada2 potentiates the Gcn5 catalytic activity and that Ada3 facilitates nucleosomal acetylation and an expanded lysine specificity.

J Biol Chem, 2002 Mar 8, 277(10), 8474 - 81 Epub 2001 Dec 28.
The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia; Touz MC et al.; Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent . Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts . Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization . Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia . Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa) . Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization . Although highly similar to each other at the amino terminus, CWP2 includes a COOH-terminal 121-amino acid extension . Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs . Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation . We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C . Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail . These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.

J Biol Chem, 2002 Mar 1, 277(9), 7222 - 30 Epub 2001 Dec 28.
Unusual polypeptide synthesis in the kinetoplast-mitochondria from Leishmania tarentolae . Identification of individual de novo translation products; Horvath A et al.; The de novo synthesis of cytochrome c oxidase subunits I, II (COI and COII), and apocytochrome b (Cyb) was investigated in kinetoplast-mitochondria of Leishmania . The organelles were isolated after breaking whole cells with nitrogen cavitation . Individual COI, COII, and Cyb polypeptides were identified by fractionation of the kinetoplast membranes, labeled with {(35)S}methionine and cysteine, using two-dimensional (9 versus 14% and 20 versus 11%) denaturing gel electrophoresis . The reaction did not require exogenous energy sources or amino acids . On the contrary, the presence of amino acids other than methionine somewhat inhibited the labeling reaction probably by competing with the uptake of labeled amino acids . The synthesis reaction was insensitive to 100 microg/ml chloramphenicol, gentamycin, paromomycin, lincomycin, hygromycin, and tetracycline, as well as cycloheximide . The process showed a linear increase in the amount of synthesized polypeptides during the first 2 h of incubation, followed by a slower accumulation of products for up to 4 h . The de novo synthesized polypeptides were stable for several additional hours . Their assembly into respiratory complexes, investigated using two-dimensional Blue Native/N-{2-hydroxy-1,1-bis(hydroxymethyl)ethyl}glycine-SDS gels, began early during the incubation and continued throughout the course of the synthesis . This work represents the first unequivocal identification of the polypeptide synthesis in kinetoplasts.

Invest Ophthalmol Vis Sci, 2002 Jan, 43(1), 176 - 82
Protein interactions with myocilin; Wentz-Hunter K et al.; PURPOSE: To identify factors that interact in vivo with myocilin, a glaucoma gene product . METHODS: The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin . Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system . Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis . RESULTS: Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners . Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268 . Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC) . The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling . CONCLUSIONS: These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain . Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin-RLC interaction . The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.

Exp Gerontol, 2002 Jan-Mar, 37(2-3), 321 - 8
The DNA repair protein ku is involved in gp130-mediated signal transduction events in PBMC from young but not from elderly subjects; Frasca D et al.; Ku, composed of 70kDa (ku 70) and 86kDa (ku 80) proteins, is the DNA-targeting subunit of the DNA-dependent serine/threonine kinase (DNA-PK), which plays a crucial role in DNA double strand break recognition and repair in mammalian cells . We have investigated the effects of an IL-6-type cytokine (K-7/D-6), known to trigger gp130, on the expression and function of the ku protein in cytoplasmic and nuclear extracts of freshly isolated human peripheral blood mononuclear cells (PBMC) from subjects of different ages . DNA-binding of nuclear ku was found to be increased by cytokine treatment of cells from young donors but only to a negligible extent from elderly subjects . This cytokine effect was correlated with a greater amount of phosphorylated ku 80, rather than increased expression of ku 70 and ku 80 proteins . DNA-binding activity of cytoplasmic ku was hardly discernible, as compared to nuclear ku, in both young and elderly subjects and was unaffected by the cytokine treatment regardless of age . Regarding the mechanisms whereby ku and gp130 signaling are coupled in PBMC, results from co-immunoprecipitation experiments have shown that ku in the cytoplasm of PBMC from young, but not from elderly subjects, is associated with Tyk-2, a kinase involved in signal transduction events after gp130 triggering by IL-6-type cytokines . This association was independent of PHA stimulation . Moreover, the present results indicate that after gp130 signaling both Tyk-2 and ku 80 are phosphorylated, suggesting their activation by K-7/D-6.

Biochem J, 2002 Jan 15, 361(Pt 2), 243 - 54
Phosphorylation states of Cdc42 and RhoA regulate their interactions with Rho GDP dissociation inhibitor and their extraction from biological membranes; Forget MA et al.; The Rho GDP dissociation inhibitor (RhoGDI) regulates the activation-inactivation cycle of Rho small GTPases, such as Cdc42 and RhoA, by extracting them from the membrane . To study the roles of Mg(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), ionic strength and phosphorylation on the interactions of RhoGDI with Cdc42 and RhoA, we developed a new, efficient and reliable method to produce prenylated Rho proteins using the yeast Saccharomyces cerevisiae . It has been previously reported that protein kinase A (PKA)-treatment of isolated membranes increased RhoA extraction from membranes by RhoGDI {Lang, Gesbert, Delespine-Carmagnat, Stancou, Pouchelet and Bertoglio (1996) EMBO J . 16, 510-519} . In the present study, we used an in vitro affinity chromatography system to show that phosphorylation of RhoA and Cdc42 significantly increased their interaction with RhoGDI under physiological conditions of ionic strength . This increase was independent of the nucleotide (GDP or guanosine 5'-{gamma-thio}triphosphate) loaded on to the Rho proteins, as well as of Mg(2+) and PIP(2) . Moreover, dephosphorylation of rat brain membranes by alkaline phosphatase significantly decreased the extraction of RhoA and Cdc42 by RhoGDI . Subsequent re-phosphorylation by PKA restored the extraction levels, indicating the reversibility of this process . These results clearly demonstrate that the phosphorylation states of Cdc42 and RhoA regulate their interactions with RhoGDI and, consequently, their extraction from rat brain membranes . We therefore suggest that phosphorylation is a mechanism of regulation of Cdc42 and RhoA activity that is independent of GDP-GTP cycling.

Nat Neurosci, 2001 Dec, 4(12), 1199 - 206
Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene; Mack TG et al.; Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected . The degenerative and protective mechanisms are unknown . We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration . Transected distal axons survived for two weeks, and neuromuscular junctions were also protected . Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism . Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues . Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

Fresenius J Anal Chem, 2001 Dec, 371(7), 939 - 43
Novel enzymatic assay for determination of alkyl polyglycosides with short chain fatty alcohols; Bastl-Borrmann R et al.; An enzymatic assay has been developed for the quantitative detection of alkyl polyglycosides after enzymatic hydrolysis with different carbohydrolases . A three-step enzymatic method was used for the quantification of alkyl polyglycosides . In the first step the enzymatic hydrolysis of alkyl polyglycosides was performed with different carbohydrolases, or an acid hydrolysis was used . The second step was quantification of free glucose with an enzyme electrode, which was covered with an immobilized glucose oxidase membrane; glucose was used as standard . The last step was the enzymatic quantification of fatty alcohols, which are the second substrate after enzymatic hydrolysis of alkyl polyglycosides . Surprisingly, the enzyme alcohol dehydrogenase ADH (E.C . 1.1.1.1) from bakers' yeast could efficiently oxidize a wide variety of aliphatic alcohols and had the highest catalytic specificity with short and medium fatty alcohol substrates, including octanol and decanol.

Biotechniques, 2001 Dec, 31(6), 1280 - 2, 1284, 1286
Vectors for the expression of tagged proteins in Drosophila; Parker L et al.; Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns . If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies . If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue . We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS . These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

Acta Oncol, 2001, 40(6), 702 - 11
The inherited basis of human radiosensitivity; Gatti RA; Certain individuals cannot tolerate 'conventional' doses of radiation therapy . This is known to be true of patients with ataxia-telangiectasia and ligase IV deficiency . Although in vitro testing may not correlate completely with clinical radiosensitivity, fibroblasts and lymphoblasts from patients with both of these disorders have been clearly shown to be radiosensitive . Using a colony survival assay (CSA) to test lymphoblastoid cells after irradiation with 1 Gy, a variety of other genetic disorders have been identified as strong candidates for clinical radiosensitivity, such as Nijmegen breakage syndrome, Mre 11 deficiency, and Fanconi's anemia . These data are presented and considered as a starting-point for the inherited basis of human radiosensitivity.

Gene Expr, 2001, 9(6), 249 - 55
Exploring relationships in gene expressions: a partial least squares approach; Datta S; Microarray technology has revolutionized the way gene functions are monitored . Analysis of microarray data is a fast growing research area that interfaces various disciplines such as biology, biochemistry, computer science, and statistics . While various clustering and classification techniques have been successfully employed to group genes based on the similarity of their expression patterns, much is yet to be learned about the interrelationship of the expression levels among various genes . We approach this problem with a statistical technique called partial least squares that is capable of modeling a large number of variables each with relatively few observations . This property of the partial least squares methodology appears to be attractive for application to microarray data sets where the simultaneous expression levels of many genes are collected each at a few time points (or individuals) . We use it to analyze publicly available microarray data on sporulation of budding yeast (Saccharomyces cerevisiae) . We investigate a number of representative genes, one from each temporal group (based on the time of first induction) of positively expressed genes and show that in each case most of the variability was explained by only two partial regression terms based on all remaining genes . Moreover, the predicted expression levels of the representative genes from partial least squares fit very well on the average with the true expression levels over time . Finally, we compare the biological functions of the genes with largest coefficients with those of the predicted genes . In many cases, the genes are involved in similar or related biological functions including negative relationships . We show that this method can identify established gene relationships; we argue that it can be an exploratory tool for identifying potential gene relationships requiring further biological investigation.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 233 - 8 Epub 2001 Dec 26.
UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma -H2AX formation, and Mre11 relocalization; Limoli CL et al.; UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (gamma-H2AX) in large nuclear foci at sites of stalled replication forks . These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs) . This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest . After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of gamma-H2AX foci that paralleled the production of Mre11 foci . The percentage of foci-positive cells increased rapidly (10-15%) up to fluences of 10 J.(-2) before saturating at higher fluences . Frequencies of gamma-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation . This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of gamma-H2AX foci were found to precede the formation of Mre11 foci by several hours . The nuclear distributions of gamma-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation . UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and gamma-H2AX foci-positive cells . These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest . These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation.

Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 190 - 5 Epub 2001 Dec 26.
Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway; Talloczy Z et al.; The eIF2alpha kinases are a family of evolutionarily conserved serine/threonine kinases that regulate stress-induced translational arrest . Here, we demonstrate that the yeast eIF2alpha kinase, GCN2, the target phosphorylation site of Gcn2p, Ser-51 of eIF2alpha, and the eIF2alpha-regulated transcriptional transactivator, GCN4, are essential for another fundamental stress response, starvation-induced autophagy . The mammalian IFN-inducible eIF2alpha kinase, PKR, rescues starvation-induced autophagy in GCN2-disrupted yeast, and pkr null and Ser-51 nonphosphorylatable mutant eIF2alpha murine embryonic fibroblasts are defective in autophagy triggered by herpes simplex virus infection . Furthermore, PKR and eIF2alpha Ser-51-dependent autophagy is antagonized by the herpes simplex virus neurovirulence protein, ICP34.5 . Thus, autophagy is a novel evolutionarily conserved function of the eIF2alpha kinase pathway that is targeted by viral virulence gene products.

Mol Cell Biol, 2002 Jan, 22(2), 626 - 34
AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation; Rabinovich E et al.; Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway . Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome . Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones . Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD . We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast . The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants . The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response . We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.

Mol Cell Biol, 2002 Jan, 22(2), 614 - 25
Chromatin assembly factor I mutants defective for PCNA binding require Asf1/Hir proteins for silencing; Krawitz DC et al.; Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex . Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing . In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA . Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway . Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes . Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA . We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.

Mol Cell Biol, 2002 Jan, 22(2), 505 - 16
Identification of a multifunctional domain in autonomously replicating sequence-binding factor 1 required for transcriptional activation, DNA replication, and gene silencing; Miyake T et al.; Autonomously replicating sequence-binding factor 1 (ABF1) is a multifunctional, site-specific DNA binding protein that is essential for cell viability in Saccharomyces cerevisiae . ABF1 plays a direct role in transcriptional activation, stimulation of DNA replication, and gene silencing at the mating-type loci . Here we demonstrate that all three activities of ABF1 are conferred by the C terminus of the protein (amino acids {aa} 604 to 731) . Furthermore, a detailed mutational analysis has revealed two important clusters of amino acid residues in the C terminus (C-terminal sequence 1 {CS1}, aa 624 to 628; and CS2, aa 639 to 662) . While both regions play a pivotal role in supporting cell viability, they make distinct contributions to ABF1 functions in various nuclear processes . CS1 specifically participates in transcriptional silencing and/or repression in a context-dependent manner, whereas CS2 is universally required for all three functions of ABF1 . When tethered to specific regions of the genome, a 30-aa fragment that contains CS2 alone is sufficient for activation of transcription and chromosomal replication . In addition, CS2 is responsible for ABF1-mediated chromatin remodeling . Based on these results, we suggest that ABF1 may function as a chromatin-reorganizing factor to increase accessibility of the local chromatin structure, which in turn facilitates the action of additional factors to establish either an active or repressed chromatin state.

Mol Cell Biol, 2002 Jan, 22(2), 430 - 41
Characterization of the ECB binding complex responsible for the M/G(1)-specific transcription of CLN3 and SWI4; Mai B et al.; The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes . In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G(1) phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB) . Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein . Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences . These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element . Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes.

J Cell Biol, 2001 Dec 24, 155(7), 1265 - 73 Epub 2001 Dec 24.
A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition; Coscoy L et al.; Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition {MIR}1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells . This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins . Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface . MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity . In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain . Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

J Cell Biol, 2001 Dec 24, 155(7), 1173 - 84 Epub 2001 Dec 24.
Polyploids require Bik1 for kinetochore-microtubule attachment; Lin H et al.; The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells . Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface . Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids . The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton . In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids . The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics . The finding that a protein involved in kinetochore-MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

J Cell Biol, 2001 Dec 24, 155(7), 1147 - 57 Epub 2001 Dec 24.
CENP-A is phosphorylated by Aurora B kinase and plays an unexpected role in completion of cytokinesis; Zeitlin SG et al.; Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis . We investigated a role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homologue . Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue . Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation) . The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1 . Our data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell . These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis.

J Cell Biol, 2001 Dec 24, 155(7), 1137 - 45 Epub 2001 Dec 24.
Implication of a novel multiprotein Dam1p complex in outer kinetochore function; Cheeseman IM et al.; Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast . Here, we present our purification from yeast extracts of an approximately 245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p . This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle . We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of approximately 0.5 microM . To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19-green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1 . These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

J Cell Biol, 2001 Dec 24, 155(7), 1103 - 7 Epub 2001 Dec 24.
Specific tetraspanin functions; Hemler ME; Relatively little attention has been given to the large family of abundantly expressed transmembrane proteins known as tetraspanins . Now, the importance of tetraspanins is strongly supported by emerging genetic evidence, coupled with new insights into the biochemistry and functions of tetraspanin protein complexes.

J Cell Biol, 2001 Dec 24, 155(7), 1099 - 101 Epub 2001 Dec 24.
Traffic through the Golgi apparatus; Pelham HR; The role of vesicles in cargo transport through the Golgi apparatus has been controversial . Large forms of cargo such as protein aggregates are thought to progress through the Golgi stack by a process of cisternal maturation, balanced by a return flow of Golgi resident proteins in COPI-coated vesicles . However, whether this is the primary role of vesicles, or whether they also serve to transport small cargo molecules in a forward direction has been debated . Two papers (Martinez-Menarguez et al., 2001; Mironov et al., 2001, this issue) use sophisticated light and electron microscopy to provide evidence that the vesicular stomatitis virus membrane glycoprotein (VSV G)* is largely excluded from vesicles in vivo, and does not move between cisternae, whereas resident Golgi enzymes freely enter vesicles as predicted by the cisternal maturation model . Both papers conclude that vesicles are likely to play only a minor role in the anterograde transport of cargo through the Golgi apparatus in mammalian tissue culture cells.

J Biol Chem, 2002 Mar 15, 277(11), 9548 - 61 Epub 2001 Dec 27.
Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells; Mukai S et al.; We previously isolated and characterized a Chinese hamster ovary (CHO) cell mutant, ZPG207, that is defective in import of proteins carrying a peroxisome-targeting signal type 2 (PTS2) nonapeptide . Herein we have cloned Chinese hamster (Cl) PEX7 encoding the PTS2 receptor . ClPex7p consists of 318 amino acids, shorter than human Pex7p by 5 residues, showing 91 and 30% identity with Pex7p from humans and the yeast Saccharomyces cerevisiae, respectively . Expression of ClPEX7 rescued the impaired PTS2 import in pex7 ZPG207 . Mutation in ZPG207 PEX7 was determined by reverse transcription PCR; a G-to-A transition caused a 1-amino acid substitution, W221ter . We investigated the molecular dysfunction of Pex7p variants in mammals, including Pex7p-W221ter and Pex7p with one site mutation at G217R, A218V, or L292ter, which frequently occurs in the human fatal genetic peroxisomal disease rhizomelic chondrodysplasia punctata, showing a cell phenotype of PTS2 import defect . All types of the mutations affected Pex7p in binding to both PTS2 cargo protein and the longer isoform of PTS1 receptor Pex5pL that is responsible for transport of the Pex7p-PTS2 complex . Subcellular fractionation and protease protection studies demonstrated bimodal distribution of Pex7p between the cytoplasm and peroxisomes in CHO and human cells . Moreover, expression of Pex5pL, but not of the shorter isoform Pex5pS, enhanced translocation of Pex7p-PTS2 proteins into peroxisomes, thereby implying that both PTS receptors shuttle between peroxisomes and the cytosol . Furthermore, a ClPex7p mutant with a deletion of 7 amino acids from the N terminus retained peroxisome-restoring activity, whereas an 11-amino acid truncation abrogated the activity . ClPex7p with a C-terminal 9- amino acid truncation, comprising residues 1--309, maintained the activity, whereas a 14-amino acid shorter form lacking several amino acids of the sixth WD motif lost the activity . Therefore, nearly the full length of Pex7p, including all WD motifs, is required for its function.

Blood, 2002 Jan 1, 99(1), 275 - 81
The MLL fusion partner AF10 binds GAS41, a protein that interacts with the human SWI/SNF complex; Debernardi S et al.; The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus . AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy . The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10 . This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library . The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma . GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL . The interaction was confirmed in vivo . Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate . INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription . The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes.

FEBS Lett, 2002 Jan 2, 510(1-2), 27 - 30
Introns in protein-coding genes in Archaea; Watanabe Y et al.; Introns in protein-coding genes are ubiquitous in eukaryotic cells, but pre-mRNA splicing has yet to be reported in archaeal and its viral genomes . We present evidence of introns in genes encoding a homolog of eukaryotic Cbf5p (centromere-binding factor 5; a subunit of a small nucleolar ribonucleoprotein) in three Archaea; Aeropyrum pernix, Sulfolobus solfataricus and Sulfolobus tokodaii . Splicing of pre-mRNAs in vivo was demonstrated by reverse transcriptase-mediated polymerase chain reaction . The exon-intron boundaries of these genes are predicted to be folded into a structure similar to the bulge-helix-bulge motif, suggesting that splicing of these pre-mRNAs probably depends on the splicing system elucidated for archaeal pre-tRNAs and rRNAs.

Radiat Res, 2002 Jan, 157(1), 19 - 25
An antisense construct of full-length human RAD50 cDNA confers sensitivity to ionizing radiation and alkylating agents on human cell lines; Kim YC et al.; In Saccharomyces cerevisiae, Rad50 is reported to participate in the repair of double-stranded DNA breaks, and most rad50 mutants are unable to repair gamma-ray-induced DNA damage . In this study, we examined whether human RAD50 is involved in the repair of DNA damage induced by gamma radiation, radiomimetic alkylating agents, or UVB radiation in cultured human cells . Because homozygous null RAD50 mutant cells could not be isolated, human 293 embryonic kidney cells and A431 epithelial tumor cells were transfected with antisense RAD50 cDNA to obtain viable cell lines which expressed reduced RAD50 . Selected individual clones were subjected to PCR-Southern and Western blot analyses to confirm the integrity of the antisense RAD50 construct and the reduced RAD50 expression levels . The cells engineered to express reduced RAD50 levels showed significantly increased sensitivity to gamma radiation, mitomycin C and methylmethane sulfonate compared with control cells that were transfected with the vector alone . However, there were no differences in viability of cells with reduced RAD50 levels and control cells treated with UVB radiation . These results indicate that human RAD50 is involved in the repair of DNA damage induced by gamma radiation and alkylating agents in mammalian cells and suggest the possible application of antisense RAD50 cDNA transfection as a radiation sensitizer in radiation oncology.

J Nat Prod, 2001 Dec, 64(12), 1576 - 8
Four new bioactive pyrrole-derived alkaloids from the marine sponge Axinella brevistyla; Tsukamoto S et al.; Four new alkaloids (1-4) were isolated from the marine sponge Axinella brevistyla, and their structures were determined on the basis of spectroscopic analysis . The alkaloids 1-4 were antifungal against the yeast Saccharomyces cerevisiae at <1.0, <1.0, 30, and 100 microg/disk, respectively . Compounds 1-3 also exhibited cytotoxicity against L1210 cells with IC(50) values of 1.1, 0.66, and 2.5 microg/mL, respectively.

Nat Biotechnol, 2002 Jan, 20(1), 83 - 7
Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed); Bevis BJ et al.; The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP) . But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility . To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10-15 times faster than the wild-type protein . An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission . Additional amino acid substitutions suppress this green emission while further accelerating the maturation . To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein . The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.

Sci STKE . 2001 Jul 31;2001(93):PE1.
HOG on the promoter: regulation of the osmotic stress response; Chellappan SP; Members of the mitogen-activated protein (MAP) kinase family regulate transcription through phosphorylation of specific transcription factors . New studies indicate that this process may be more complex than previously anticipated . The yeast Hog1 protein kinase (a homolog of the mammalian p38 MAP kinase) interacts with transcription factors and perhaps with the general transcription machinery at target promoters . Chellappan discusses the recent results and their implications for understanding control of transcription by stress-activated MAP kinases.

Sci STKE . 2000 Oct 03;2000(52):PE1.
What do scaffold proteins really do?
Ferrell JE Jr.
Scaffold proteins play an important role in coordinating signal transduction cascades . However, their exact mechanism of action and the ultimate effect they have on the signal output remain unclear . Ferrell discusses how computer simulations have provided insight into the multiple possible functions that scaffold proteins may have . What remains is to test the predictions in real cells to determine what difference the presence of a scaffold really makes in the output of a signaling pathway.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15360 - 5
Tetrahydrofolate biosynthesis in plants: molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana; Ravanel S et al.; Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated . In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule . In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana . The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate . DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell . In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast . Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes . The compartmentation of FPGS isoforms is in agreement with the predominance of gamma-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids . Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism . Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15113 - 8
Identification of a small molecule inhibitor of Sir2p; Bedalov A et al.; Sir2p is an NAD(+)-dependent histone deacetylase required for chromatin-dependent silencing in yeast . In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae . Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA . In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p . In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein . Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein . By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.

Plant Cell, 2001 Dec, 13(12), 2703 - 17
New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis; Perez-Amador MA et al.; In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis . The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts . As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes . These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype . Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants . In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1 . RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants . These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses . Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

Plant Cell, 2001 Dec, 13(12), 2687 - 702
Tomato SP-interacting proteins define a conserved signaling system that regulates shoot architecture and flowering; Pnueli L et al.; Divergent architecture of shoot models in flowering plants reflects the pattern of production of vegetative and reproductive organs from the apical meristem . The SELF-PRUNING (SP) gene of tomato is a member of a novel CETS family of regulatory genes (CEN, TFL1, and FT) that controls this process . We have identified and describe here several proteins that interact with SP (SIPs) and with its homologs from other species: a NIMA-like kinase (SPAK), a bZIP factor, a novel 10-kD protein, and 14-3-3 isoforms . SPAK, by analogy with Raf1, has two potential binding sites for 14-3-3 proteins, one of which is shared with SP . Surprisingly, overexpression of 14-3-3 proteins partially ameliorates the effect of the sp mutation . Analysis of the binding potential of chosen mutant SP variants, in relation to conformational features known to be conserved in this new family of regulatory proteins, suggests that associations with other proteins are required for the biological function of SP and that ligand binding and protein-protein association domains of SP may be separated . We suggest that CETS genes encode a family of modulator proteins with the potential to interact with a variety of signaling proteins in a manner analogous to that of 14-3-3 proteins.

Plant Cell, 2001 Dec, 13(12), 2671 - 86
Expression and stability of Arabidopsis CDC6 are associated with endoreplication; Castellano MM et al.; Studies on the CDC6 protein, which is crucial to the control of DNA replication in yeast and animal cells, are lacking in plants . We have isolated an Arabidopsis cDNA encoding the AtCDC6 protein and studied its possible connection to the occurrence of developmentally regulated endoreplication cycles . The AtCDC6 gene is expressed maximally in early S-phase, and its promoter contains an E2F consensus site that mediates the binding of a plant E2F/DP complex . Transgenic plants carrying an AtCDC6 promoter-beta-glucuronidase fusion revealed that it is active in proliferating cells and, interestingly, in endoreplicating cells . In particular, the extra endoreplication cycle that occurs in dark-grown hypocotyl cells is associated with upregulation of the AtCDC6 gene . This was corroborated using ctr1 Arabidopsis mutants altered in their endoreplication pattern . The ectopic expression of AtCDC6 in transgenic plants induced endoreplication and produced a change in the somatic ploidy level . AtCDC6 was degraded in a ubiquitin- and proteosome-dependent manner by extracts from proliferating cells, but it was degraded poorly by extracts from dark-grown hypocotyl endoreplicating cells . Our results indicate that endoreplication is associated with expression of the AtCDC6 gene and, most likely, the stability of its product; it also apparently requires activation of the retinoblastoma/E2F/DP pathway . These conclusions may apply to endoreplicating cells in other tissues of the plant and to endoreplicating cells in other eukaryotes.

Plant Cell, 2001 Dec, 13(12), 2573 - 87
The signaling mechanism of Arabidopsis CRY1 involves direct interaction with COP1; Yang HQ et al.; Dark-grown transgenic Arabidopsis seedlings expressing the C-terminal domains (CCT) of the cryptochrome (CRY) blue light photoreceptors exhibit features that are normally associated only with light-grown seedlings, indicating that the signaling mechanism of Arabidopsis CRY is mediated through CCT . The phenotypic properties mediated by CCT are remarkably similar to those of the constitutive photomorphogenic1 (cop1) mutants . Here we show that Arabidopsis cryptochrome 1 (CRY1) and its C-terminal domain (CCT1) interacted strongly with the COP1 protein . Coimmunoprecipitation studies showed that CRY1 was bound to COP1 in extracts from both dark- and light-grown Arabidopsis . An interaction also was observed between the C-terminal domain of Arabidopsis phytochrome B and COP1, suggesting that phytochrome signaling also proceeds, at least in part, through direct interaction with COP1 . These findings give new insight into the initial step in light signaling in Arabidopsis, providing a molecular link between the blue light receptor, CRY1, and COP1, a negative regulator of photomorphogenesis.

Mol Biol Evol, 2002 Jan, 19(1), 39 - 48
A homologue of CROC-1 in a ciliated protist (Sterkiella histriomuscorum) testifies to the ancient origin of the ubiquitin-conjugating enzyme variant family; Villalobo E et al.; Resting cysts of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) have been shown to contain messenger RNA, one of which codes for a protein significantly similar to CROC-1 . CROC-1 is a human regulatory protein capable of transactivating the promoter of c-fos and belongs to a newly characterized family of ubiquitin-conjugating enzyme (E2) variants (UEV) . We have determined the corresponding macronuclear gene sequence, which is the first protistan UEV sequence available . The phylogenetic analysis indicates the deep separation and solid clustering of all the UEV sequences within the E2 tree showing the ancient origin of these regulatory genes and their high structural conservation during evolution . Furthermore, overexpression of the ciliate UEV is able to rescue the Saccharomyces cerevisiae mms2 null mutant from killing by DNA damaging agents, implying that the UEV family proteins are functionally conserved . In S . histriomuscorum, expression of UEV is correlated with the growth of the cells as transcripts are present in excysting and vegetative cells but are rapidly down-regulated during starvation . These data support the high conservation of the UEV family in eukaryotes, and a regulatory role of the gene is discussed in relation to known functions of UEVs . This analysis may promote the search for homologues of other regulatory genes (metazoan regulators of differentiation) in ciliates.

J Biol Chem, 2002 Feb 22, 277(8), 6719 - 25 Epub 2002 Jan 07.
GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor; Booth RA et al.; Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway . We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP) . Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC . Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation . While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes . This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.

Genes Dev, 2001 Dec 15, 15(24), 3319 - 29
Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain; Cho EJ et al.; The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription . Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription . At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH . However, in coding regions, the CTD is predominantly phosphorylated at Ser 2 . Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb . Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation . Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase . Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation . Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.

Genes Dev, 2001 Dec 15, 15(24), 3308 - 18
Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange; Sigurdsson S et al.; Five Rad51-like proteins, referred to as Rad51 paralogs, have been described in vertebrates . We show that two of them, Rad51B and Rad51C, are associated in a stable complex . Rad51B-Rad51C complex has ssDNA binding and ssDNA-stimulated ATPase activities . We also examined the functional interaction of Rad51B-Rad51C with Rad51 and RPA . Even though RPA enhances Rad51-catalyzed DNA joint formation via removal of secondary structure in the ssDNA substrate, it can also compete with Rad51 for binding to the substrate, leading to suppressed reaction efficiency . The competition by RPA for substrate binding can be partially alleviated by Rad51B-Rad51C . This recombination mediator function of Rad51B-Rad51C is likely required for the assembly of the Rad51-ssDNA nucleoprotein filament in vivo.

Genes Dev, 2001 Dec 15, 15(24), 3278 - 85
Emi1 regulates the anaphase-promoting complex by a different mechanism than Mad2 proteins; Reimann JD et al.; The anaphase-promoting complex/cyclosome (APC) ubiquitin ligase is activated by Cdc20 and Cdh1 and inhibited by Mad2 and the spindle assembly checkpoint complex, Mad2B, and the early mitotic inhibitor Emi1 . Mad2 inhibits APC(Cdc20), whereas Mad2B preferentially inhibits APC(Cdh1) . We have examined the mechanism of APC inhibition by Emi1 and find that unlike Mad2 proteins, Emi1 binds and inhibits both APC(Cdh1) and APC(Cdc20) . Also unlike Mad2, Emi1 stabilizes cyclin A in the embryo and requires zinc for its APC inhibitory activity . We find that Emi1 binds the substrate-binding region of Cdc20 and prevents substrate binding to the APC, illustrating a novel mechanism of APC inhibition.

Genes Dev, 2001 Dec 15, 15(24), 3237 - 42
Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells; Pierce AJ et al.; Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site . To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components . We find that the HDR frequency is enhanced in Ku70(-/-), XRCC4(-/-), and DNA-PKcs(-/-) cells, with the increase being particularly striking in Ku70(-/-) cells . Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins . A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.

EMBO Rep, 2002 Jan, 3(1), 50 - 5 Epub 2001 Dec 19.
HAT activity is essential for CBP-1-dependent transcription and differentiation in Caenorhabditis elegans; Victor M et al.; The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains . A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling . However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear . We addressed the importance of the HAT activity of CBP-1, the worm ortholog of p300/CBP, in Caenorhabditis elegans with three different and complementary approaches . These include allele-specific RNA-mediated interference (RNAi), genetic rescue and the use of a specific chemical inhibitor of the p300/CBP HAT . Our findings demonstrate that HAT activity is of primary importance for CBP-1 to regulate transcription and to promote differentiation during C . elegans embryogenesis.

Cancer Res, 2001 Dec 15, 61(24), 8723 - 9
Interaction of p53 and DNA-PK in response to nucleoside analogues: potential role as a sensor complex for DNA damage; Achanta G et al.; Therapeutic nucleoside analogues such as ara-C, gemcitabine, and fludarabine exert their cytotoxic activity against cancer cells mainly by incorporation into DNA and disruption of further DNA synthesis, resulting in the triggering of apoptosis . However, the molecules that recognize the incorporated analogues in DNA and subsequently initiate the downstream cellular responses remain to be identified . Here, we report that the DNA-dependent protein kinase (DNA-PK) and p53 are able to form a protein complex that interacts with the gemcitabine-containing DNA and plays a role in signaling to apoptotic pathways . DNA-PK/Ku and p53 were copurified in a protein fraction that binds to gemcitabine-containing DNA in preference to normal DNA . Immunoprecipitation experiments revealed that the two proteins physically associate in a complex . Treatment with gemcitabine resulted in an increase of DNA-PK and p53 protein and an increase in the phosphorylation of p53 at Ser15 . Furthermore, confocal microscopy demonstrated a colocalization of DNA-PK and p53 to the nucleus in cells treated with gemcitabine . The nuclear localization of the DNA-PK/p53 complex was coincident with the induction of apoptosis in these cells . Although the wild-type p53 present in the protein complex exhibited 3'-5' exonuclease activity, it was incapable of excising the incorporated gemcitabine from DNA . The binding of the p53/DNA-PK complex to DNA substantially blocked further DNA synthesis by DNA polymerases alpha and epsilon in vitro, indicating a stalling of this complex at the site of drug incorporation . These data suggest that DNA-PK and p53 may form a sensor complex that detects the disruption of DNA replication caused by nucleoside analogue incorporation and may subsequently signal for apoptosis.

Biophys J, 2002 Jan, 82(1 Pt 1), 335 - 44
Molecular stretching of long DNA in agarose gel using alternating current electric fields; Kaji N et al.; We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields . The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy . The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated . Stretching was only observed in the AC field when a frequency of approximately 10 Hz was used . The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used . Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3% . Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose . Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb) . These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell.

Biophys J, 2002 Jan, 82(1 Pt 1), 99 - 108
Control analysis for autonomously oscillating biochemical networks; Reijenga KA et al.; It has hitherto not been possible to analyze the control of oscillatory dynamic cellular processes in other than qualitative ways . The control coefficients, used in metabolic control analyses of steady states, cannot be applied directly to dynamic systems . We here illustrate a way out of this limitation that uses Fourier transforms to convert the time domain into the stationary frequency domain, and then analyses the control of limit cycle oscillations . In addition to the already known summation theorems for frequency and amplitude, we reveal summation theorems that apply to the control of average value, waveform, and phase differences of the oscillations . The approach is made fully operational in an analysis of yeast glycolytic oscillations . It follows an experimental approach, sampling from the model output and using discrete Fourier transforms of this data set . It quantifies the control of various aspects of the oscillations by the external glucose concentration and by various internal molecular processes . We show that the control of various oscillatory properties is distributed over the system enzymes in ways that differ among those properties . The models that are described in this paper can be accessed on http://jjj.biochem.sun.ac.za.

Bioinformatics, 2001 Dec, 17(12), 1143 - 51
Statistical estimation of cluster boundaries in gene expression profile data; Horimoto K et al.; MOTIVATION: Gene expression profile data are rapidly accumulating due to advances in microarray techniques . The abundant data are analyzed by clustering procedures to extract the useful information about the genes inherent in the data . In the clustering analyses, the systematic determination of the boundaries of gene clusters, instead of by visual inspection and biological knowledge, still remains challenging . RESULTS: We propose a statistical procedure to estimate the number of clusters in the hierarchical clustering of the expression profiles . Following the hierarchical clustering, the statistical property of the profiles at the node in the dendrogram is evaluated by a statistics-based value: the variance inflation factor in the multiple regression analysis . The evaluation leads to an automatic determination of the cluster boundaries without any additional analyses and any biological knowledge of the measured genes . The performance of the present procedure is demonstrated on the profiles of 2467 yeast genes, with very promising results . AVAILABILITY: A set of programs will be electronically sent upon request . CONTACT: horimoto@post.saga-med.ac.jp; toh@beri.co.jp

Toxicology, 2002 Jan 15, 170(1-2), 153 - 61
Estrogenic and anti-estrogenic activities of two types of diesel exhaust particles; Taneda S et al.; In an earlier study using a recombinant yeast screen we found that a suspension of diesel exhaust particles (DEP) and some extracts of DEP are not estrogenic but possess anti-estrogenic activity . In the present study, estrogenic and anti-estrogenic activities of two types of DEP, type-1 (old type) and type-2 (new type) were compared . Whole DEP of both types were found to possess estrogenic and anti-estrogenic activities . The DEP were serially extracted with organic solvents and then with 1 M ammonia and 1 M HCl . In type-2 DEP, the ratio of dry weight of a hexane extract was higher than those of methanol and ammonia extracts, which were lower than those in type-1 DEP . In the hexane extract, estrogenic activity was found in both types of DEP . In the benzene and dichloromethane extracts, estrogenic and anti-estrogenic activities were found in both types of DEP . In the methanol extract, estrogenic activity was found in type-2 DEP, and extracts of both types decreased the activity of estrogen . Anti-estrogenic activity was found in extracts of ammonia and HCl from both types of DEP . It was found that both type-1 and type-2 DEP possess estrogenic and anti-estrogenic activities.

Biochim Biophys Acta, 2001 Dec 3, 1522(2), 122 - 9
Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2); Pirtle IL et al.; Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Delta-12 fatty acid desaturase (FAD2-3) gene . A partial FAD2-3 cDNA clone was also analyzed . The FAD2-3 gene has one large intron of 2967 bp entirely within its 5'-untranslated region, only 12 bp upstream from the ATG initiation codon . Several potential promoter elements, including several light-responsive motifs, occur in the 5'-flanking region . The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids . The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum . Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

FEBS Lett, 2001 Dec 14, 509(3), 370 - 4
Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At); Mohlmann T et al.; Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine . This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1 . Yeast cells expressing ENT1,At are able to grow on adenosine-containing media, adenosine import exhibited an apparent affinity (K(M)) of 3.6 microM, and led to accumulation of this nucleoside within the yeast cell . Transport is inhibited by various nucleosides . Typical inhibitors of ENT-type nucleoside transporters do not inhibit (3)H-adenosine import . The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton-dependent adenosine transport . This is the first functional characterisation of a plant nucleoside transport protein.

J Biol Chem, 2002 Feb 8, 277(6), 3813 - 22 Epub 2001 Dec 03.
Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin; Woo MH et al.; DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT) . Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development . However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear . To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast . Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3 . This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes . Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722 . The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected . Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3 . These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT . Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.

J Biol Chem, 2002 Feb 1, 277(5), 3412 - 8 Epub 2001 Dec 03.
A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5; Saito K et al.; The small GTPase Rab family, which cycles between GTP-bound active and GDP-bound inactive states, plays an important role in membrane trafficking . Among them, Rab5 is involved in early endocytic pathway, and several Rab5-binding proteins have been identified as regulators or effectors to coordinate the docking and fusion processes of endocytic vesicles . We describe a novel binding protein exhibiting unique biochemical properties for Rab5 . The Rab5-binding protein enhances GDP-GTP exchange reaction on Rab5 but preferentially interacts with its GTP-bound form . Gel filtration and immunoprecipitation analyses indicate that the Rab5-binding protein functions as a tetramer composed of anti-parallel linkage of two parallel dimers . These results suggest that the newly identified protein may function as an upstream activator and/or downstream effector for Rab5 in endocytic pathway . Possible roles of the quaternary structure have been discussed in terms of the Rab5-mediated signaling.

J Biol Chem, 2002 Feb 15, 277(7), 4918 - 24 Epub 2001 Nov 30.
Ku represses the HIV-1 transcription: identification of a putative Ku binding site homologous to the mouse mammary tumor virus NRE1 sequence in the HIV-1 long terminal repeat; Jeanson L et al.; Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance . Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression . Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses . The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription . First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80 . LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells . This enhancement was suppressed after re-expression of Ku80 . Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA . Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells . These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression . Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

Eur J Biochem, 2001 Dec, 268(23), 6311 - 7
Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid; Hayashi H et al.; An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem . by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase . Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene . This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells . The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases . The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures . Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages . Induction of this mRNA preceded accumulation of bryonolic acid . In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle . These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

Yeast, 2001 Dec, 18(16), 1525 - 36
Characterization of the sec1-1 and sec1-11 mutations; Brummer MH et al.; Sec1 proteins are implicated in positive and negative regulation of SNARE complex formation . To better understand the function of Sec1 proteins we have identified the nature of the temperature-sensitive mutations in sec1-1 and sec1-11 . The sec1-1 mutation changes a conserved glycine(443) to glutamic acid . The sec1-11 mutation changes a highly conserved arginine(432) to proline . Based on homology and the crystal structure of the mammalian nSec1p, the corresponding amino acids localize to the 3b domain of nSec1p . Compared to the wild-type Sec1p the mutant proteins are less abundant even at the permissive temperature . Thus, the R432P and G443E mutations may cause structural alterations that affect folding and make the mutant proteins more susceptible to degradation . The remaining part is sufficient for growth and protein secretion at 24 degrees C and thus is likely to be properly folded . At 37 degrees C the mutant proteins become non-functional . In pulse-chase-type experiments the newly synthesized Sec1-1 and Sec1-11 proteins decayed similarly with the wild-type protein . Thus, the non-functionality of the mutant proteins cannot be explained by denaturation-induced degradation only . It is possible that the newly synthesized mutant proteins fold slowly and are susceptible to degradation before they have managed to fold and associate with other proteins . The mutant proteins were unable to interact with the Sec1p-interacting proteins Mso1p and Sso2p in the two-hybrid assay, even at the permissive temperature . These results localize sec1-1 and sec1-11 mutations to a domain of Sec1p and suggest a mechanism by which sec1-1 and sec1-11 cells become temperature-sensitive .

Anat Rec, 2002 Jan 1, 266(1), 1 - 4
Comparative analysis of Prx1 and Prx2 expression in mice provides evidence for incomplete compensation; Chesterman ES et al.; The family of paired-related homeobox genes to which Prx1 and Prx2 belong plays an integral role in limb and craniofacial development, as evidenced by both transgenic mice and in situ hybridization data . However, little is known about protein expression of these homeodomain transcription factors . Recent studies in our lab have established the pattern of Prx1 protein expression during normal mouse embryogenesis . Here we present a comparative analysis of Prx1 and Prx2 protein expression in the developing mouse using a novel anti-Prx2 antibody reagent .

J Biol Chem, 2002 Mar 1, 277(9), 7029 - 36 Epub 2001 Dec 17.
Functional interaction between coactivators CBP/p300, PCAF, and transcription factor FKLF2; Song CZ et al.; The Sp1/KLF family of factors regulates diverse cellular processes, including growth and development . Fetal Kruppel-like factor (FKLF2) is a new member of this family . In this study, we characterized the coactivators involved in FKLF2 transcriptional activation . Our results show that both CBP/p300 and p300/CBP-associated factor (PCAF) enhance FKLF2 transcriptional activity . We demonstrate that the acetyltransferase activity of PCAF but not that of CBP/p300 is required for stimulating FKLF2 transcription activity . We further show that p300 and PCAF act cooperatively in stimulating FKLF2 transcriptional activation . FKLF2 interacts with both CBP and PCAF through specific domains, and CBP and PCAF acetylate FKLF2 . Both CBP/p300 and PCAF stimulate FKLF2 DNA binding activity . The integrity of the acetyltransferase domain of PCAF but not that of CBP/p300 is required for stimulating FKLF2 DNA binding activity . These results demonstrate that CBP/p300 and PCAF stimulate FKLF2 transcriptional activity at least by enhancing its DNA binding . The acetyltransferase activities of CBP/p300 and PCAF play a distinct role in stimulating FKLF2 transcription and DNA binding.

J Biol Chem, 2002 Mar 1, 277(9), 7002 - 9 Epub 2001 Dec 17.
In vivo changes of nucleosome positioning in the pretranscription state; Di Mauro E et al.; The involvement of chromatin structure and organization in transcriptional regulatory pathways has become evident . One unsolved question concerns the molecular mechanisms of chromatin remodeling during in vivo promoter activation . By using a high resolution in vivo analysis we show that when yeast cells are exposed to a regulatory signal the positions of specific nucleosomes change . The system analyzed consists of the basic elements of the Saccharomyces cerevisiae ADH2 promoter, two nucleosomes of which are shown to change the distribution of their positions by few nucleotides in the direction of transcription when the glucose content of the medium is lowered . Such repositioning does not occur in the absence of the ADH2 transcriptional activator Adr1 or in the presence of its DNA-binding domain alone . A construct consisting of the DNA-binding domain plus a 43-amino acid peptide containing the Adr1 activation domain is sufficient to induce the same effect of the full-length protein . Nucleosome repositioning occurs even when the catalytic activity of the RNA polymerase II is impaired, suggesting that the Adr1 activation domain mediates the recruitment of some factor to correctly preset the relevant sequences for the subsequent transcription steps.

Cell, 2001 Dec 14, 107(6), 751 - 62
Functional link between the mammalian exosome and mRNA decapping; Wang Z et al.; Mechanistic understanding of mammalian mRNA turnover remains incomplete . We demonstrate that the 3' to 5' exoribonuclease decay pathway is a major contributor to mRNA decay both in cells and in cell extract . An exoribonuclease-dependent scavenger decapping activity was identified that follows decay of the mRNA and hydrolyzes the residual cap . The decapping activity is associated with a subset of the exosome proteins in vivo, implying a higher-order degradation complex consisting of exoribonucleases and a decapping activity, which together coordinate the decay of an mRNA . These findings indicate that following deadenylation of mammal mRNA, degradation proceeds by a coupled 3' to 5' exoribonucleolytic activity and subsequent hydrolysis of the cap structure by a scavenger decapping activity.

J Comput Biol, 2001, 8(6), 625 - 37
Assessing gene significance from cDNA microarray expression data via mixed models; Wolfinger RD et al.; The determination of a list of differentially expressed genes is a basic objective in many cDNA microarray experiments . We present a statistical approach that allows direct control over the percentage of false positives in such a list and, under certain reasonable assumptions, improves on existing methods with respect to the percentage of false negatives . The method accommodates a wide variety of experimental designs and can simultaneously assess significant differences between multiple types of biological samples . Two interconnected mixed linear models are central to the method and provide a flexible means to properly account for variability both across and within genes . The mixed model also provides a convenient framework for evaluating the statistical power of any particular experimental design and thus enables a researcher to a priori select an appropriate number of replicates . We also suggest some basic graphics for visualizing lists of significant genes . Analyses of published experiments studying human cancer and yeast cells illustrate the results.

Biochemistry, 2001 Dec 25, 40(51), 15846 - 55
EPR-detected folding kinetics of externally located cysteine-directed spin-labeled mutants of iso-1-cytochrome c; DeWeerd K et al.; We report the application of our newly developed dielectric resonator-based flow and stopped-flow kinetic EPR systematically to probe protein folding in yeast iso-1-cytochrome c at cysteine-directed spin-labeled locations . The locations studied have not been previously directly probed by other techniques, and we observe them on a time scale stretching from 50 micros to seconds . On the basis of crystal structure and homology information, the following mutation-tolerant, externally located cysteine labeling sites were chosen (in helices, T8C, E66C, and N92C; in loops, E21C, V28C, H39C, D50C, and K79C), and labeling at these sites was not destabilizing . Dilution of denaturant was used to induce folding and thereby to cause a change in the spin label EPR signal as folding altered the motion of the spin label . Under folding conditions, including the presence of imidazole to eliminate kinetic trapping due to heme misligation, a phase of folding on the 20-30 ms time scale was found . This phase occurred not only at the T8C and N92C labeling sites in the N- and C-terminal helices, where such a phase has been associated with folding in these helices, but overall at labeling sites throughout the protein . In the absence of imidazole the 20-30 ms phase disappeared, and another phase having the time scale of 1 s appeared throughout the protein . There was evidence under all conditions for a burst phase on a scale of less than several milliseconds which occurred at labeling positions V28C, H39C, D50C, E66C, and K79C in the middle of the protein sequence . At spin-labeled D50C rapid-mix flow EPR indicated a very short approximately 50 micros phase possibly associated with the prefolding or compaction of the loop to which D50 belongs . Spin labels have been criticized as perturbing the phenomena which they measure, but our spin labeling strategy has reported common kinetic themes and not perturbed, disconnected kinetic events.

Biochemistry, 2001 Dec 25, 40(51), 15725 - 32
Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis; Dougherty CA et al.; We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin . In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-tubulin exchangeable E site . Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by at least 15-fold, compared to tubulin isolated from control wild-type cells . The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs . The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with the effects of the tub2-T143G mutation in haploid cells . The average length of MT spindles in large-budded mutant cells was only 3.7 +/- 0.2 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells . There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures (30% versus 12% for wild-type cells), again suggesting such a delay . The results show that beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras) . The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.

Biochemistry, 2001 Dec 25, 40(51), 15699 - 706
Determinants of the broad recognition of exocytic Rab GTPases by Mss4; Zhu Z et al.; Rab GTPases function as essential regulators of vesicle transport between subcellular compartments of eukaryotic cells . Mss4, an evolutionarily conserved Rab accessory factor, facilitates nucleotide release and binds tightly to the nucleotide-free form of exocytic but not endocytic Rab GTPases . A structure-based mutational analysis of residues that are conserved only in exocytic Rab GTPases reveals three residues that are critical determinants of the broad specificity recognition of exocytic Rab GTPases by Mss4 . One of these residues is located at the N-terminus of the switch I region near the nucleotide binding site whereas the other two flank an exposed hydrophobic triad previously implicated in effector recognition . The spatial disposition of these residues with respect to the structure of Rab3A correlates with the dimensions of the elongated Rab interaction epitope in Mss4 and supports a mode of interaction similar to that of other exchange factor-GTPase complexes . The complementarity of the corresponding interaction surfaces suggests a hypothetical structural model for the complex between Mss4 and Rab GTPases.

Biochemistry, 2001 Dec 25, 40(51), 15456 - 63
Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions; Sauve AA et al.; The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation . New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2 . The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3') . The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods . The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs . This mechanism is unprecedented in ADP-ribosyl transferase enzymology . The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.

Exp Eye Res, 2001 Nov, 73(5), 703 - 10
Regulatory elements in the rat betaB2-crystallin promoter; Doerwald L et al.; The suggested common regulator of the eye lens crystallin genes is c-Maf . Maf responsive elements have been detected in a number of crystallin promoters including that of the rat betaB2-crystallin gene . The betaB2-crystallin gene is active in the post-natal lens and its mRNA reaches its maximal level in the rat lens 6 months after birth . Yet c-Maf has been reported to be present in the rat lens only up to 3 months of age . This discrepancy prompted an investigation into the role of the Maf responsive element (MARE) in the regulation of activity of the rat betaB2-crystallin promoter in rat lens fiber cells . Although betaB2 promoter activity is enhanced by c-Maf in both in vitro differentiating rat lens fiber cells and CHO cells, deletion of the betaB2 MARE, which was mapped to -143/-123, does not decrease betaB2 promoter activity in lens fiber cells . Furthermore, a dominant negative c-Maf construct did not inhibit activity of the betaB2 promoter in lens fiber cells . The data suggest that the betaB2 MARE does not play a major role in regulating activity of the betaB2 promoter . Rather, a putative Sox binding site at -164/-159 and a positive element at -14/-7 seem to be the prime regulatory elements . (C) 2001 Academic Press.

Arch Biochem Biophys, 2002 Jan 1, 397(1), 40 - 50
Maize seryl-tRNA synthetase: specificity of substrate recognition by the organellar enzyme; Rokov-Plavec J et al.; In our study of seryl-tRNA formation in maize, we investigated the enzymes involved in serylation . Only two dissimilar seryl-tRNA synthetase (SerRS) cDNA clones were identified in the Zea mays EST (expressed sequence tag) databases . One encodes a seryl-tRNA synthetase, which presumably functions in the organelles (SerZMm), while the other synthetase product is more similar to eukaryotic cytosolic counterparts (SerZMc) . The expression of SerZMm in Saccharomyces cerevisiae resulted in complementation of mutant respiratory phenotype, caused by a disruption of the nuclear gene, presumably encoding yeast mitochondrial seryl-tRNA synthetase (SerSCm) . Purified mature SerZMm displays tRNA-assisted serine activation and aminoacylates maize mitochondrial and chloroplast tRNA(Ser) transcripts with similar efficiencies, raising the possibility that only two isoforms of seryl-tRNA synthetase may be sufficient to catalyze seryl-tRNA(Ser) formation in three cellular compartments of Zea mays . Phylogenetic analysis suggests that SerZMm is of mitochondrial origin.

Genes Chromosomes Cancer, 2002 Jan, 33(1), 36 - 46
Identification and functional characterization of the promoter region of the human MSH6 gene; Szadkowski M et al.; Postreplicative mismatch repair (MMR) corrects polymerase errors arising during DNA replication . Consistent with this role, the Saccharomyces cerevisiae MMR genes MSH2, MSH6, and PMS1 were reported to be transcriptionally upregulated during late G(1) phase of the cell cycle . Surprisingly, despite the high degree of conservation of the MMR system in evolution, the human MMR genes studied to date, MSH2, MLH1, and PMS2, appear to be transcribed from classical housekeeping promoters, and the amounts of the polypeptides encoded by them fluctuate little during the cell cycle . Only the amounts of the 160-kDa MSH6 protein were reported to vary, both during development and following stimulation of cell growth . Moreover, transcription of this gene was found to be downregulated by CpG methylation of the promoter region in a subset of clones treated with alkylating agents . In an attempt to understand the molecular basis underlying these phenomena, we isolated the 5' region of the MSH6 gene and subjected it to functional analysis . We now show that the MSH6 gene is also transcribed from a classical housekeeping gene promoter . Despite housing putative binding sites for the transcription factors AP1, NF-kappaB, and MTF-1, the MSH6 promoter failed to respond to ionizing radiation or heavy metals . Interestingly, MSH6 transcription was upregulated during late G(1) phase, even though the levels of the protein remained essentially constant during the cell cycle.

Environ Mol Mutagen, 2001, 38(2-3), 166 - 74
Improved method for measuring the ensemble average of strand breaks in genomic DNA; Bespalov VA et al.; The cis-syn cyclobutane pyrimidine dimer (CPD) is the major photoproduct induced in DNA by low wavelength ultraviolet radiation . An improved method was developed to detect CPD formation and removal in genomic DNA that avoids the problems encountered with the standard method of endonuclease detection of these photoproducts . Since CPD-specific endonucleases make single-strand cuts at CPD sites, quantification of the frequency of CPDs in DNA is usually done by denaturing gel electrophoresis . The standard method of ethidium bromide staining and gel photography requires more than 10 microg of DNA per gel lane, and correction of the photographic signal for the nonlinear film response . To simplify this procedure, a standard Southern blot protocol, coupled with phosphorimage analysis, was developed . This method uses random hybridization probes to detect genomic sequences with minimal sequence bias . Because of the vast linearity range of phosphorimage detection, scans of the signal profiles for the heterogeneous population of DNA fragments can be integrated directly to determine the number-average size of the population .

Proteins, 2002 Jan 1, 46(1), 41 - 60
Calculation of protein ionization equilibria with conformational sampling: pK(a) of a model leucine zipper, GCN4 and barnase; Gorfe AA et al.; The use of conformational ensembles provided by nuclear magnetic resonance (NMR) experiments or generated by molecular dynamics (MD) simulations has been regarded as a useful approach to account for protein motions in the context of pK(a) calculations, yet the idea has been tested occasionally . This is the first report of systematic comparison of pK(a) estimates computed from long multiple MD simulations and NMR ensembles . As model systems, a synthetic leucine zipper, the naturally occurring coiled coil GCN4, and barnase were used . A variety of conformational averaging and titration curve-averaging techniques, or combination thereof, was adopted and/or modified to investigate the effect of extensive global conformational sampling on the accuracy of pK(a) calculations . Clustering of coordinates is proposed as an approach to reduce the vast diversity of MD ensembles to a few structures representative of the average electrostatic properties of the system in solution . Remarkable improvement of the accuracy of pK(a) predictions was achieved by the use of multiple MD simulations . By using multiple trajectories the absolute error in pK(a) predictions for the model leucine zipper was reduced to as low as approximately 0.25 pK(a) units . The validity, advantages, and limitations of explicit conformational sampling by MD, compared with the use of an average structure and a high internal protein dielectric value as means to improve the accuracy of pK(a) calculations, are discussed .

J Cell Biochem, 2001, 83(4), 660 - 70
Trichostatin A inhibits beta-casein expression in mammary epithelial cells; Pujuguet P et al.; Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors . When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein . We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element . We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA . We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct . As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells . These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types .

Bioessays, 2001 Dec, 23(12), 1176 - 9
Analysis of genetic control elements in eukaryotes: transcriptional activity or nuclear hitchhiking?
Zohar M, Mesika A, Reich Z.
A common way to analyse basal and stimulated activity of eukaryotic genetic control elements, such as promoters and enhancers, is to introduce them into cells via DNA vectors containing an easily assayable reporter gene . Activity is then studied by measurement of transiently produced mRNA or reporter protein . In such assays, it is assumed that the variable measured is proportional to the transcriptional activity of the control element under investigation . Here we question the validity of this generally accepted assumption . Specifically, recent observations indicate that control elements, in addition to modulating transgene transcription, can facilitate the nuclear uptake of their carrier plasmids . This process is mediated by transcription factors or other nuclear proteins harbouring nuclear localisation signals, which bind to the control elements in the cytoplasm and transport the DNA into the nucleus through the protein nuclear import machinery . As the number of mRNA transcripts produced for an epi-chromosomally expressed transgene is directly related to its copy number inside the nucleus, such transport activity may lead to substantial overestimation of the transcriptional potency of the control element(s) studied .

Cancer, 2001 Nov 1, 92(9), 2484 - 92
Expression and DNA binding activity of the Ku heterodimer in bladder carcinoma; Stronati L et al.; BACKGROUND: The Ku protein is a tightly associated heterodimer, comprised of 70-kilodalton (kD) and 86-kD subunits, that forms the DNA-dependent protein kinase (DNA-PK) complex together with the 470-kD DNA-PKcs catalytic subunit, and is involved mainly in DNA double-strand breaks (DSBs) repair . The objective of the current study was to investigate the expression and DNA-binding activity of the Ku protein in fresh tissues from patients with bladder carcinoma and to compare it with that in nontumor tissues obtained from the same organ . Moreover, the DNA-binding activity of Ku was assessed after exposure of the tumor cells to 1 or 2 grays (Gy) of X-rays . Furthermore, the level of phosphorylated Ku was analyzed in both the nuclear and cytoplasmic compartment of normal tissue after exposure to 2 Gy of X-rays . METHODS: The expression and DNA-binding activity of Ku protein were assessed in tumor samples from patients who all were diagnosed with transitional cell carcinoma (TCC) of the bladder using Western blot analysis and the electrophoretic mobility shift assay, respectively . RESULTS: Enhanced Ku activity and expression were found in tumor tissue compared with normal tissue for each patient . Moreover, variations in Ku activity were found in a dose-dependent manner after the tumor cells were exposed to 1 or 2 Gy of X-rays . A decrease in phosphorylated Ku in the cytoplasm and a parallel increase in the nucleus of normal tissue cells were observed after exposure to X-rays . CONCLUSIONS: The results of the current study suggest a possible role of Ku in regulating the DNA-PK activity of DSBs repair in bladder tumors .

Planta Med, 2001 Dec, 67(9), 787 - 90
Differential activation by daphnetoxin and mezerein of PKC-isotypes alpha, beta I, delta and zeta; Saraiva L et al.; Daphnetoxin, a mezerein derivative, was isolated from the stem bark of Daphne gnidium . Mezerein is a PKC activator that exhibits antileukemic properties . However, daphnetoxin and its analogue 12-hydroxydaphnetoxin were described as being devoid of this effect . In the present study daphnetoxin and mezerein were compared as PKC activators on classical (alpha and beta I), novel (delta) and atypical (zeta) isoforms, using an alternative in vivo yeast phenotypic assay . The aim was to clarify if daphnetoxin is a PKC activator and if the differences between the antiproliferative effect of mezerein and of its analogue daphnetoxin may be ascribed to differences on their potency or selectivity as PKC activators . Yeast samples expressing each of the mammalian PKC isoforms tested were incubated with daphnetoxin or mezerein . Growth inhibition caused by these drugs was assumed to be due to PKC activation since it did not occur when expression was not induced . Mezerein inhibited the growth of yeast expressing PKC alpha (IC(50) = 1190 +/- 237 nM; n = 20), PKC beta I (IC(50) = 908 +/- 46 nM; n = 20), and PKC delta (IC(50) = 141 +/- 25 nM; n = 20) but not of yeast expressing PKC zeta . Daphnetoxin also inhibited the growth of yeast expressing isoforms alpha, beta I and delta, being more potent than mezerein on PKC alpha (IC(50) = 536 +/- 183 nM; n = 20; P < 0.05), as potent as mezerein on PKC beta I (IC(50) = 902 +/- 129 nM; n = 20) and less potent than mezerein upon PKC delta (IC(50) = 3370 +/- 492 nM; n = 20; P < 0.05) . These results show that daphnetoxin is a potent PKC activator but with a selectivity different from that of mezerein . It is suggested that the lack of antileukemic and antiproliferative effects of daphnetoxin may be due to its lower potency to activate PKC delta.

J Biol Chem, 2002 Mar 8, 277(10), 7808 - 15 Epub 2001 Dec 13.
Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1; McDermott EP et al.; Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap . Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1 . Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK . Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1 . Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway . Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6 . In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were affected . Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88 . In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras . Our study suggests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.

J Biol Chem, 2002 Feb 15, 277(7), 5168 - 74 Epub 2001 Dec 13.
Transcriptional regulation of the transforming growth factor beta type II receptor gene by histone acetyltransferase and deacetylase is mediated by NF-Y in human breast cancer cells; Park SH et al.; Transcriptional repression of the transforming growth factor-beta (TGF-beta) type II receptor (TbetaRII) gene is one of several mechanisms leading to TGF-beta resistance . Previously, we have shown that MS-275, a synthetic inhibitor of histone deacetylase (HDAC), specifically induces the expression of the TbetaRII gene and restores the TGF-beta signaling in human breast cancer cell lines . However, little is known about the mechanism by which inhibition of HDAC activates TbetaRII expression . MS-275 treatment of cells expressing a wild-type TbetaRII promoter/luciferase construct resulted in a 10-fold induction of the promoter activity . DNA transfection and an electrophoretic mobility shift assay showed that the induction of the TbetaRII promoter by MS-275 requires the inverted CCAAT box and its cognate binding protein, NF-Y . In addition, a DNA affinity pull-down assay indicated that the PCAF protein, a transcriptional coactivator with intrinsic histone acetyltransferase (HAT) activity, is specifically recruited to the NF-Y complex in the presence of either MS-275 or trichostatin A . Based on these results, we suggest that treatment with the HDAC inhibitor induces TbetaRII promoter activity by the recruitment of the PCAF protein to the NF-Y complex, interacting with the inverted CCAAT box in the TbetaRII promoter.

FASEB J, 2002 Feb, 16(2), 219 - 21 Epub 2001 Dec 14.
Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis; Inoki I et al.; Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions . The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage . To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two-hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate . The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners . A competitive affinity-binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 +/- 11 nM and 125 +/- 38 nM) . Binding assay using deletion mutants of CTGF indicated that the thrombospondin type-1 repeat (TSP-1) domain of CTGF binds to the exon 7-coded region of VEGF165 and that the COOH-terminal domain preserves the affinity to both VEGF165 and VEGF121 . The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor . By in vitro tube formation assay of endothelial cells, full-length CTGF and the deletion mutant possessing the TSP-1 domain inhibited VEGF165-induced angiogenesis significantly in the complex form . This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice . These data demonstrate for the first time that VEGF165 binds to CTGF through a protein-to-protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

J Mol Biol, 2001 Dec 14, 314(5), 1053 - 66
Beyond synexpression relationships: local clustering of time-shifted and inverted gene expression profiles identifies new, biologically relevant interactions; Qian J et al.; The complexity of biological systems provides for a great diversity of relationships between genes . The current analysis of whole-genome expression data focuses on relationships based on global correlation over a whole time-course, identifying clusters of genes whose expression levels simultaneously rise and fall . There are, of course, other potential relationships between genes, which are missed by such global clustering . These include activation, where one expects a time-delay between related expression profiles, and inhibition, where one expects an inverted relationship . Here, we propose a new method, which we call local clustering, for identifying these time-delayed and inverted relationships . It is related to conventional gene-expression clustering in a fashion analogous to the way local sequence alignment (the Smith-Waterman algorithm) is derived from global alignment (Needleman-Wunsch) . An integral part of our method is the use of random score distributions to assess the statistical significance of each cluster . We applied our method to the yeast cell-cycle expression dataset and were able to detect a considerable number of additional biological relationships between genes, beyond those resulting from conventional correlation . We related these new relationships between genes to their similarity in function (as determined from the MIPS scheme) or their having known protein-protein interactions (as determined from the large-scale two-hybrid experiment); we found that genes strongly related by local clustering were considerably more likely than random to have a known interaction or a similar cellular role . This suggests that local clustering may be useful in functional annotation of uncharacterized genes . We examined many of the new relationships in detail . Some of them were already well-documented examples of inhibition or activation, which provide corroboration for our results . For instance, we found an inverted expression profile relationship between genes YME1 and YNT20, where the latter has been experimentally documented as a bypass suppressor of the former . We also found new relationships involving uncharacterized yeast genes and were able to suggest functions for many of them . In particular, we found a time-delayed expression relationship between J0544 (which has not yet been functionally characterized) and four genes associated with the mitochondria . This suggests that J0544 may be involved in the control or activation of mitochondrial genes . We have also looked at other, less extensive datasets than the yeast cell-cycle and found further interesting relationships . Our clustering program and a detailed website of clustering results is available at (or

Science, 2001 Dec 14, 294(5550), 2301 - 4
DNA replication . Genomic views of genome duplication; Stillman B; DNA replication is initiated at numerous origins of replication (oris) within the chromosomes . In a pair of ambitious studies, two groups have used different techniques to pinpoint the locations of all of the oris throughout the yeast genome at different times during S phase (Raghuraman et al., Wyrick et al.) . Stillman, in his Perspective, compares and contrasts the different methods and their findings, and speculates on the value of combining these techniques to look at oris in the human genome.

Science, 2002 Jan 11, 295(5553), 321 - 4 Epub 2001 Dec 13.
A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules; Tong AH et al.; Peptide recognition modules mediate many protein-protein interactions critical for the assembly of macromolecular complexes . Complete genome sequences have revealed thousands of these domains, requiring improved methods for identifying their physiologically relevant binding partners . We have developed a strategy combining computational prediction of interactions from phage-display ligand consensus sequences with large-scale two-hybrid physical interaction tests . Application to yeast SH3 domains generated a phage-display network containing 394 interactions among 206 proteins and a two-hybrid network containing 233 interactions among 145 proteins . Graph theoretic analysis identified 59 highly likely interactions common to both networks . Las17 (Bee1), a member of the Wiskott-Aldrich Syndrome protein (WASP) family of actin-assembly proteins, showed multiple SH3 interactions, many of which were confirmed in vivo by coimmunoprecipitation.

Plant Physiol, 2001 Dec, 127(4), 1556 - 67
An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine; Ciavatta VT et al.; In contrast to angiosperms, pines and other gymnosperms form well-developed suspensors in somatic embryogenic cultures . This creates a useful system to study suspensor biology . In a study of gene expression during the early stages of conifer embryogenesis, we identified a transcript, PtNIP1;1, that is abundant in immature loblolly pine (Pinus taeda) zygotic and somatic embryos, but is undetectable in later-stage embryos, megagametophytes, and roots, stems, and needles from 1 year-old seedlings . Analysis of PtNIP1;1 transcript in embryo proper and suspensor tissues by reverse transcription-polymerase chain reaction suggests preferential expression in the suspensor . Based on comparisons of derived amino acid sequences, PtNIP1;1 belongs to the nodulin-like members of the major intrinsic protein superfamily branch of the aquaporin (major intrinsic protein) superfamily . Through heterologous expression in Xenopus laevis oocytes and the yeast (Saccharomyces cerevisiae) fps1(-) mutant, PtNIP1;1 has been shown to be an active aquaglyceroporin.

EMBO J, 2001 Dec 17, 20(24), 7294 - 302
Nucleosome positioning at the replication fork; Lucchini R et al.; In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension . Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery . Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.

EMBO J, 2001 Dec 17, 20(24), 7209 - 19
RTG-dependent mitochondria to nucleus signaling is negatively regulated by the seven WD-repeat protein Lst8p; Liu Z et al.; In cells with reduced mitochondrial function, RTG1, 2 and 3 are required for expression of genes involved in glutamate synthesis . Glutamate negatively regulates RTG-dependent gene expression upstream of Rtg2p, which, in turn, acts upstream of the bHLH/Zip transcription factors, Rtg1p and Rtg3p . Here we report that some mutations {lst8-(2-5)} in LST8, an essential gene encoding a seven WD40-repeat protein required for targeting of amino acid permeases (AAPs) to the plasma membrane, bypass the requirement for Rtg2p and abolish glutamate repression of RTG-dependent gene expression . The lst8-1 mutation, however, which reduces plasma membrane expression of AAP, cannot bypass the Rtg2p requirement, but still suppresses glutamate repression of RTG target gene expression . We show that Lst8p negatively regulates RTG gene function, acting at two sites, one upstream of Rtg2p, affecting glutamate repression of RTG-dependent gene expression through Ssy1p, an AAP-like sensor of external amino acids, and the other between Rtg2p and Rtg1p-Rtg3p . These data, together with genome-wide transcription profiling, reveal pathways regulated by glutamate, and provide insight into the regulation of cellular responses to mitochondrial dysfunction.

EMBO J, 2001 Dec 17, 20(24), 7096 - 107
Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome; Fu H et al.; The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins . Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes . To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis . Within the base, a Rpt4/5/3/6 interaction cluster was evident . Within the lid, a structural cluster formed around Rpn5/11/9/8 . Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement . No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly . Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly . A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association . Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.

EMBO J, 2001 Dec 17, 20(24), 6979 - 89
Functions of Vrp1p in cytokinesis and actin patches are distinct and neither requires a WH2/V domain; Thanabalu T et al.; Vrp1 (verprolin, End5) is a Saccharomyces cerevisiae actin-associated protein and is related to mammalian Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) . Vrp1-deficient (vrp1 Delta) cells are inviable at high temperature, have partially depolarized cortical actin patches and have defects in both actomyosin ring-dependent and Hof1 (Cyk2)-dependent pathways of cytokinesis . We demonstrate here that N-Vrp1(1-364) and C-Vrp1(364-817) are each sufficient to restore viability, actomyosin ring constriction and Hof1 localization at 37 degrees C to vrp1 Delta . C-Vrp1, like Vrp1, partially co-localizes with cortical actin patches and restores actin patch polarization to vrp1 Delta . Cortical localization of C-Vrp1, but not Vrp1, requires Las17 . N-Vrp1 exhibits diffuse cytoplasmic localization and functions in cytokinesis without efficiently restoring polarization of cortical actin patches . N-Vrp1 function is not abolished by mutations affecting the WASP homology 2 (WH2) {verprolin homology (V)} actin-binding domain . N-Vrp1 may function through the type I myosins and actin, while C-Vrp1 may function through both Las17 (Bee1) and type I myosins . The functions of Vrp1 in viability at 37 degrees C and cytokinesis do not require efficient localization to, and function in, the cortical actin cytoskeleton.

EMBO J, 2001 Dec 17, 20(24), 6946 - 57
Prospore membrane formation linked to the leading edge protein (LEP) coat assembly; Moreno-Borchart AC et al.; In yeast, the differentiation process at the end of meiosis generates four daughter cells inside the boundaries of the mother cell . A meiosis-specific plaque (MP) at the spindle pole bodies (SPBs) serves as the starting site for the formation of the prospore membranes (PSMs) that are destined to encapsulate the post-meiotic nuclei . Here we report the identification of Ady3p and Ssp1p, which are functional components of the leading edge protein (LEP) coat, that covers the ring-shaped opening of the PSMs . Ssp1p is required for the assembly of the LEP coat, which consists of at least three proteins (Ssp1p, Ady3p and Don1p) . The assembly of the LEP coat starts with the formation of cytosolic precursors, which then bind in an Ady3p-dependent manner to the SPBs . Subsequent processes at the SPBs leading to functional LEP coats require Ssp1p and the MP components . During growth of the PSMs, the LEP coat functions in formation of the cup-shaped membrane structure that is indispensable for the regulated cellularization of the cytoplasm around the post-meiotic nuclei.

J Theor Biol, 2001 Dec 21, 213(4), 599 - 608
Profiles of random change during aging contain hidden information about longevity and the aging process; Jazwinski SM et al.; Many different morphological and physiological changes occur during the yeast replicative lifespan . It has been proposed that change is a cause rather than an effect of aging . It is difficult to ascribe causality to processes that manifest themselves at the level of the entire organism, because of their global nature . Although causal connections can be established for processes that occur at the molecular level, their exact contributions are obscured, because they are immersed in a highly interactive network of processes . A top-down approach that can isolate crucial features of aging processes for further study may be a productive avenue . We have mathematically depicted the complicated and random changes that occur in cellular spatial organization during the lifespan of individual yeast cells . We call them budding profiles . This has allowed us to demonstrate that budding profiles are a highly individual characteristic, and that they are correlated with an individual cell's longevity . Additional information can be extracted from our model, indicating that random budding is associated with longevity . This expectation was confirmed, providing new avenues for exploring causal factors in yeast aging . The methodology described here can be readily applied to other aspects of aging in yeast and in higher organisms .

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14979 - 84 Epub 2001 Dec 11.
The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus; Gao Y et al.; Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease . The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma) . This processing resembles regulated intramembrane proteolysis of transmembrane proteins such as Notch, where the released cytoplasmic fragments enter the nucleus and modulate gene expression . Here, we examined whether the analogous Cgamma fragments of APP also exert effects in the nucleus . We find that ectopically expressed Cgamma is present both in the cytoplasm and in the nucleus . Interestingly, expression of Cgamma59 causes disappearance of PAT1, a protein that interacts with the APP cytoplasmic domain, from the nucleus and induces its proteosomal degradation . Treatment of cells with lactacystin prevents PAT1 degradation and retains its nuclear localization . By contrast, Cgamma57, a minor product of gamma-cleavage, is only marginally effective in PAT1 degradation . Furthermore, Cgamma59 but not Cgamma57 potently represses retinoic acid-responsive gene expression . Thus, our studies provide the evidence that, as predicted by the regulated intramembrane proteolysis mechanism, Cgamma seems to function in the nucleus.

J Biol Chem, 2002 Feb 22, 277(8), 6303 - 10 Epub 2001 Dec 11.
Heterogeneous nuclear ribonucleoprotein K protein associates with multiple mitochondrial transcripts within the organelle; Ostrowski J et al.; Heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein interacts with a subset of cellular RNAs . We used K protein as a bait in the yeast three-hybrid screen to identify RNAs that bind K protein in vivo . A large number of K protein-binding RNA clones were identified from a human hybrid RNA library . These sequences consisted of C-rich patches and were G-poor . Unexpectedly, several of the RNA clones were encoded by the mitochondrial genome . In a subsequent three-hybrid screen of a hybrid RNA library generated from a mouse liver mitochondrial genome, K protein bound RNA sequences encoded by different loci spanning nearly the entire mitochondrial genome . Western blot analysis of extracts from mitochondria and mitochondrial fractions showed that K protein is localized within mitoplasts . Reverse transcriptase PCR of RNA co-immunoprecipitated with K protein from lysates of isolated mitochondria showed that K protein is associated with several processed mitochondrial transcripts . In contrast, in the same assay, the polycistronic nascent mtRNA bound K protein weakly or not at all . Results of this study suggest that K protein acts within functional modules that are responsible for expression of genes in mitochondria.

J Biol Chem, 2002 Mar 1, 277(9), 7483 - 92 Epub 2001 Dec 10.
The BNIP-2 and Cdc42GAP homology/Sec14p-like domain of BNIP-Salpha is a novel apoptosis-inducing sequence; Zhou YT et al.; We have cloned the cDNAs for two novel human proteins, designated BNIP-Salpha and beta (for BNIP-2 Similar) that are homologous to BNIP-2, a previously known Bcl-2 and E1B-associated protein . The BNIP-S gene encodes two protein isoforms; the longer protein (BNIP-Salpha) contains a complete BNIP-2 and Cdc42GAP Homology (BCH) domain, a novel protein domain that we recently identified, whereas its shorter variant (BNIP-Sbeta) lacks the full BCH domain as a result of an alternative RNA splicing that introduces a nonsense intron . Primer-specific reverse-transcription PCR revealed that both BNIP-Salpha and BNIP-Sbeta mRNA are differentially expressed in various cells and tissues . The expression of BNIP-Salpha or the complete BCH domain, but not BNIP-Sbeta, causes extensive apoptosis in cells . Furthermore, BNIP-Salpha can form a homophilic complex via a unique sequence motif within its BCH domain, and deletion of this interacting motif prevents its pro-apoptotic effect . These results indicate the presence of two BNIP-S splicing variants as cellular regulators and that the BCH domain of BNIP-Salpha confers a novel apoptotic function . The significance of this is discussed.

J Biol Chem, 2002 Feb 22, 277(8), 5929 - 39 Epub 2001 Dec 06.
Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties; Dan I et al.; We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase) . MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region . Like other GCK-III subfamily kinases, MASK does not activate any mitogen-activated protein kinase pathways . Wild type MASK, but not a form lacking the C terminus, exhibits homophilic binding in the yeast two-hybrid system and in coimmunoprecipitation experiments . Additionally, deletion of this C-terminal region of MASK leads to an increased kinase activity toward itself as well as toward an exogenous substrate, myelin basic protein . A potential caspase 3 cleavage site (DESDS) is present in the C-terminal region of MASK, and we show that MASK is cleaved in vitro by caspase 3 . Finally, wild type and C-terminally truncated forms of MASK can both induce apoptosis upon overexpression in mammalian cells that is abrogated by CrmA, suggesting involvement of MASK in the apoptotic machinery in mammalian cells.

Hum Mol Genet, 2001 Dec 1, 10(25), 2953 - 60
Interaction between krit1 and icap1alpha infers perturbation of integrin beta1-mediated angiogenesis in the pathogenesis of cerebral cavernous malformation; Zhang J et al.; Cerebral cavernous malformation (CCM) is a common autosomal dominant disorder characterized by venous sinusoids that predispose to intracranial hemorrhage . CCM is genetically heterogeneous, with loci at 7q, 7p and 3q . Mutations in KRIT1 account for all cases linked to 7q (CCM1), but the pathogenesis of CCM is not understood . Krev Interaction Trapped 1 (krit1) was originally identified through its interaction with the Ras-family GTPase krev1/rap1a in a two-hybrid screen, inferring a role in GTPase signaling cascades . We demonstrated additional 5'-coding exons for krit1, extending the N-terminus by 207 amino acids compared to the previously reported protein . Remarkably, by two-hybrid analysis and co-immunoprecipitation, full-length krit1 fails to interact with krev1/rap1a but shows strong interaction with integrin cytoplasmic domain-associated protein-1 (icap1) . Icap1 binds to a NPXY motif in the cytoplasmic domain of beta1 integrin and participates in beta1-mediated cell adhesion and migration . The novel N-terminus of krit1 contains a NPXY motif that it is required for icap1 interaction . Like beta1 integrin, krit1 interacts with the 200 amino acid isoform of icap1 (icap1alpha), but not a 150 amino acid form that results from alternative splicing (icap1beta) . In a competition assay, induced expression of krit1 diminishes the interaction between icap1alpha and beta1 integrin . Taken together, these data suggest that beta1 integrin and krit1 compete for the same site on icap1alpha, perhaps constituting a regulatory mechanism . Loss-of-function KRIT1 mutations, as observed in CCM1, would shift the balance with predicted consequences for endothelial cell performance during integrin beta1-dependent angiogenesis.

FEBS Lett, 2001 Dec 7, 509(2), 230 - 4
Mining DNA microarray data using a novel approach based on graph theory; del Rio G et al.; The recent demonstration that biochemical pathways from diverse organisms are arranged in scale-free, rather than random, systems {Jeong et al., Nature 407 (2000) 651-654}, emphasizes the importance of developing methods for the identification of biochemical nexuses--the nodes within biochemical pathways that serve as the major input/output hubs, and therefore represent potentially important targets for modulation . Here we describe a bioinformatics approach that identifies candidate nexuses for biochemical pathways without requiring functional gene annotation; we also provide proof-of-principle experiments to support this technique . This approach, called Nexxus, may lead to the identification of new signal transduction pathways and targets for drug design.

Mol Cell, 2001 Nov, 8(5), 1129 - 35
Human Rad50/Mre11 is a flexible complex that can tether DNA ends; de Jager M et al.; The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways . To find out what the common function of R/M in these pathways might be, we investigated its architecture . Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members . R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain . DNA end-bound R/M oligomers could tether linear DNA molecules . These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.

Mol Cell, 2001 Nov, 8(5), 1027 - 39
The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction; Wan Y et al.; Degradation of SnoN is thought to play an important role in the transactivation of TGF-beta responsive genes . We demonstrate that the anaphase-promoting complex (APC) is a ubiquitin ligase required for the destruction of SnoN and that the APC pathway is regulated by TGF-beta . The destruction box of SnoN is required for its degradation in response to TGF-beta signaling . Furthermore, the APC activator CDH1 and Smad3 synergistically regulate SnoN degradation . Under these circumstances, CDH1 forms a quaternary complex with SnoN, Smad3, and APC . These results suggest that APC(CDH1) and SnoN play central roles in regulating growth through the TGF-beta signaling system.

Mol Cell, 2001 Nov, 8(5), 1017 - 26
TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway; Jacinto E et al.; In Saccharomyces cerevisiae, the rapamycin-sensitive TOR kinases negatively regulate the type 2A-related phosphatase SIT4 by promoting the association of this phosphatase with the inhibitor TAP42 . Here, we describe TIP41, a conserved TAP42-interacting protein involved in the regulation of SIT4 . Deletion of the TIP41 gene confers rapamycin resistance, suppresses a tap42 mutation, and prevents dissociation of SIT4 from TAP42 . Furthermore, a TIP41 deletion prevents SIT4-dependent events such as dephosphorylation of the kinase NPR1 and nuclear translocation of the transcription factor GLN3 . Thus, TIP41 negatively regulates the TOR pathway by binding and inhibiting TAP42 . The binding of TIP41 to TAP42 is stimulated upon rapamycin treatment via SIT4-dependent dephosphorylation of TIP41, suggesting that TIP41 is part of a feedback loop that rapidly amplifies SIT4 phosphatase activity under TOR-inactivating conditions.

Mol Cell, 2001 Nov, 8(5), 931 - 2
Regulating microtubule properties by modifying their organizing minus ends; Usui T et al.; In the November 2001 issue of Developmental Cell, Vogel et al . describe that the budding yeast gamma-tubulin, Tub4p, is phosphorylated at a conserved tyrosine in G1 phase of the cell cycle . The results suggest that gamma-tubulin phosphorylation regulates the number and dynamics of microtubules.

Radiat Res, 2001 Dec, 156(6), 767 - 74
Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells; Beck BD et al.; XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70) . DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends . Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100 . In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells . Such decreases in extractability are indicative of protein aggregation within nuclei . Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock . The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more . Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells . When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical . The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein . Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times . Heat has been shown to inactivate XRCC5 . We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 1314 - 9
Mitochondrial AAA-type protease Yme1p is involved in Bax effects on cytochrome c oxidase; Manon S et al.; Expression of the pro-apoptotic protein Bax in yeast Saccharomyces cerevisiae induces a release of cytochrome c accompanied by a decrease of the amount of cytochrome c oxidase . Here we show that the decrease of cytochrome c oxidase is due to the activation of mitochondrial protease Yme1p, of which cytochrome c oxidase subunit 2 (Cox2p) is a substrate . The absence of Yme1p slightly delays Bax-induced cell death, suggesting a role of this protease in yeast cell death and thus of its mammalian homologue in apoptosis.

Biochem Biophys Res Commun, 2001 Dec 21, 289(5), 942 - 9
Expression of CCAAT/enhancer binding protein family genes in monolayer and sandwich culture of hepatocytes: induction of stress-inducible GADD153; Wilkinson RC et al.; Removal of hepatocytes from their physiological environment for experimentation in vitro activates loss of liver-specific phenotype . Hepatocytes cultured in a sandwich configuration reportedly maintain greater expression of certain liver-specific genes than hepatocytes in monolayer cultures . We show that sandwich culture of rat hepatocytes improves retention of expression of a liver-enriched transcription factor, C/EBPalpha (CCAAT/enhancer binding protein alpha), which regulates many liver-specific genes . However, we also demonstrate increased expression of a stress-responsive C/EBP homologue, GADD153 (growth arrest and DNA damage gene 153), during monolayer culture, which may promote dedifferentiation . Induction of GADD153 was not prevented in sandwich cultured hepatocytes . Activation of a homologue of the mouse GADD153 target gene, doc1, was observed in monolayer and sandwich culture, suggesting that GADD153 was transcriptionally active . We suggest that the capability of sandwich cultures to maintain hepatocyte phenotype may be limited by the altered profile of transcription factor activity.

Nat Struct Biol, 2002 Jan, 9(1), 61 - 7
A new FAD-binding fold and intersubunit disulfide shuttle in the thiol oxidase Erv2p; Gross E et al.; Erv2p is an FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation during protein biosynthesis in the yeast endoplasmic reticulum . The structure of Erv2p, determined by X-ray crystallography to 1.5 A resolution, reveals a helix-rich dimer with no global resemblance to other known FAD-binding proteins or thiol oxidoreductases . Two pairs of cysteine residues are required for Erv2p activity . The first (Cys-Gly-Glu-Cys) is adjacent to the isoalloxazine ring of the FAD . The second (Cys-Gly-Cys) is part of a flexible C-terminal segment that can swing into the vicinity of the first cysteine pair in the opposite subunit of the dimer and may shuttle electrons between substrate protein dithiols and the FAD-proximal disulfide.

J Nutr, 2001 Dec, 131(12), 3166 - 74
Oxidized frying oil up-regulates hepatic acyl-CoA oxidase and cytochrome P450 4 A1 genes in rats and activates PPARalpha; Chao PM et al.; Oxidized LDL (oxLDL) and its component hydroxy fatty acids were shown to activate peroxisome proliferator-activating receptor alpha (PPARalpha) and gamma (PPARgamma) . To test the hypothesis that lipid oxidation products in oxidized frying oil (OFO) can activate PPARalpha and up-regulate its target genes, a feeding experiment and a transactivation experiment were conducted . Based on a 2 x 2 factorial design, four groups of Sprague-Dawley male weanling rats were fed diets containing either high (20 g/100 g, HO and HF) or low (5 g/100 g, LO and LF) levels of oxidized frying soybean oil (HO and LO) or fresh soybean oil (HF and LF) for 6 wk . The OFO sample was prepared by frying wheat dough sheets in soybean oil at 205 +/- 5 degrees C for 24 h . OFO dose dependently and significantly increased (P < 0.05) mRNA of acyl-CoA oxidase (ACO) and cytochrome P(450) 4A1(CYP4A1) in liver of rats . Dietary OFO also dose dependently increased liver microsomal CYP4A protein (P < 0.05) . The activity of hepatic ACO of the HO group was sixfold that of the HF group (P < 0.05) . Plasma total lipids, liver triglycerides, cholesterol and total lipids were reduced in rats fed the LO and HO diets (P < 0.05) . Through the ligand binding domain of PPARalpha, the hydrolyzed OFO enhanced the expression of alkaline phosphatase (ALP) reporter gene to a significantly greater extent (P < 0.05) than the hydrolyzed fresh soybean oil in a transactivation assay using a clone of CHO K1 cells stably expressing Gal4-PPARalpha chimeric receptor and UAS4-ALP reporter . The results support our hypothesis that dietary OFO, by activating PPARalpha, up-regulates the expression of PPARalpha downstream genes and alters lipid metabolism in rats.

Mol Biol Cell, 2001 Dec, 12(12), 4114 - 28
HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins; Bays NW et al.; We isolated a temperature-sensitive mutant, hrd4-1, deficient in ER-associated degradation (ERAD) . The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport . Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome . Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes . We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD.

Mol Cell Biol, 2002 Jan, 22(1), 138 - 47
Protection from free beta-tubulin by the beta-tubulin binding protein Rbl2p; Abruzzi KC et al.; Free beta-tubulin not in heterodimers with alpha-tubulin can be toxic, disrupting microtubule assembly and function . We are interested in the mechanisms by which cells protect themselves from free beta-tubulin . This study focused specifically on the function of Rbl2p, which, like alpha-tubulin, can rescue cells from free beta-tubulin . In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding . Here we show that Rbl2p becomes essential in cells containing a modest excess of beta-tubulin relative to alpha-tubulin . However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay . Rescue of beta-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to beta-tubulin . The data suggest that Rbl2p binds transiently to free beta-tubulin, which then passes into an aggregated form that is not toxic.

J Cell Sci, 2001 Nov, 114(Pt 22), 4105 - 15
Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus; Yoshimura SI et al.; The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches . First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation . Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions . Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence . When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization . Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro . In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes . The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane . These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

J Cell Sci, 2001 Nov, 114(Pt 22), 3967 - 78
The Ste5p scaffold; Elion EA; An emerging theme of mitogen-activated protein kinase (MAPK) cascades is that they form molecular assemblies within cells; the spatial organization of which is provided by scaffold proteins . Yeast Ste5p was the first MAPK cascade scaffold to be described . Early work demonstrated that Ste5p selectively tethers the MAPKKK, MAPKK and MAPK of the yeast mating pathway and is essential for efficient activation of the MAPK by the pheromone stimulus . Recent work indicates that Ste5p is not a passive scaffold but plays a direct role in the activation of the MAPKKK by a heterotrimeric G protein and PAK-type kinase . This activation event requires the formation of an active Ste5p oligomer and proper recruitment of Ste5p to a Gbetagamma dimer at the submembrane of the cell cortex, which suggests that Ste5p forms a stable Ste5p signalosome linked to a G protein . Additional studies underscore the importance of regulated localization of Ste5p to the plasma membrane and have revealed nuclear shuttling as a regulatory device that controls the access of Ste5p to the plasma membrane . A model that links Ste5p oligomerization with stable membrane recruitment is presented . In this model, pathway activation is coordinated with the conversion of a less active closed form of Ste5 containing a protected RING-H2 domain into an active Ste5p dimer that can bind to Gbetagamma and form a multimeric scaffold lattice upon which the MAPK cascade can assemble.

J Cell Biol, 2001 Dec 10, 155(6), 969 - 78 Epub 2001 Dec 10.
The Tlg SNARE complex is required for TGN homotypic fusion; Brickner JH et al.; Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN) . Fusion was temperature, ATP, and cytosol dependent . It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent . Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer . Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction . Membrane fusion also required the rab protein Vps21p . Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction . These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.

J Cell Biol, 2001 Dec 10, 155(6), 961 - 8 Epub 2001 Dec 10.
A t-SNARE of the endocytic pathway must be activated for fusion; Paumet F et al.; The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast . We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p . In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion . Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell . Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

J Cell Biol, 2001 Dec 10, 155(6), 937 - 48 Epub 2001 Dec 10.
Crystal structure of Sar1-GDP at 1.7 A resolution and the role of the NH2 terminus in ER export; Huang M et al.; The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the ER . We report the 1.7-A structure of Sar1 and find that consistent with the sequence divergence of Sar1 from Arf family GTPases, Sar1 is structurally distinct . In particular, we show that the Sar1 NH2 terminus contains two regions: an NH2-terminal extension containing an evolutionary conserved hydrophobic motif that facilitates membrane recruitment and activation by the mammalian Sec12 guanine nucleotide exchange factor, and an alpha1' amphipathic helix that contributes to interaction with the Sec23/24 complex that is responsible for cargo selection during ER export . We propose that the hydrophobic Sar1 NH2-terminal activation/recruitment motif, in conjunction with the alpha1' helix, mediates the initial steps in COPII coat assembly for export from the ER.

J Cell Biol, 2001 Dec 10, 155(6), 923 - 36 Epub 2001 Dec 10.
Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex; Gleizes PE et al.; To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae . In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus . In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus . This phenotype is reproduced with pre-60S particles in cells defective in pre-rRNA processing, whereas pre-40S particles only accumulate in the nucleolus, suggesting a tight control of the exit of the small subunit from the nucleolus . Examination of nucleoporin mutants reveals that preribosome nuclear export requires the Nup82p-Nup159p-Nsp1p complex . In contrast, mutations in the nucleoporins forming the Nup84p complex yield very mild or no nuclear accumulation of preribosome . Interestingly, domains of Nup159p required for mRNP trafficking are not necessary for preribosome export . Furthermore, the RNA helicase Dbp5p and the protein Gle1p, which interact with Nup159p and are involved in mRNP trafficking, are dispensable for ribosomal transport . Thus, the Nup82p-Nup159p-Nsp1p nucleoporin complex is part of the nuclear export pathways of preribosomes and mRNPs, but with distinct functions in these two processes.

J Endocrinol, 2001 Dec, 171(3), 445 - 53
Characterization of topology-, gestation- and labor-related changes of a cassette of myometrial contraction-associated protein mRNA in the pregnant baboon myometrium; Wu WX et al.; Our objective was to examine the topology-, gestation- and labor-related changes of estrogen receptor (ER)alpha, progesterone receptor (PR), oxytocin receptor (OTR) and thrombospondin-1 (TSP1) mRNA in pregnant baboon myometrium . ER alpha, PR, OTR and TSP1 mRNAs extracted from the lower uterine segment and fundal myometrium of pregnant baboons not in labor between 121 and 180 days of gestational age (n=9) and in established spontaneous labor between 164 and 193 days of gestational age (n=5) were analyzed by Northern blot . There were no topology-, gestation- or labor-related changes of ER alpha and PR mRNA in or between the lower uterine segment or/and the fundus . OTR mRNA was the same in the lower uterine segment and the fundus from baboons not in labor and non-labor fundal, but not lower uterine segment, myometrial OTR mRNA increased with gestation (R(2)=0.81, P<0.05) . Fundal OTR mRNA rose significantly compared with the lower uterine segment during spontaneous labor . TSP1 mRNA increased significantly in both the fundus and lower uterine segment during labor . TSP1 mRNA in the lower uterine segment during spontaneous labor was higher than in the fundus during spontaneous labor . In conclusion, fundal and lower uterine segment ER alpha and PR mRNA remained unchanged in late gestation and spontaneous labor . The increased OTR mRNA may serve as a mechanism to increase uterine sensitivity to OT during late gestation . The higher fundal OTR mRNA compared with the lower uterine segment provides polarity which assists fetal expulsion by uterine contractions during labor . The significance of increased TSP1 mRNA during labor may relate to homeostasis and merits further study.

Gene, 2001 Dec 12, 280(1-2), 135 - 44
Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins; Zhang SX et al.; Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD) . In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway . To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins . Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important . Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis . In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin . Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue . Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.

Vaccine, 2001 Dec 12, 20(5-6), 763 - 70
Antibodies to Plasmodium vivax transmission-blocking vaccine candidate antigens Pvs25 and Pvs28 do not show synergism; Hisaeda H et al.; Transmission-blocking vaccines against malaria parasites target molecules expressed by sexual stage parasites to elicit antibodies that prevent the infection of the mosquito vector . Pvs25 and Pvs28, expressed on the surface of ookinetes, are potential candidates for such a vaccine and induce antibodies that block the infectivity of Plasmodium vivax in immunized animals . To improve the ability to induce transmission-blocking antibodies, Pvs25 and Pvs28 were produced as a single fusion protein by the yeast Saccharomyces cerevisiae . Mice immunized with a low dose of the chimeric molecule (Pvs25-28) developed higher antibody responses compared with mice immunized with either Pvs25 or Pvs28 . In membrane feeding assays, both anti-Pvs25-28 and anti-Pvs25 antisera had similarly potent transmission-blocking activities (and both were much greater than anti-Pvs28) . Furthermore, serum from mice simultaneously immunized with both Pvs25 and Pvs28, or serum mixtures of anti-Pvs25 alone and anti-Pvs28 alone did not enhance the efficacy over anti-Pvs25 serum alone, demonstrating that there is no synergism in the ability to block transmission of P . vivax between anti-Pvs25 and anti-Pvs28 antibodies.

Trends Plant Sci, 2001 Dec, 6(12), 577 - 85
Strategies to identify transport systems in plants; Barbier-Brygoo H et al.; Since the first molecular structures of plant transporters were discovered over a decade ago, considerable advances have been made in the study of plant membrane transport, but we still do not understand transport regulation . The genes encoding the transport systems in the various cell membranes are still to be identified, as are the physiological roles of most transport systems . A wide variety of complementary strategies are now available to study transport systems in plants, including forward and reverse genetics, proteomics, and in silico exploitation of the huge amount of information contained in the completely known genomic sequence of Arabidopsis.

Genome Biol . 2001;2(11):REVIEWS3012 . Epub 2001 Oct 24.
The syntaxins; Teng FY et al.; SUMMARY: The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place . Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains . There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae . In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway . They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.

J Mol Evol, 2002 Jan, 54(1), 35 - 41
Correlations between mRNA expression levels and GC contents of coding and untranslated regions of genes in rodents; Konu O et al.; Gene expression is regulated by a highly coordinated network of events whose efficiency may constrain the level of expression . Among other factors, natural selection for increased translational efficiency and/or fidelity may shape nucleotide composition and, hence, codon usage during evolution . Previous studies have shown that highly expressed genes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster have relatively higher codon usage biases . However, in the case of mammals, results have been equivocal . In this study, we assessed the correlation between nucleotide composition and mRNA expression levels of rodent genes measured by cDNA microarray and serial analysis of gene expression (SAGE) techniques . We found that mRNA expression levels were correlated with the third nucleotide position GC (GC3) content for both Rattus norvegicus (r = 0.246, p = 0.01; N = 110) and Mus musculus (r = 0.21, p = 0.0026; N = 203) genes . However, no significant correlation was evident between mRNA expression level and GC contents of 5'- and 3'-untranslated regions (UTRs) for either species . This suggests that, in rodents, nucleotide composition of coding sequences and UTRs might evolve differentially when considered along an expression gradient . Accordingly, it is possible that higher GC levels may present the rodent genes with a selective advantage for translational efficiency . However, the increase in GC3 content seems to level off above an expressional threshold (e.g., >or=threefold the median expression for R . norvegicus), suggesting that conflicting demands posed by different aspects of transcriptional and translational machineries (e.g., efficiency versus fidelity) may set an upper limit for GC3.

Nature, 2001 Nov 29, 414(6863), 514 - 21
Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication; Nash P et al.; SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins . The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication . The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding . The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication . Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein-protein interactions.

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 14991 - 6 Epub 2001 Dec 04.
A genetic screen identifies a cellular regulator of adeno-associated virus; Cathomen T et al.; Adeno-associated virus type 2 (AAV2) is a human parvovirus that has attracted attention as a vector for gene transfer . Replication and site-specific integration of the wild-type virus requires binding of the AAV2 Rep proteins to a cis-regulatory element named the Rep recognition sequence (RRS) . RRS motifs are found within the cellular AAVS1 integration locus, the viral p5 promoter, and the inverted terminal repeats (ITRs) . Here we report the design of a genetic screen based on the yeast one-hybrid assay to identify cellular RRS-binding proteins . We show that the human zinc finger 5 protein (ZF5) binds specifically to RRS motifs in vitro and in vivo . ZF5 is a highly conserved and ubiquitously expressed transcription factor that contains five C-terminal zinc fingers and an N-terminal POZ domain . Ectopic expression of ZF5 leads to an ITR-dependent repression of the autologous p5 promoter and reduces both AAV2 replication and the production of recombinant AAV2 . By using deletion and substitution mutants we show that two different domains of ZF5 contribute to AAV2 repression . Negative regulation of the p5 promoter requires the POZ domain, whereas viral replication is inhibited by the zinc finger domain, likely by competing with Rep for binding to the ITR . Identification and characterization of proteins that bind the ITR, the only viral genetic element retained in AAV2 vectors, will lead to new insights into the unique life cycle of AAV2 and will suggest improvements important for its application as a gene therapy vector.

Hum Mol Genet, 2001 Nov 15, 10(24), 2737 - 43
A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency; Ji HP et al.; The coding sequences of multiple human tumor suppressor genes include microsatellite sequences that are prone to mutations . Saccharomyces cerevisiae strains deficient in DNA mismatch repair (MMR) can be used to determine de novo mutation rates of these human tumor suppressor genes as well as any other gene sequence . Microsatellites in human TGFBR2, PTEN and APC genes were placed in yeast vectors and analyzed in isogenic yeast strains that were wild-type or deletion mutants for MSH2 or MLH1 . In MMR-deficient strains, the vector containing the (A)(10) microsatellite sequence of TGFBR2 had a mutation rate (mutations/cell division) of 1.4 x 10(-4), compared to a mutation rate of 1.7 x 10(-6) in the wild-type strain . In MMR-deficient strains, mutation rates in PTEN and APC were also elevated above background levels . PTEN mutation rates were higher in both msh2 (4.4 x 10-5) and mlh1 strains (2.3 x 10-5) . APC mutation rates in the msh2 strain (2.4 x 10-6) and the mlh1 strain (1.7 x 10-6) were also significantly, but less dramatically, elevated over background . Mutations selected for in the yeast screen were identical to those previously observed in human tumor samples with microsatellite instability (MSI) . This functional assay has applicability in providing quantitative data about microsatellite mutation rates caused by MMR deficiency in any human tumor suppressor gene sequence . It can also be applied as a genetic screen to identify new genes that are vulnerable to such microsatellite mutations and thus may be involved in the neoplastic development of tumors with MSI.

FEBS Lett, 2001 Nov 30, 509(1), 77 - 80
Activity of human Delta5 and Delta6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids; de Antueno RJ et al.; Yeast co-expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n-3 and n-6 fatty acids with the same or different carbon chain length . The results clearly demonstrated that a single human Delta5 desaturase is active on 20:3n-6 and 20:4n-3 . Endogenous Delta6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n-6 or 20:5n-3 which, through two sequential elongations, produced 24:4n-6 and 24:5n-3, respectively . Overall, our data suggest that a single human Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.

J Mol Biol, 2001 Dec 7, 314(4), 901 - 10
Importance of two ATP-binding sites for oligomerization, ATPase activity and chaperone function of mitochondrial Hsp78 protein; Krzewska J et al.; The yeast mitochondrial chaperone Hsp78, a homologue of yeast cytosolic Hsp104 and bacterial ClpB, is required for maintenance of mitochondrial functions under heat stress . Here, Hsp78 was purified to homogeneity and shown to form a homo-hexameric complex, with an apparent molecular mass of approximately 440 kDa, in an ATP-dependent manner . Analysis of its ATPase activity reveals that the observed positive cooperativity effect depends both on Hsp78 and ATP concentration . Site-directed mutagenesis of the two putative Hsp78 nucleotide-binding domains suggest that the first nucleotide-binding domain is responsible for ATP hydrolysis and the second one for protein oligomerization . Studies on the chaperone activity of Hsp78 show that its cooperation with the mitochondrial Hsp70 system, consisting of Ssc1p, Mdj1p and Mge1p, is needed for the efficient reactivation of substrate proteins . These studies also suggest that the oligomerization but not the Hsp78 ATPase activity is essential for its chaperone activity .

J Mol Biol, 2001 Dec 7, 314(4), 717 - 33
DNA recombination and RNA cleavage activities of the Flp protein: roles of two histidine residues in the orientation and activation of the nucleophile for strand cleavage; Grainge I et al.; Using a combination of DNA and hybrid DNA-RNA substrates, we have analyzed the mechanism of phosphoryl transfer by the Flp site-specific recombinase in three different reactions: DNA strand breakage and joining, and two types of RNA cleavage activities . These reactions were then used to characterize Flp variants altered at His309 and His345, amino acid residues that are in close proximity to two key catalytic residues (Arg308 and Tyr343) . These histidine residues are important for strand cutting by Tyr343, the active-site nucleophile of Flp, but neither residue contributes to the type II RNA cleavage activity or to the strand-joining reaction in a pre-cleaved substrate . Strand cleavage reactions using small, diffusible nucleophiles indicate that this histidine pair contributes to the correct positioning and activation of Tyr343 within the shared active site of Flp . The implications of these results are evaluated against the recently solved crystal structure of Flp in association with a Holliday junction .

J Mol Biol, 2001 Dec 7, 314(4), 683 - 94
Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex; Chen J et al.; The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation . The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9x10(6) Da and 1.0x10(6) Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation . As a first step in characterizing the functional roles of the 1.0 MDa complex, we have purified it to near homogeneity . Mass spectrometric analysis was subsequently used to identify all the components of the complex . The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130 . CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes . Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes . Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins . Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex . Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes . The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex .

Gynecol Oncol, 2001 Dec, 83(3), 485 - 90
Dominant-negative mutation of p53 tumor suppressor gene in endometrial carcinoma; Sakuragi N et al.; BACKGROUND: It has been suggested that mutation of the TP53 tumor suppressor gene is involved in endometrial carcinogenesis . However, the status of p53 function in endometrial cancers has not yet been investigated in detail . METHODS: We surveyed inactivating p53 mutations in endometrial carcinomas using the yeast p53 functional assay, which can evaluate the transcriptional activity of p53 in vivo in yeast . To the detected p53 mutants, we also applied a transdominance assay, which assesses the dominant-negative property of mutants . RESULTS: Of 23 endometrial carcinomas, 9 tumors (39.1%) were found to harbor p53 mutations . Only 1 of the 6 mutants in 18 endometrioid-type tumors showed dominant-negative capacity . In contrast to the endometrioid-type tumor, all 3 mutations in 5 serous-type tumors (R273H, 9-bp deletion in codons 240-243, and R248W) showed dominant-negative capacity and presented in a homozygous state in the tumors, indicating a complete functional inactivation . CONCLUSIONS: Although this study included a relatively small number of cases and therefore is a preliminary study, these results suggest that the dominant-negative mutation of the TP53 gene is related to serous adenocarcinoma . The role of the dominant-negative status of p53 mutants in endometrial carcinogenesis and progression of this disease should be further investigated . (c)2001 Elsevier Science.

Cell, 2001 Nov 30, 107(5), 667 - 77
Mobilization of processed, membrane-tethered SPT23 transcription factor by CDC48(UFD1/NPL4), a ubiquitin-selective chaperone; Rape M et al.; The OLE pathway of yeast regulates the level of the ER-bound enzyme Delta9-fatty acid desaturase OLE1, thereby controlling membrane fluidity . A central component of this regulon is the transcription factor SPT23, a homolog of mammalian NF-kappaB . SPT23 is synthesized as an inactive, ER membrane-anchored precursor that is activated by regulated ubiquitin/proteasome-dependent processing (RUP) . We now show that SPT23 dimerizes prior to processing and that the processed molecule, p90, retains its ubiquitin modification and initially remains tethered to its unprocessed, membrane-bound SPT23 partner . Subsequently, p90 is liberated from its partner for nuclear targeting by the activity of the chaperone-like CDC48(UFD1/NPL4) complex . Remarkably, this enzyme binds preferentially ubiquitinated substrates, suggesting that CDC48(UFD1/NPL4) is qualified to selectively remove ubiquitin conjugates from protein complexes.

Cell, 2001 Nov 30, 107(5), 655 - 65
Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints; Hu F et al.; During mitosis, a ras-related GTPase (Tem1) binds GTP and activates a signal transduction pathway to allow mitotic exit . During most of the cell cycle, Tem1 function is antagonized by a GTPase-activating protein complex, Bfa1/Bub2 . How the Bfa1/Bub2 complex is regulated is not well understood . We find that Polo/Cdc5 kinase acts upstream of Bfa1/Bub2 in the mitotic exit network . Cdc5 phosphorylates Bfa1 and acts to antagonize Bfa1 function to promote mitotic exit . Bfa1 is regulated by multiple cell cycle checkpoints . The spindle assembly and spindle orientation checkpoints inhibit Bfa1 phosphorylation . DNA damage does not inhibit Bfa1 phosphorylation and instead causes a Rad53- and Dun1-dependent modification of Bfa1 . Regulation of Bfa1 may therefore be a key step controlled by multiple checkpoint pathways to ensure a mitotic arrest.

Cell, 2001 Nov 30, 107(5), 551 - 4
The fuss about Mus81; Haber JE et al.; Endonucleolytic cleavage of Holliday junctions is important in recombination and replication . Mus81 proteins in yeasts and humans appear to have many, but not all, of the expected properties of eukaryotic Holliday junction resolvases, with intriguing connections to DNA replication checkpoints.

Biochemistry, 2001 Dec 11, 40(49), 14839 - 46
Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A; Nandigama RK et al.; To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae . All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate . The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes . On purification and further characterization, these two mutants were found to each contain covalent FAD . Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A . Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin . In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme . Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05 . The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77 . These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues . The mechanism of C-H abstraction is therefore unaltered . The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.

Genome Res, 2001 Dec, 11(12), 1971 - 3
Is there a bias in proteome research?
Mrowka R, Patzak A, Herzel H.
Advances in technology have enabled us to take a fresh look at data acquired by traditional single experiments and to compare them with genomewide data . The differences can be tremendous, as we show here, in the field of proteomics . We have compared data sets of protein-protein interactions in Saccharomyces cerevisiae that were detected by an identical underlying technical method, the yeast two-hybrid system . We found that the individually identified protein-protein interactions are considerably different from those identified by two genomewide scans . Interacting proteins in the pooled database from single publications are much more closely related to each other with respect to transcription profiles when compared to genomewide data . This difference may have been introduced by two factors: by a selection process in individual publications and by false positives in the whole-genome scans . If we assume that the differences are a result of false positives in the whole-genome data, the scans would contain 47%, 44%, and 91% of false positives for the UETZ, ITO-core, and ITO-full data, respectively . If, however, the true fraction of false positives is considerably lower than estimated here, the data from hypothesis-driven experiments must have been subjected to a serious selection process.

Genes Dev, 2001 Dec 1, 15(23), 3144 - 54
Histone H3 specific acetyltransferases are essential for cell cycle progression; Howe L et al.; Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells . For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death . In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability . To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes . We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation . Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity . This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases . Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G(2)/M phase of the cell cycle . The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.

Cancer Res, 2001 Dec 1, 61(23), 8429 - 34
Fatty acid CoA ligase 4 is up-regulated in colon adenocarcinoma; Cao Y et al.; Arachidonic acid metabolism plays an important role in colon carcinogenesis . Cyclooxygenase-2 (COX-2), which catalyzes the rate-limiting step in the synthesis of prostaglandins from arachidonic acids, is known to be up-regulated in colon cancer, and multiple lines of evidence indicate that it is a critical early step in colon carcinogenesis . Recently, 15-lipoxygenase-1, the enzyme that converts arachidonic acid to 15(S)-HETE, was also found to be up-regulated in colon carcinoma . In our previous studies, we cloned a gene that encodes another arachidonic acid-using enzyme, fatty acid CoA ligase 4 (FACL4), and showed that overexpression of this enzyme prevents apoptosis . We have also showed that FACL4 and COX-2 synergistically inhibit apoptosis by reducing the intracellular level of free arachidonic acid . Here, we report that expression of FACL4 is significantly increased in colon adenocarcinoma compared with adjacent normal tissue at both the mRNA and protein levels by quantitative RT-PCR (paired t test, P < 0.015), immunoblot, and immunohistochemical staining . We found that the increase in expression level of FACL4 mRNA relative to control ranged between 2.4- and 54.5-fold; the average fold-increase was 13.4 . The increase in FACL4 protein expression is between 2.4- and 65.0-fold . In addition, we found that a higher level of increased FACL4 expression was correlated with well and moderately differentiated adenocarcinoma, whereas no similar correlation was observed with COX-2 expression . The in situ hybridization results indicate that expression of FACL4 is localized predominantly in the colon epithelium but not in the stroma . The onset of FACL4 up-regulation appears to occur during the transformation from adenoma to adenocarcinoma because FACL4 expression was not increased above normal in the three colon adenomas examined . Finally, we observed that a tumor promoter significantly induced FACL4 expression . These findings suggest that the FACL4 pathway may be important in colon carcinogenesis, and that the development of selective inhibitors for FACL4 may be a worthy effort in the prevention and treatment of colon cancer.

Cancer Res, 2001 Dec 1, 61(23), 8381 - 4
Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis; Rigas B et al.; DNA-dependent protein kinase (DNA-PK), consisting of a catalytic subunit (DNA-PKcs) and the Ku70 and Ku86 proteins, participates in the repair of DNA double-strand breaks (DSBs) . We assessed its expression immunohistochemically in normal human colon tissue, colon adenomas, colon carcinomas, and normal tissue distant from carcinomas . Normal colonocytes expressed all DNA-PK proteins . Compared with the expression in normal tissue {176.62 +/- 18.56 (the intensity of expression x the percentage of cells expressing this protein), mean + SE}, the expression of Ku70 was significantly reduced in adenomas (36.62 +/- 11.09; P < 0.001) and carcinomas (85.68 +/- 15.76; P < 0.01), as was the expression of Ku86 {(113.10 +/- 10.22 versus 41.66 +/- 14.71 in adenomas (P < 0.01) or versus 85.68 +/- 15.76 in carcinomas (P < 0.05)} . The expression of DNA-PKcs was not significantly changed . The marked underexpression of Ku70 and Ku86 starting at the adenoma stage may be crucial to the development of colon cancer.

Curr Opin Microbiol, 2001 Dec, 4(6), 696 - 702
The social life of actin and microtubules: interaction versus cooperation; Yarm F et al.; The actin and microtubule cytoskeletons play key roles in cell polarity, spindle orientation and nuclear movement . Recent work in fungal systems has identified potential "functional links" between these cytoskeletal systems . This review discusses molecular mechanisms through which these links may be established.

Science, 2001 Nov 30, 294(5548), 1841 - 2
Cell biology . TAPping into mRNA export; Moore MJ et al.; There seem to be numerous pathways for exporting mRNAs from the nucleus to the cytoplasm . But working out which set of export adaptors and receptors transport individual mRNAs has been very difficult . In a Perspective, Moore and Rosbash discuss a new strategy using cell-penetrating peptide inhibitors for unraveling the routes of mRNA export in living cells (Gallouzi and Steitz).

Microbiol Mol Biol Rev, 2001 Dec, 65(4), 570 - 94, table of contents
Transport into and out of the nucleus; Macara IG; A defining characteristic of eukaryotic cells is the possession of a nuclear envelope . Transport of macromolecules between the nuclear and cytoplasmic compartments occurs through nuclear pore complexes that span the double membrane of this envelope . The molecular basis for transport has been revealed only within the last few years . The transport mechanism lacks motors and pumps and instead operates by a process of facilitated diffusion of soluble carrier proteins, in which vectoriality is provided by compartment-specific assembly and disassembly of cargo-carrier complexes . The carriers recognize localization signals on the cargo and can bind to pore proteins . They also bind a small GTPase, Ran, whose GTP-bound form is predominantly nuclear . Ran-GTP dissociates import carriers from their cargo and promotes the assembly of export carriers with cargo . The ongoing discovery of numerous carriers, Ran-independent transport mechanisms, and cofactors highlights the complexity of the nuclear transport process . Multiple regulatory mechanisms are also being identified that control cargo-carrier interactions . Circadian rhythms, cell cycle, transcription, RNA processing, and signal transduction are all regulated at the level of nucleocytoplasmic transport . This review focuses on recent discoveries in the field, with an emphasis on the carriers and cofactors involved in transport and on possible mechanisms for movement through the nuclear pores.

Genetics, 2001 Nov, 159(3), 1291 - 8
Probabilistic prediction of unknown metabolic and signal-transduction networks; Gomez SM et al.; Regulatory networks provide control over complex cell behavior in all kingdoms of life . Here we describe a statistical model, based on representing proteins as collections of domains or motifs, which predicts unknown molecular interactions within these biological networks . Using known protein-protein interactions of Saccharomyces cerevisiae as training data, we were able to predict the links within this network with only 7% false-negative and 10% false-positive error rates . We also use Markov chain Monte Carlo simulation for the prediction of networks with maximum probability under our model . This model can be applied across species, where interaction data from one (or several) species can be used to infer interactions in another . In addition, the model is extensible and can be analogously applied to other molecular data (e.g., DNA sequences).

Biochimie, 2001 Oct, 83(10), 969 - 71
JAB1/CSN5 interacts with the GAL4 DNA binding domain: a note of caution about two-hybrid interactions; Nordgard O et al.; The Jun activation domain binding protein 1 (JAB1) was first identified as an interaction partner and coactivator of c-Jun . Subsequently, it was found to be a subunit of the COP9 signalosome (CSN) and termed CSN subunit 5 (CSN5) . This complex regulates light-mediated development in plants and plays an essential role in a variety of organisms . A striking feature of JAB1/CSN5 is its reported interaction with a wide range of proteins and its modulation of their activity or stability . We applied the yeast two-hybrid system to screen for proteins interacting with the DNA-binding domain of the transcription factor c-Myb and found JAB1/CSN5 among the double-positive clones . To our surprise JAB1/CSN5 was shown to interact with the DNA-binding domain of GAL4 alone and had to be rejected as a false positive in the GAL4-based two-hybrid system . This finding emphasizes the necessity of particular caution when JAB1/CSN5 is found in two-hybrid screenings.

Biochimie, 2001 Oct, 83(10), 953 - 6
Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate; Meng FG et al.; The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration . Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration . However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM . The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio . Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded . The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.

FEBS Lett, 2001 Nov 23, 508(3), 443 - 6
Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis; Meinhard M et al.; Protein phosphatases 2C (PP2Cs) exhibit diverse regulatory functions in signalling pathways of animals, yeast and plants . ABI1 is a PP2C of Arabidopsis that exerts negative control on signalling of the phytohormone abscissic acid (ABA) . Characterisation of the redox sensitivity of ABI1 revealed a strong enzymatic inactivation by hydrogen peroxide (H2O2) which has recently been implicated as a secondary messenger of ABA signalling . H2O2 reversibly inhibited ABI1 activity in vitro with an IC(50) of approximately 140 microM in the presence of physiological concentrations of glutathione . In addition, ABI1 was highly susceptible to inactivation by phenylarsine oxide (IC(50)=3-4 microM) indicative for the facile oxidation of vicinal cysteine residues . Thus, H2O2 generated during ABA signalling seems to inactivate the negative regulator of the ABA response.

Immunity, 2001 Nov, 15(5), 687 - 90
Resistance is futile: assimilation of cellular machinery by HIV-1; Perez OD et al.; HIV-1 budding appears to require Vps4 and Tsg101-two proteins that have links to endosomal sorting machinery . A picture emerges wherein divergent viruses recruit endosomal proteins like Tsg101 to gain access to ubiquitin processes that play a crucial role during viral budding.

Curr Biol, 2001 Nov 27, 11(23), R957 - 60
Genome instability: McClintock revisited; Lundblad V; Recent studies in yeast have shed light on the molecular mechanisms by which telomere dysfunction leads to chromosome fusions . Furthermore, examination of the consequences of telomerase loss in mice suggests that only a few critically short telomeres may be sufficient to promote genomic instability.

Curr Biol, 2001 Nov 27, 11(23), 1815 - 24
Role of scaffolds in MAP kinase pathway specificity revealed by custom design of pathway-dedicated signaling proteins; Harris K et al.; BACKGROUND: Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus . Scaffold proteins are thought to contribute to this specificity by binding select substrates . RESULTS: We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG . We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2 . Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates . This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11 . We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific . Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway . CONCLUSIONS: The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration . Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins . Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

Plant Cell Physiol, 2001 Nov, 42(11), 1274 - 81
Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis; Tamura K et al.; Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis . In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT . The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids . The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding . Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT . Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis . Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum . AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.

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