G2Cdb::Gene report

Gene id
G00001754
Gene symbol
EEF1A1 (HGNC)
Species
Homo sapiens
Description
eukaryotic translation elongation factor 1 alpha 1
Orthologue
G00000505 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000015031 (Vega human gene)
Gene
ENSG00000156508 (Ensembl human gene)
1915 (Entrez Gene)
897 (G2Cdb plasticity & disease)
EEF1A1 (GeneCards)
Literature
130590 (OMIM)
Marker Symbol
HGNC:3189 (HGNC)
Protein Sequence
P68104 (UniProt)

Synonyms (1)

  • EE1A1

Literature (92)

Pubmed - other

  • Interaction of elongation factor 1-alpha with leucine-rich repeat kinase 2 impairs kinase activity and microtubule bundling in vitro.

    Gillardon F

    Boehringer-Ingelheim Pharma GmbH & Co. KG, CNS Research, Birkendorfer St. 65, 88397 Biberach an der Riss, Germany. frank.gillardon@bc.boehringer-ingelheim.com

    Autosomal dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset Parkinson's disease. However, the regulators/effectors contributing to the (patho-)physiological functions of LRRK2 remain poorly defined. Here we show that human LRRK2 co-purifies/co-immunoprecipitates with elongation factor 1-alpha (EF1A). Co-incubation of recombinant LRRK2 and EF1A significantly reduces the kinase activity of LRRK2, whereas its GTPase activity remains unchanged. In addition to its canonical role in mRNA translation, EF1A maintains stability of the microtubule cytoskeleton. In the present study, EF1A promotes microtubule assembly in an in vitro tubulin polymerization assay which is impaired by co-incubation with LRRK2 at sub-stoichiometric concentrations. These findings suggest that the interaction between LRRK2 and EF1A may reciprocally modulate their physiological function.

    Neuroscience 2009;163;2;533-9

  • Structural models of human eEF1A1 and eEF1A2 reveal two distinct surface clusters of sequence variation and potential differences in phosphorylation.

    Soares DC, Barlow PN, Newbery HJ, Porteous DJ and Abbott CM

    Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, United Kingdom. Dinesh.Soares@ed.ac.uk

    Background: Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome.

    To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Balpha and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins.

    Conclusions: The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.

    Funded by: Wellcome Trust

    PloS one 2009;4;7;e6315

  • Region of elongation factor 1A1 involved in substrate recognition by Legionella pneumophila glucosyltransferase Lgt1: identification of Lgt1 as a retaining glucosyltransferase.

    Belyi Y, Stahl M, Sovkova I, Kaden P, Luy B and Aktories K

    Gamaleya Research Institute, Moscow 123098, Russia.

    Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide (50)GKGSFKYAWV(59) of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A' of eEF1A and is part of the first turn of helix A'. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide (50)GKGSFKYAWV(59) with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment ((303)GKASFAYAWV(312)) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an alpha-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.

    The Journal of biological chemistry 2009;284;30;20167-74

  • The hepatitis delta virus RNA genome interacts with eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2.

    Sikora D, Greco-Stewart VS, Miron P and Pelchat M

    Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Room 4111A, Ottawa, Ontario, Canada, K1H 8M5.

    Because of its extremely limited coding capacity, the hepatitis delta virus (HDV) takes over cellular machineries for its replication and propagation. Despite the functional importance of host factors in both HDV biology and pathogenicity, little is known about proteins that associate with its RNA genome. Here, we report the identification of several host proteins interacting with an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA, using mass spectrometry on a UV crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation was used to confirm the interactions of eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2 with the right terminal stem-loop domain of HDV genomic RNA in vitro, and with both polarities of HDV RNA within HeLa cells. Our discovery that HDV RNA associates with RNA-processing pathways and translation machinery during its replication provides new insights into HDV biology and its pathogenicity.

    Virology 2009;390;1;71-8

  • Cathepsin D and eukaryotic translation elongation factor 1 as promising markers of cellular senescence.

    Byun HO, Han NK, Lee HJ, Kim KB, Ko YG, Yoon G, Lee YS, Hong SI and Lee JS

    Divisions of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.

    Induction of premature senescence may be a promising strategy for cancer treatment. However, biomarkers for senescent cancer cells are lacking. To identify such biomarkers, we performed comparative proteomic analysis of MCF7 human breast cancer cells undergoing cellular senescence in response to ionizing radiation (IR). IR-induced senescence was associated with up-regulation of cathepsin D (CD) and down-regulation of eukaryotic translation elongation factor 1beta2 (eEF1B2), as confirmed by Western blot. The other elongation factor, eukaryotic translation elongation factor 1alpha1 (eEF1A1), was also down-regulated. IR-induced senescence was associated with similar changes of CD and eEF1 (eEF1A1 and eEF1B2) levels in the HCT116 colon cancer cell line and the H460 lung cancer cell line. Up-regulation of CD and down-regulation of eEF1 seemed to be specific to senescence, as they were observed during cellular senescence induced by hydrogen peroxide or anticancer drugs (camptothecin, etoposide, or 50 ng doxorubicin) but not during apoptosis induced by Taxol or 10 microg doxorubicin or autophagy induced by tamoxifen. The same alterations in CD and eEF1A1 levels were observed during replicative senescence and Ras oncogene-induced senescence. Transient cell cycle arrest did not alter levels of eEF1 or CD. Chemical inhibition of CD (pepstatin A) and small interfering RNA-mediated knockdown of CD and eEF1 revealed that these factors participate in cell proliferation. Finally, the senescence-associated alteration in CD and eEF1 levels observed in cell lines was also observed in IR-exposed xenografted tumors. These findings show that CD and eEF1 are promising markers for the detection of cellular senescence induced by a variety of treatments.

    Cancer research 2009;69;11;4638-47

  • The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration.

    Zhong D, Zhang J, Yang S, Soh UJ, Buschdorf JP, Zhou YT, Yang D and Low BC

    Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore.

    Deleted in liver cancer 1 (DLC1) is a multi-modular Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor. Besides its RhoGAP domain, functions of other domains in DLC1 remain largely unknown. By protein precipitation and mass spectrometry, we identified eukaryotic elongation factor 1A1 (EF1A1) as a novel partner for the sterile alpha motif (SAM) domain of DLC1 but not the SAM domain of DLC2. The solution structure of DLC1 SAM revealed a new monomeric fold with four parallel helices, similar to that of DLC2 SAM but cc distinct from other SAM domains. Mutating F38, L39 and F40 within a hydrophobic patch retained its overall structure but abolished its interaction with EF1A1 with F38 and L39 forming an indispensable inte 1e6c racting motif. DLC1 SAM did not localize to and was not required for DLC1 to suppress the turnover of focal adhesions. Instead, DLC1 SAM facilitated EF1A1 distribution to the membrane periphery and ruffles upon growth factor stimulation. Compared with wild-type DLC1, the non-interactive DLC1 mutant is less potent in suppressing cell migration, whereas overexpression of the DLC1 SAM domain alone, but not the non-interactive mutant SAM or DLC2 SAM, greatly enhanced cell migration. This finding reveals a novel contribution of the SAM-EF1A1 interaction as a potentially important GAP-independent modulation of cell migration by DLC1.

    Journal of cell science 2009;122;Pt 3;414-24

  • EF1A1-actin interactions alter mRNA stability to determine differential osteopontin expression in HepG2 and Hep3B cells.

    Zhang J, Guo H, Mi Z, Gao C, Bhattacharya S, Li J and Kuo PC

    Department of Surgery, 110 Bell Building, Duke University Medical Center, Box 3522, Durham, NC 27710, USA.

    Cancer progression depends on an accumulation of metastasis-supporting physiological changes which are regulated by cell signaling molecules. One such molecule, osteopontin (OPN), is a secreted phosphoprotein which mediates increased cellular migratory and invasive behavior, increased metastasis, protection from apoptosis, promotion of colony formation and 3D growth ability, induction of tumor-associated inflammatory cells, and induction of expression of angiogenic factors. Studies show that OPN expression is controlled by complex regulatory pathways at the transcriptional level in several cancers, but the molecular mechanisms which determine expression of OPN in HCC are largely unknown. In HepG2 and Hep3B tumor cell lines that differentially express OPN mRNA and protein, we identify elongation translation factor-1A1 (EF1A1) to be the trans-acting factor regulating differential OPN mRNA stability between HepG2 and Hep3B cell lines and characterize its interactions with G- and F-actin. EF1A1 binds to the OPN 5'-UTR to regulate OPN mRNA half-life. EF1A1 binds to actin in Hep3B cells. Pharmacologic manipulation to increase the G:F actin ratio in Hep3B increases OPN mRNA half-life and protein expression with simultaneous decrease in EF1A1 binding to OPN 5'-UTR. The converse findings were noted in HepG2 cells. Overall, our results suggest that EF1A1 regulation of OPN mRNA stability is actin dependent. EF1A1 has not been previously identified as a regulatory factor in OPN expression in cancer.

    Experimental cell research 2009;315;2;304-12

  • Novel cell death by downregulation of eEF1A1 expression in tetraploids.

    Kobayashi Y and Yonehara S

    Laboratory of Molecular and Cellular Biology, Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.

    When duplicated sister chromatids are not properly compacted in mitosis, chromosomes are mis-segregated, inducing genetically unstable tetraploidy known to facilitate aneuploid malignancies. Here, we show that tetraploid cells produced by impaired chromosomal condensation are eliminated by a novel type of cell death different from caspase-dependent apoptosis. The cell death was associated with downregulation of eukaryotic translation elongation factor-1 alpha 1 (eEF1A1/EF-1alpha) expression in conjunction with accumulation of its mRNA in processing bodies (P bodies). Importantly, expression of exogenous eEF1A1 was shown to inhibit the caspase-independent cell death, and a similar cell death was observed after inducing the expression of short hairpin RNA specific for eEF1A1. Furthermore, the number of spontaneously arising binucleated cells was indicated to increase several fold during 1- to 2-week cultivation after initiation of exogenous eEF1A expression. Taken together, the novel cell death machinery should help to eliminate abnormal tetraploid cells and inhibit tumorigenesis.

    Cell death and differentiation 2009;16;1;139-50

  • Eukaryotic translation initiator protein 1A isoform, CCS-3, enhances the transcriptional repression of p21CIP1 by proto-oncogene FBI-1 (Pokemon/ZBTB7A).

    Choi WI, Kim Y, Kim Y, Yu MY, Park J, Lee CE, Jeon BN, Koh DI and Hur MW

    Department of Biochemistry and Molecular Biology, Brain Korea 21 Project for Medical Science, Institute of Genetic Science, Yonsei University School of Medicine, Seoul, Korea.

    FBI-1, a member of the POK (POZ and Kruppel) family of transcription factors, plays a role in differentiation, oncogenesis, and adipogenesis. eEF1A is a eukaryotic translation elongation factor involved in several cellular processes including embryogenesis, oncogenic transformation, cell proliferation, and cytoskeletal organization. CCS-3, a potential cervical cancer suppressor, is an isoform of eEF1A. We found that eEF1A forms a complex with FBI-1 by co-immunoprecipitation, SDS-PAGE, and MALDI-TOF Mass analysis of the immunoprecipitate. GST fusion protein pull-downs showed that FBI-1 directly interacts with eEF1A and CCS-3 via the zinc finger and POZ-domain of FBI-1. FBI-1 co-localizes with either eEF1A or CCS-3 at the nuclear periplasm. CCS-3 enhances transcriptional repression of the p21CIP1 gene (hereafter referred to as p21) by FBI-1. The POZ-domain of FBI-1 interacts with the co-repressors, SMRT and BCoR. We found that CCS-3 also interacts with the co-repressors independently. The molecular interaction between the co-repressors and CCS-3 at the POZ-domain of FBI-1 appears to enhance FBI-1 mediated transcriptional repression. Our data suggest that CCS-3 may be important in cell differentiation, tumorigenesis, and oncogenesis by interacting with the proto-oncogene FBI-1 and transcriptional co-repressors.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2009;23;4-6;359-70

  • Prostate-tumor-inducing gene-1 analysis in human prostate cancer cells and tissue in relation to Mycoplasma infection.

    Scaggiante B, Bonin S, Cristiano L, Siracusano S, Stanta G, Dapas B, Giansante C, Fiotti N and Grassi G

    Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Trieste, Italy. bscaggiante@units.it

    The potential role of PTI-1, in the natural story of prostate adenocarcinoma remains to be fully determined. PTI-1 expression was evaluated in human prostate cancer cell lines and in paraffin-embedded archive tissues. PTI-1 expression was found in Mycoplasma infected but not in non-infected cells. The lack of PTI-1 expression was also confirmed in fixed and paraffin-embedded human cancer prostate biopsies. The overall data indicate that, in prostate tumor cell lines, PTI-1 presence parallels Mycoplasma infection suggesting that PTI-1 might not necessarily play a major role in the onset of prostate tumorigenesis.

    Cancer investigation 2008;26;8;800-8

  • Tumor necrosis factor-alpha downregulates endothelial nitric oxide synthase mRNA stability via translation elongation factor 1-alpha 1.

    Yan G, You B, Chen SP, Liao JK and Sun J

    Department of Cell Biology & Molecular Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA.

    Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-alpha at the posttranscriptional level. To elucidate the molecular basis of TNF-alpha-mediated eNOS mRNA instability, eNOS 3' untranslated region (3'-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3'-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-alpha. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-alpha 1 (eEF1A1). In HUVECs, TNF-alpha rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase-eEF1A1 fusion protein specifically binds to a UC-rich sequence in 11dd the 3'-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3'-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3'-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-alpha-induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3'-UTR binding protein that plays a critical role in mediating TNF-alpha-induced decrease in eNOS mRNA stability.

    Funded by: NHLBI NIH HHS: HL52233, HL70274, R01 HL052233, R01 HL052233-12, R01 HL070274, R01 HL070274-05, R01 HL080187-03; NIDDK NIH HHS: DK62729, R01 DK062729, R01 DK062729-01A1, R01 DK062729-05; NINDS NIH HHS: P01 NS010828-330036

    Circulation research 2008;103;6;591-7

  • The nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha.

    Zhou B, Liu J, Wang Q, Liu X, Li X, Li P, Ma Q and Cao C

    State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing, China.

    Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. Using a yeast two-hybrid screen with the nucleocapsid (N) protein of SARS-CoV as a bait, the C terminus (amino acids 251 to 422) of the N protein was found to interact with human elongation factor 1-alpha (EF1alpha), an essential component of the translational machinery with an important role in cytokinesis, promoting the bundling of filamentous actin (F-actin). In vitro and in vivo interaction was then confirmed by immuno-coprecipitation, far-Western blotting, and surface plasmon resonance. It was demonstrated that the N protein of SARS-CoV induces aggregation of EF1alpha, inhibiting protein translation and cytokinesis by blocking F-actin bundling. Proliferation of human peripheral blood lymphocytes and other human cell lines was significantly inhibited by the infection of recombinant retrovirus expressing SARS-CoV N protein.

    Funded by: NIAAA NIH HHS: R21 AA022203

    Journal of virology 2008;82;14;6962-71

  • BPOZ-2 directly binds to eEF1A1 to promote eEF1A1 ubiquitylation and degradation and prevent translation.

    Koiwai K, Maezawa S, Hayano T, Iitsuka M and Koiwai O

    Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan. koiwai@rs.noda.tus.ac.jp

    Bood POZ containing gene type 2 (BPOZ-2), which contains ankyrin repeats, NLS, BTB/POZ domains and LXXLL motifs, is an adaptor protein for the E3 ubiquitin ligase scaffold protein CUL3. We isolated a cDNA encoding eukaryotic elongation factor 1A1 (eEF1A1) as a BPOZ-2 binding protein by screening a human thymus cDNA library using a yeast two-hybrid system. eEF1A1 is essential for translation and is also involved in the 26S proteasome-dependent degradation of misfolded or unfolded proteins. The binding between BPOZ-2 and eEF1A1 was confirmed by pull-down and immunoprecipitation assays in vitro and in vivo, respectively. BPOZ-2 binds to eEF1A1 through the ankyrin repeats and both BTB/POZ domains in BPOZ-2 and Domains I and III in eEF1A1. BPOZ-2 and eEF1A1 over-expressed in HEK 293T cells co-localized as speckles within the cytoplasm. BPOZ-2 promoted eEF1A1 ubiquitylation and degradation, suggesting that eEF1A1 is a substrate of BPOZ-2. BPOZ-2 inhibited GTP binding to eEF1A1 and prevented translation in in vitro translation assay using rabbit reticulocytes.

    Genes to cells : devoted to molecular & cellular mechanisms 2008;13;6;593-607

  • The novel gene AngRem104 downregulates glucocorticoid receptor expression and activates NF-kappaB in human mesangial cells.

    Zhang Y, Zhang H, Hou P, Liang X, Li Z and Wang H

    Renal Division, Peking University First Hospital, Institute of Nephrology, Peking University, No. 8, Xishiku Street, Beijing 100034, China.

    AngRem104 [angiotensin II (Ang II)-related genes in human mesangial cells (MCs), clone104], a novel gene in human MCs induced by Ang II, was previously identified in human MCs and found to interact with several proteins. The current study used a yeast two-hybrid system and co-immunoprecipitation to investigate the interaction between AngRem104 and glucocorticoid receptor (GR) AF-1-specific elongation factor (GR-EF). GR expression was downregulated and the number of MCs positive for activated nuclear factor kappaB (NF-kappaB) was increased when AngRem104 was overexpressed. Transfection with antisense AngRem104 vector resulted in the upregulation of GR protein and reduced numbers of MCs with activated NF-kappaB. These results indicate that the novel gene AngRem104 is involved in the in vivo regulation of GR expression and the activation of NF-kappaB through interaction with GR-EF in human MCs.

    Biochemical and biophysical research communications 2008;369;4;1057-60

  • Eukaryotic elongation factor 1A interacts with sphingosine kinase and directly enhances its catalytic activity.

    Leclercq TM, Moretti PA, Vadas MA and Pitson SM

    Hanson Institute, Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia.

    Sphingosine 1-phosphate (S1P) has many important roles in mammalian cells, including contributing to the control of cell survival and proliferation. S1P is generated by sphingosine kinases (SKs), of which two mammalian isoforms have been identified (SK1 and SK2). To gain a better understanding of SK regulation, we have used a yeast two-hybrid screen to identify SK1-interacting proteins and established elongation factor 1A (eEF1A) as one such protein that associates with both SK1 and SK2. We show the direct interaction of eEF1A with the SKs in vitro, whereas the physiological relevance of this association was demonstrated by co-immunoprecipitation of the endogenous proteins from cell lysates. Although the canonical role of eEF1A resides in protein synthesis, it has also been implicated in other roles, including regulating the activity of some signaling enzymes. Thus, we examined the potential role of eEF1A in regulation of the SKs and show that eEF1A is able to directly increase the activity of SK1 and SK2 approximately 3-fold in vitro. Substrate kinetics demonstrated that eEF1A increased the catalytic rate of both SKs, while having no observable effect on substrate affinities of these enzymes for either ATP or sphingosine. Overexpression of eEF1A in quiescent Chinese hamster ovary cells increased cellular SK activity, whereas a small interfering RNA-mediated decrease in eEF1A levels in MCF7 cells substantially reduced cellular SK activity and S1P levels, supporting the in vivo physiological relevance of this interaction. Thus, this study has established a novel mechanism of regulation of both SK1 and SK2 that is mediated by their interaction with eEF1A.

    The Journal of biological chemistry 2008;283;15;9606-14

  • Green tea polyphenol epigallocatechin-3-gallate signaling pathway through 67-kDa laminin receptor.

    Umeda D, Yano S, Yamada K and Tachibana H

    Laboratory of Food Chemistry, Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.

    (-)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to be a potent chemopreventive agent. Recently, 67-kDa laminin receptor (67LR) has been identified as a cell surface receptor for EGCG that mediates the anticancer activity of EGCG. Indeed, expression of 67LR confers EGCG responsiveness to tumor cells; however, the molecular basis for the anticancer activity of EGCG in vivo is not entirely understood. Here we show that (i) using a direct genetic screen, eukaryotic translation elongation factor 1A (eEF1A) is identified as a component responsible for the anticancer activity of EGCG; (ii) through both eEF1A and 67LR, EGCG induces the dephosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) at Thr-696 and activates myosin phosphatase; and (iii) silencing of 67LR, eEF1A, or MYPT1 in tumor cells results in abrogation of EGCG-induced tumor growth inhibition in vivo. Additionally, we found that eEF1A is up-regulated by EGCG through 67LR. Overall, these findings implicate both eEF1A and MYPT1 in EGCG signaling for cancer prevention through 67LR.

    The Journal of biological chemistry 2008;283;6;3050-8

  • Novel interaction partners of Bardet-Biedl syndrome proteins.

    Oeffner F, Moch C, Neundorf A, Hofmann J, Koch M and Grzeschik KH

    Center of Human Genetics, University of Marburg, 35037 Marburg, Germany. oeffner@staff.uni-marburg.de

    Bardet-Biedl syndrome (BBS) is a rare, developmental disorder characterized by six major symptoms: rod-cone dystrophy, obesity, polydactyly, renal abnormalities, learning difficulties, and hypogonadism. Secondary features include cardiac and hepatic anomalies, metabolic disturbancies, and hearing loss. BBS is genetically heterogeneous with 12 disease genes (BBS1-BBS12) described thus far. Current data suggest a functional disturbance in ciliary function and intraflagellar transport being associated with the phenotype. However, the precise functions of the BBS proteins have yet to be elucidated. This study focuses on the detection of protein factors interacting with BBS proteins. Applying yeast two-hybrid (Y2H) technology we found a series of novel, functionally potentially plausible binding partners of BBS1, BBS2, BBS4, and BBS7. Protein interactions were supported by coimmunoprecipitation analyses (ALDOB, EPAS1) and substantiated by colocalization studies at the subcellular level (ALDOB, EXOC7, FLOT1, KRT18, PAX2). Our work provides new insights into the understanding of BBS interactions and thus their biological function.

    Cell motility and the cytoskeleton 2008;65;2;143-55

  • Multiple molecular dynamics simulation of the isoforms of human translation elongation factor 1A reveals reversible fluctuations between "open" and "closed" conformations and suggests specific for eEF1A1 affinity for Ca2+-calmodulin.

    Kanibolotsky DS, Novosyl'na OV, Abbott CM, Negrutskii BS and El'skaya AV

    Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Academician Zabolotny Street, 03680 Kiev, Ukraine. kanibolotsky@univ.kiev.ua

    Background: Eukaryotic translation elongation factor eEF1A directs the correct aminoacyl-tRNA to ribosomal A-site. In addition, eEF1A is involved in carcinogenesis and apoptosis and can interact with large number of non-translational ligands. There are two isoforms of eEF1A, which are 98% similar. Despite the strong similarity, the isoforms differ in some properties. Importantly, the appearance of eEF1A2 in tissues in which the variant is not normally expressed can be coupled to cancer development.We reasoned that the background for the functional difference of eEF1A1 and eEF1A2 might lie in changes of dynamics of the isoforms.

    Results: It has been determined by multiple MD simulation that eEF1A1 shows increased reciprocal flexibility of structural domains I and II and less average distance between the domains, while increased non-correlated diffusive atom motions within protein domains characterize eEF1A2. The divergence in the dynamic properties of eEF1A1 and eEF1A2 is caused by interactions of amino acid residues that differ between the two variants with neighboring residues and water environment. The main correlated motion of both protein isoforms is the change in proximity of domains I and II which can lead to disappearance of the gap between the domains and transition of the protein into a "closed" conformation. Such a transition is reversible and the protein can adopt an "open" conformation again. This finding is in line with our earlier experimental observation that the transition between "open" and "closed" conformations of eEF1A could be essential for binding of tRNA and/or other biological ligands. The putative calmodulin-binding region Asn311-Gly327 is less flexible in eEF1A1 implying its increased affinity for calmodulin. The ability of eEF1A1 rather than eEF1A2 to interact with Ca2+/calmodulin is shown experimentally in an ELISA-based test.

    Conclusion: We have found that reversible transitions between "open" and "close" conformations of eEF1A provide a molecular background for the earlier observation that the eEF1A molecule is able to change the shape upon interaction with tRNA. The ability of eEF1A1 rather than eEF1A2 to interact with calmodulin is predicted by MD analysis and showed experimentally. The differential ability of the eEF1A isoforms to interact with signaling molecules discovered in this study could be associated with cancer-related properties of eEF1A2.

    Funded by: Wellcome Trust: 074742/Z04/Z

    BMC structural biology 2008;8;4

  • The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade.

    Grassi G, Scaggiante B, Farra R, Dapas B, Agostini F, Baiz D, Rosso N and Tiribelli C

    Department of Clinical, Morphological and Technological Sciences, Division of Internal Medicine, University of Trieste, Via Giorgieri, 1, 34127 Trieste, Italy.

    Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.

    Biochimie 2007;89;12;1544-52

  • Overexpression of the elongation factor 1A1 relates to muscle proteolysis and proapoptotic p66(ShcA) gene transcription in hypercatabolic trauma patients.

    Bosutti A, Scaggiante B, Grassi G, Guarnieri G and Biolo G

    Division of Internal Medicine, Department of Clinical, Morphological and Technological Sciences, University of Trieste, 34127, Trieste, Italy.

    The eukaryotic elongation factors (eEF1A2 and eEF1A1) play a key role in translation of messenger RNA (mRNA) to protein. In skeletal muscle of healthy humans, EEF1A2 is overexpressed and selected over EEF1A1. In cellular stress models, muscle EEF1A1 expression increased and was associated with apoptosis and catabolism. We have determined mRNA levels of EEF1A1 and EEF1A2, as well as those of other proapoptotic genes, such as p66(ShcA) and c-MYC, in skeletal muscle of severely traumatized patients and healthy volunteers. Muscle protein kinetic was determined by stable isotopes and the arteriovenous technique. The patients were in a hypercatabolic condition because the rate of muscle proteolysis exceeded that of synthesis. Mean mRNA levels of EEF1A1 and EEF1A2 were 165- and 29-fold greater (P < .01) in patients than in the control group, respectively. Mean p66(ShcA) mRNA levels were 3-fold greater (P < .05) in patients than in the controls. In contrast, c-MYC mRNA levels were not significantly different in patients and healthy controls. In patients, muscle mRNA levels of EEF1A1 and p66(ShcA) directly correlated (P < .05) with the rate of proteolysis (R = 0.901 and R = 0.826, respectively). This is in agreement with a reduction in actin and tubulin protein content, both markers of cytoskeletal and sarcomeric disorganization, and with an increased poly(adenosine diphosphate-ribose) polymerase cleavage, a marker of apoptosis. In conclusion, in hypercatabolic traumatized patients, an up-regulation of muscle EEF1A1 and p66(ShcA) relates to proteolysis rate, suggesting an involvement of these genes in muscle catabolic response.

    Metabolism: clinical and experimental 2007;56;12;1629-34

  • Subtractive hybridisation screen identifies genes regulated by glucose deprivation in human neuroblastoma cells.

    Kobayashi K, Xin Y, Ymer SI, Werther GA and Russo VC

    Centre for Hormone Research, Murdoch Children's Research Institute, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, 3052 Parkville, Australia.

    Glucose is the major source of energy for the brain and inadequate glucose supply causes damage of neuronal cells. In this study we employed the human neuroblastoma cell line SH-SY5Y, as an in vitro model for neuronal cells, to identify genes regulated by glucose deprivation. Using subtractive hybridisation screen, validated by Northern analysis, we identify for the first time specific targets of the glucopenic response. These genes are involved in key cellular process including gene transcription, protein synthesis, mitochondrial metabolism, neuronal development, neuroprotection and neuronal apoptosis. Our findings suggest that the fate of neuronal cells undergoing glucose starvation relies on complex gene interactions. Modulation of the expression of these genes in vivo will enable determination of the precise role of each gene and possibly identify key elements and potential therapeutic targets of the glucopenic response.

    Brain research 2007;1170;129-39

  • Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells.

    Han J, Kim D and Morris KV

    Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    siRNAs targeted to gene promoters can direct epigenetic modifications that result in transcriptional gene silencing in human cells. It is not clear whether the antisense strand of the siRNAs bind directly to DNA or to a sense-stranded RNA transcript corresponding to the known promoter region. We present evidence that an RNA polymerase II expressed mRNA containing an extended 5' untranslated region that overlaps the gene promoter is required for RNA-directed epigenetic modifications and transcriptional silencing of the RNA-targeted promoter. These promoter-associated RNAs were detected by their hybridization to the antisense strand of the complementary promoter-directed siRNA. Antisense phosphorothioate oligodeoxynucleotides were used to degrade the promoter-associated RNA transcripts, the loss of which abrogated the effect of siRNA-mediated transcriptional gene silencing, as well as the complexing of the siRNA with the silent state histone methyl mark and the promoter-associated RNA. These data demonstrate that low-copy promoter-associated RNAs transcribed through RNAPII promoters are recognized by the antisense strand of the siRNA and function as a recognition motif to direct epigenetic silencing complexes to the corresponding targeted promoters to mediate transcriptional silencing in human cells.

    Funded by: NHLBI NIH HHS: HL83473, R01 HL083473

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;30;12422-7

  • C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A.

    Lamberti A, Longo O, Marra M, Tagliaferri P, Bismuto E, Fiengo A, Viscomi C, Budillon A, Rapp UR, Wang E, Venuta S, Abbruzzese A, Arcari P and Caraglia M

    Department of Biochemistry and Medical Biotechnology, Federico II University of Naples, Naples, Italy.

    Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

    Cell death and differentiation 2007;14;5;952-62

  • HDM2-binding partners: interaction with translation elongation factor EF1alpha.

    Frum R, Busby SA, Ramamoorthy M, Deb S, Shabanowitz J, Hunt DF and Deb SP

    Department of Biochemistry and the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

    To understand the cellular functions of HDM2, we attempted to identify novel HDM2-interacting proteins by proteomic analysis. Along with previously identified interactions with the ribosomal proteins, our analysis reveals interactions of HDM2 with the ribosomal translation elongation factor EF1alpha, 40S ribosomal protein S20, tubulins, glyceraldehyde 3-phosphate dehydrogenase, and a proteolysis-inducing factor dermicidin in the absence of tumor suppressor p53. Because a CTCL tumor antigen HD-CL-08 has high degree of homology with EF1alpha, we confirmed interaction of HDM2 with EF1alpha by immunoprecipitation and Western blot analysis in transformed as well as near normal diploid cells. Endogenous HDM2- EF1alpha complex was detected in cancer cells overexpressing HDM2, suggesting a possible role of this interaction in HDM2-mediated oncogenesis. Consistent with their interaction, colocalization of HDM2 and EF1alpha can be detected in the cytoplasm of normal or transformed cells. Amino acid residues 1-58 and 221-325 of HDM2 were found to be essential for its interaction with EF1alpha, suggesting that the interaction is independent of its other ribosomal interacting proteins L5, L11, and L23. Overexpression of HDM2 did not affect translation. Because EF1alpha has been implicated in DNA replication and severing of microtubules, interaction of HDM2 with EF1alpha may signify a p53-independent cell growth regulatory role of HDM2.

    Funded by: NCI NIH HHS: CA70712, CA74172; NIGMS NIH HHS: R01 GM037537

    Journal of proteome research 2007;6;4;1410-7

  • 14-3-3sigma controls mitotic translation to facilitate cytokinesis.

    Wilker EW, van Vugt MA, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM and Yaffe MB

    Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis.

    Nature 2007;446;7133;329-32

  • Identification of the ligands of protein interaction domains through a functional approach.

    Caratù G, Allegra D, Bimonte M, Schiattarella GG, D'Ambrosio C, Scaloni A, Napolitano M, Russo T and Zambrano N

    CEINGE Biotecnologie Avanzate, Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy.

    The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

    Molecular & cellular proteomics : MCP 2007;6;2;333-45

  • Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.

    Eckhardt K, Troger J, Reissmann J, Katschinski DM, Wagner KF, Stengel P, Paasch U, Hunziker P, Borter E, Barth S, Schlafli P, Spielmann P, Stiehl DP, Camenisch G and Wenger RH

    Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.

    PASKIN links energy flux and protein synthesis in yeast, regulates glycogen synthesis in mammals, and has been implicated in glucose-stimulated insulin production in pancreatic beta-cells. Using newly generated monoclonal antibodies, PASKIN was localized in the nuclei of human testis germ cells and in the midpiece of human sperm tails. A speckle-like nuclear pattern was observed for endogenous PASKIN in HeLa cells in addition to its cytoplasmic localization. By yeast two-hybrid screening, we identified the multifunctional eukaryotic translation elongation factor eEF1A1 as a novel interaction partner of PASKIN. This interaction was mapped to the PAS A and kinase domains of PASKIN and to the C-terminus of eEF1A1 using mammalian two-hybrid and GST pull-down assays. Kinase assays, mass spectrometry and site-directed mutagenesis revealed PASKIN auto-phosphorylation as well as eEF1A1 target phosphorylation mainly but not exclusively at Thr432. Wild-type but not kinase-inactive PASKIN increased the in vitro translation of a reporter cRNA. Whereas eEF1A1 did not localize to the nucleus, it co-localizes with PASKIN to the cytoplasm of HeLa cells. The two proteins also showed a remarkably similar localization in the midpiece of the sperm tail. These data suggest regulation of eEF1A1 by PASKIN-dependent phosphorylation in somatic as well as in sperm cells.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2007;20;1-4;227-40

  • Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.

    Kim SC, Sprung R, Chen Y, Xu Y, Ball H, Pei J, Cheng T, Kho Y, Xiao H, Xiao L, Grishin NV, White M, Yang XJ and Zhao Y

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    Acetylation of proteins on lysine residues is a dynamic posttranslational modification that is known to play a key role in regulating transcription and other DNA-dependent nuclear processes. However, the extent of this modification in diverse cellular proteins remains largely unknown, presenting a major bottleneck for lysine-acetylation biology. Here we report the first proteomic survey of this modification, identifying 388 acetylation sites in 195 proteins among proteins derived from HeLa cells and mouse liver mitochondria. In addition to regulators of chromatin-based cellular processes, nonnuclear localized proteins with diverse functions were identified. Most strikingly, acetyllysine was found in more than 20% of mitochondrial proteins, including many longevity regulators and metabolism enzymes. Our study reveals previously unappreciated roles for lysine acetylation in the regulation of diverse cellular pathways outside of the nucleus. The combined data sets offer a rich source for further characterization of the contribution of this modification to cellular physiology and human diseases.

    Funded by: NCI NIH HHS: CA107943

    Molecular cell 2006;23;4;607-18

  • A novel cervical cancer suppressor 3 (CCS-3) interacts with the BTB domain of PLZF and inhibits the cell growth by inducing apoptosis.

    Rho SB, Park YG, Park K, Lee SH and Lee JH

    Molecular Therapy Research Center, Sungkyunkwan University, Samsung Medical Center Annex 8F, 50 Ilwon-Dong, Kangnam-Ku, Seoul 135-710, South Korea.

    Promyelocytic leukemia zinc finger protein (PLZF) is a sequence-specific, DNA binding, transcriptional repressor differentially expressed during embryogenesis and in adult tissues. PLZF is known to be a negative regulator of cell cycle progression. We used PLZF as bait in a yeast two-hybrid screen with a cDNA library from the human ovary tissue. A novel cervical cancer suppressor 3 (CCS-3) was identified as a PLZF interacting partner. Further characterization revealed the BTB domain as an interacting domain of PLZF. Interaction of CCS-3 with PLZF in mammalian cells was also confirmed by co-immunoprecipitation and in vitro binding assays. It was found that, although CCS-3 shares similar homology with eEF1A, the study determined CCS-3 to be an isoform. CCS-3 was observed to be downregulated in human cervical cell lines as well as in cervical tumors when compared to those from normal tissues. Overexpression of CCS-3 in human cervical cell lines inhibits cell growth by inducing apoptosis and suppressing human cyclin A2 promoter activity. These combined results suggest that the potential tumor suppressor activity of CCS-3 may be mediated by its interaction with PLZF.

    FEBS letters 2006;580;17;4073-80

  • The identification of myocilin-associated proteins in the human trabecular meshwork.

    Fautsch MP, Vrabel AM and Johnson DH

    Department of Ophthalmology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. fautsch.michael@mayo.edu

    Myocilin forms high molecular weight complexes in vivo presumably due to interaction with itself and other myocilin binding proteins. To identify myocilin interacting proteins, yeast 2-hybrid analysis was performed on >1x10(6) human trabecular meshwork cDNA clones. Coimmunoprecipitation and Far Western analysis were also performed on cell lysates obtained from fresh human trabecular meshworks or cultured human monolayer trabecular cell lines. Among the different methods, 46 candidate myocilin-associated proteins were identified, including molecules associated with the extracellular matrix, cytoskeleton, signaling, and metabolism. The most consistent interaction was myocilin-myocilin binding. Yeast-2 hybrid and Far Western analysis also found an association between myocilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). None of the other candidate myocilin interacting proteins were identified in more than one method. Characterization of these potential interacting proteins may help to better understand the function of myocilin in the trabecular meshwork and aqueous outflow pathway.

    Funded by: NEI NIH HHS: EY 07065

    Experimental eye research 2006;82;6;1046-52

  • Interaction of G-rich GT oligonucleotides with nuclear-associated eEF1A is correlated with their antiproliferative effect in haematopoietic human cancer cell lines.

    Scaggiante B, Dapas B, Grassi G and Manzini G

    Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy. scaggiante@bbcm.units.it

    G-rich GT oligonucleotides with a different content of G clusters have been evaluated for their ability to exert cytotoxicity and to bind to nuclear-associated proteins in T-lymphoblast CCRF-CEM cells. Only the oligomers that did not form G-based structures or had a poor structure, under physiological conditions, were able to exert significant cellular growth inhibition effect. The cytotoxicity of these oligomers was related to their binding to the nuclear-associated eEF1A protein, but not to the recognition of nucleolin or other proteins. In particular, GT oligomers adopting a conformation compatible with G-quadruplex, did not exert cytotoxicity and did not bind to eEF1A. The overall results suggest that the ability of oligomers to adopt a G-quadruplex-type secondary structure in a physiological buffer containing 150 mM NaCl is not a prerequisite for antiproliferative effect in haematopoietic cancer cells. The cytotoxicity of G-rich GT oligomers was shown to be tightly related to their binding affinity for eEF1A protein.

    The FEBS journal 2006;273;7;1350-61

  • [Identification of the proteins interacting with neuroprotective peptide humanin in a yeast two-hybrid system].

    Maksimov VV, Arman IP and Tarantul VZ

    Humanine is a human neuroprotective peptide with a wide action spectrum. To analyze molecular mechanisms of humanin functioning, a search for proteins interacting with this peptide was conducted using yeast two-hybrid system. Screening of human fetal brain cDNA library identified seven proteins with different functions that specifically interacted with humanin.

    Genetika 2006;42;2;274-7

  • Molecular genetic analysis of two functional candidate genes in the autosomal recessive retinitis pigmentosa, RP25, locus.

    Abd El-Aziz MM, El-Ashry MF, Barragan I, Marcos I, Borrego S, Antiñolo G and Bhattacharya SS

    Department of Ophthalmology, Tanta University Hospital, Tanta, Egypt. m.saad@ucl.ac.uk

    Purpose: To identify the disease gene in five Spanish families with autosomal recessive retinitis pigmentosa (arRP) linked to the RP25 locus. Two candidate genes, EEF1A1 and IMPG1, were selected from the region between D6S280 and D6S1644 markers where the families are linked. The genes were selected as good candidates on the basis of their function, tissue expression pattern, and/or genetic data.

    Methods: A molecular genetic study was performed on DNA extracted from one parent and one affected member of each studied family. The coding exons, splice sites, and the 5' UTR of the genes were amplified by polymerase chain reaction (PCR). For mutation detection, direct sequence analysis was performed using the ABI 3100 automated sequencer. Segregation of an IMPG1 single nucleotide polymorphism (SNP) in all the families studied was analyzed by restriction enzyme digest of the amplified gene fragments.

    Results: In total, 15 SNPs were identified of which 7 were novel. Of the identified SNPs, one was insertion, two were deletions, five were intronic, six were missense, and one was located in the 5' UTR. These changes, however, were also identified in unaffected members of the families and/or 50 control Caucasians. The examined known IMPG1 SNP was not segregating with the disease phenotype but was correlating with the genetic data in all families studied.

    Conclusions: Our results indicate that neither EEF1A1 nor IMPG1 could be responsible for RP25 in the studied families due to absence of any pathogenic variants. However, it is important to notice that the methodology used in this study cannot detect larger deletions that lie outside the screened regions or primer site mutations that exist in the heterozygous state. A role of both genes in other inherited forms of RP and/or retinal degenerations needs to be elucidated.

    Current eye research 2005;30;12;1081-7

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Protein profiling of human pancreatic islets by two-dimensional gel electrophoresis and mass spectrometry.

    Ahmed M, Forsberg J and Bergsten P

    Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. meftun.khandker@drl.ox.ac.uk

    Completion of the human genome sequence has provided scientists with powerful resources with which to explore the molecular events associated with disease states such as diabetes. Understanding the relative levels of expression of gene products, especially of proteins, and their post-translational modifications will be critical. However, though the pancreatic islets play a key role in glucose homeostasis, global protein expression data in human are decidedly lacking. We here report the two-dimensional protein map and database of human pancreatic islets. A high level of reproducibility was obtained among the gels and a total of 744 protein spots were detected. We have successfully identified 130 spots corresponding to 66 different protein entries and generated a reference map of human islets. The functionally characterized proteins include enzymes, chaperones, cellular structural proteins, cellular defense proteins, signaling molecules, and transport proteins. A number of proteins identified in this study (e.g., annexin A2, elongation factor 1-alpha 2, histone H2B.a/g/k, heat shock protein 90 beta, heat shock 27 kDa protein, cyclophilin B, peroxiredoxin 4, cytokeratins 7, 18, and 19) have not been previously described in the database of mouse pancreatic islets. In addition, altered expression of several proteins, like GRP78, GRP94, PDI, calreticulin, annexin, cytokeratins, profilin, heat shock proteins, and ORP150 have been associated with the development of diabetes. The data presented in this study provides a first-draft reference map of the human islet proteome, that will pave the way for further proteome analysis of pancreatic islets in both healthy and diabetic individuals, generating insights into the pathophysiology of this condition.

    Journal of proteome research 2005;4;3;931-40

  • Proteomic analysis of mammalian oligosaccharyltransferase reveals multiple subcomplexes that contain Sec61, TRAP, and two potential new subunits.

    Shibatani T, David LL, McCormack AL, Frueh K and Skach WR

    Division of Molecular Medicine, Oregon Health and Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201, USA.

    Oligosaccharyltransferase (OST) catalyzes the cotranslational transfer of high-mannose sugars to nascent polypeptides during N-linked glycosylation in the rough endoplasmic reticulum lumen. Nine OST subunits have been identified in yeast. However, the composition and organization of mammalian OST remain unclear. Using two-dimensional Blue Native polyacrylamide gel electrophoresis/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry, we now demonstrate that mammalian OST can be isolated from solubilized, actively engaged ribosomes as multiple distinct protein complexes that range in size from approximately 500 to 700 kDa. These complexes exhibit different ribosome affinities and subunit compositions. The major complex, OSTC(I), had an apparent size of approximately 500 kDa and was readily released from ribosome translocon complexes after puromycin treatment under physiological salt conditions. Two additional complexes were released only after treatment with high salt: OSTC(II) ( approximately 600 kDa) and OSTC(III) ( approximately 700 kDa). Both remained stably associated with heterotrimeric Sec61alphabetagamma, while OSTC(III) also contained the tetrameric TRAP complex. All known mammalian OST subunits (STT3-A, ribophorin I, ribophorin II, OST48, and DAD1) were present in all complexes. In addition, two previously uncharacterized proteins were also copurified with OST. Mass spectrometry identified a 17 kDa protein as DC2 which is weakly homologous to the C-terminal half of yeast Ost3p and Ost6p. The second protein (14 kDa) was tentatively identified as keratinocyte-associated protein 2 (KCP2) and has no previously known function. Our results identify two potential new subunits of mammalian OST and demonstrate a remarkable heterogeneity in OST composition that may reflect a means for controlling nascent chain glycosylation.

    Funded by: NEI NIH HHS: EY10572; NHLBI NIH HHS: T32HL07781; NIDDK NIH HHS: DK51818; NIGMS NIH HHS: GM53457

    Biochemistry 2005;44;16;5982-92

  • Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast.

    Chiron S, Suleau A and Bonnefoy N

    Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France.

    The translation elongation factor EF-Tu is a GTPase that delivers amino-acylated tRNAs to the ribosome during the elongation step of translation. EF-Tu/GDP is recycled by the guanine nucleotide exchange factor EF-Ts. Whereas EF-Ts is lacking in S. cerevisiae, both translation factors are found in S. pombe and H. sapiens mitochondria, consistent with the known similarity between fission yeast and human cell mitochondrial physiology. We constructed yeast mutants lacking these elongation factors. We show that mitochondrial translation is vital for S. pombe, as it is for human cells. In a genetic background allowing the loss of mitochondrial functions, a block in mitochondrial translation in S. pombe leads to a major depletion of mtDNA. The relationships between EF-Ts and EF-Tu from both yeasts and humans were investigated through functional complementation and coexpression experiments and by a search for suppressors of the absence of the S. pombe EF-Ts. We find that S. cerevisiae EF-Tu is functionally equivalent to the S. pombe EF-Tu/EF-Ts couple. Point mutations in the S. pombe EF-Tu can render it independent of its exchange factor, thereby mimicking the situation in S. cerevisiae.

    Genetics 2005;169;4;1891-901

  • Deconstructing PTI-1: PTI-1 is a truncated, but not mutated, form of translation elongatin factor 1A1, eEF1A1.

    Mansilla F, Hansen LL, Jakobsen H, Kjeldgaard NO, Clark BF and Knudsen CR

    Department of Molecular Biology, University of Aarhus, Arhus C, Denmark. crk@imsb.au.dk

    The prostate tumor-inducing gene 1 (PTI-1) transcript is detected in various human carcinoma cells. PTI-1 is reported to consist of a 5' untranslated region (5' UTR) homologous to mycoplasma 23S rRNA and a coding region corresponding to a truncated and mutated form of the translation elongation factor 1A, eEF1A. We have found that the PTI-1 transcript may encode a truncated, but not mutated, form of the human isoform eEF1A1. Additionally, the 5' UTR sequence of PTI-1 from genomic DNA of different cell lines and blood samples varies from the original sequence. This 5' -UTR region of PTI-1 presents a fusion of E. coli and Mycoplasma hyorhinis 23S rRNA. We have overexpressed the potential PTI-1 protein in E. coli and various human cell lines. The resulting protein could be detected by western blotting using anti-eEF1A antibodies. However, we were unable to detect the PTI-1 protein in LNCaP cell extracts. The potential roles of the PTI-1 protein in carcinogenesis and the origin of the PTI-1 gene in the human genome are discussed.

    Biochimica et biophysica acta 2005;1727;2;116-24

  • Yeast two-hybrid identification of prostatic proteins interacting with human sex hormone-binding globulin.

    Pope SN and Lee IR

    School of Biomedical Sciences, Curtin University of Technology, G.P.O. Box U1987, Perth, WA 6845, Australia.

    Yeast two-hybrid (Y2H) screening of a prostate cDNA library with the cDNA for sex hormone-binding globulin (SHBG) has been used to identify proteins through which SHBG may exert autocrine or paracrine effects on sex steroid target tissues. The library screen gave 230 positive interactions of which around 60 have been sequenced. Of the proteins identified to date from database (BLAST) searches of these sequences, SHBG is one of those occurring most frequently. Amongst the proteins of interest are the membrane-associated proteins flotillin-1 and PRV-1, the enzymes cathepsin D, kallikrein 4 and acid phosphatase, various metallothioneins and translation elongation factor 1 alpha. The significance of the interaction of SHBG with these proteins is discussed.

    The Journal of steroid biochemistry and molecular biology 2005;94;1-3;203-8

  • Nucleolar proteome dynamics.

    Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI and Mann M

    Department of Biochemistry and Molecular Biology, Campusvej 55, DK-5230 Odense M, Denmark.

    The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

    Funded by: Wellcome Trust: 073980

    Nature 2005;433;7021;77-83

  • Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.

    Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, Zhang H, Zha XM, Polakiewicz RD and Comb MJ

    Cell Signaling Technology Inc., 166B Cummings Center, Beverly, Massachusetts 01915, USA.

    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.

    Funded by: NCI NIH HHS: 1R43CA101106

    Nature biotechnology 2005;23;1;94-101

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.

    Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, Worm U, Droege A, Lindenberg KS, Knoblich M, Haenig C, Herbst M, Suopanki J, Scherzinger E, Abraham C, Bauer B, Hasenbank R, Fritzsche A, Ludewig AH, Büssow K, Buessow K, Coleman SH, Gutekunst CA, Landwehrmeyer BG, Lehrach H and Wanker EE

    Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin-Buch, Germany.

    Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

    Funded by: NINDS NIH HHS: NS31862

    Molecular cell 2004;15;6;853-65

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • A protein interaction framework for human mRNA degradation.

    Lehner B and Sanderson CM

    MRC Rosalind Franklin Centre for Genomics Research, Hinxton, Cambridge CB10 1SB, United Kingdom.

    The degradation of mRNA is an important regulatory step in the control of gene expression. However, mammalian RNA decay pathways remain poorly characterized. To provide a framework for studying mammalian RNA decay, a two-hybrid protein interaction map was generated using 54 constructs from 38 human proteins predicted to function in mRNA decay. The results provide evidence for interactions between many different proteins required for mRNA decay. Of particular interest are interactions between the poly(A) ribonuclease and the exosome and between the Lsm complex, decapping factors, and 5'-->3' exonucleases. Moreover, multiple interactions connect 5'-->3' and 3'-->5' decay proteins to each other and to nonsense-mediated decay factors, providing the opportunity for coordination between decay pathways. The interaction network also predicts the internal organization of the exosome and Lsm complexes. Additional interactions connect mRNA decay factors to many novel proteins and to proteins required for other steps in gene expression. These results provide an experimental insight into the organization of proteins required for mRNA decay and their coupling to other cellular processes, and the physiological relevance of many of these interactions are supported by their evolutionary conservation. The interactions also provide a wealth of hypotheses to guide future research on mRNA degradation and demonstrate the power of exhaustive protein interaction mapping in aiding understanding of uncharacterized protein complexes and pathways.

    Genome research 2004;14;7;1315-23

  • Functional proteomics mapping of a human signaling pathway.

    Colland F, Jacq X, Trouplin V, Mougin C, Groizeleau C, Hamburger A, Meil A, Wojcik J, Legrain P and Gauthier JM

    Hybrigenics SA, 75014 Paris, France. fcolland@hybrigenics.fr

    Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

    Genome research 2004;14;7;1324-32

  • A simple device to maintain in vitro cultures of Leishmania in tropical countries.

    RL, Liyanage CP and Karunaweera ND

    Department of Parasitology, Faculty of Medicine, Kynsey Road, Colombo 08, Sri Lanka.

    A thermo-stable clay device to maintain in vitro Leishmania cultures in tropical countries within the optimal temperature range of 22 to 27 degrees C was developed. This being simple and economical could immensely benefit diagnosis and research on leishmaniasis in these countries.

    Transactions of the Royal Society of Tropical Medicine and Hygiene 2004;98;5;315-7

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

  • SEREX identification of new tumour-associated antigens in cutaneous T-cell lymphoma.

    Hartmann TB, Thiel D, Dummer R, Schadendorf D and Eichmüller S

    German Cancer Research Center, Skin Cancer Unit (D070), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

    Background: Cutaneous T-cell lymphoma (CTCL) is a clonal lymphoproliferative disorder of mainly CD4+ T cells, with primary manifestation in the skin.

    Objectives: To detect new CTCL-associated antigens for immunological therapies and to define their specificity in terms of RNA expression and seroreactivity.

    Methods: A newly constructed CTCL cDNA phage library was screened and cross-reactivities against the detected clones were tested using 15 mycosis fungoides and six Sézary syndrome sera. The mRNA expression of the identified genes was analysed by reverse transcription-polymerase chain reaction (RT-PCR) using 22 tumour tissues, nine cell lines and up to 29 different types of normal tissue.

    Results: We identified nine different tumour antigens (HD-CL-01 to HD-CL-09) of which seven clones had high homology to genes with known functions. Several of these genes had previously been associated with cancer, namely inositol 1,4,5-triphosphate 5-phosphatase, vimentin, aldose reductase and elongation factor-1alpha. Variations in the deduced protein sequences were observed in three cases, mostly due to variations in protein length. The individual clones were recognized by up to 56% of patients' sera, while control sera were negative except in one case. Using RT-PCR, we found a frequent expression of these new tumour antigens in tumour specimens (26-100%). In contrast to humoral specificity, specific mRNA was also detected in selected normal tissues (29-89%).

    Conclusions: SEREX (serological identification of antigens by recombinant expression cloning) identified multiple tumour-associated antigens in CTCL. The serological specificity and the high percentage of reactive sera of CTCL patients against several clones suggest these genes as potential targets for diagnostic and prognostic purposes.

    The British journal of dermatology 2004;150;2;252-8

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • The molecular mechanics of eukaryotic translation.

    Kapp LD and Lorsch JR

    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA. lkapp@jhmi.edu

    Great advances have been made in the past three decades in understanding the molecular mechanics underlying protein synthesis in bacteria, but our understanding of the corresponding events in eukaryotic organisms is only beginning to catch up. In this review we describe the current state of our knowledge and ignorance of the molecular mechanics underlying eukaryotic translation. We discuss the mechanisms conserved across the three kingdoms of life as well as the important divergences that have taken place in the pathway.

    Annual review of biochemistry 2004;73;657-704

  • Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A.

    Cans C, Passer BJ, Shalak V, Nancy-Portebois V, Crible V, Amzallag N, Allanic D, Tufino R, Argentini M, Moras D, Fiucci G, Goud B, Mirande M, Amson R and Telerman A

    Molecular Engines Laboratories, 20 Rue Bouvier, 75011 Paris, France.

    Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;13892-7

  • Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing.

    Zhang C, Dowd DR, Staal A, Gu C, Lian JB, van Wijnen AJ, Stein GS and MacDonald PN

    Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

    Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.

    Funded by: NIAMS NIH HHS: R01 AR049069; NIDDK NIH HHS: DK53980

    The Journal of biological chemistry 2003;278;37;35325-36

  • The fragile X mental retardation protein FMRP binds elongation factor 1A mRNA and negatively regulates its translation in vivo.

    Sung YJ, Dolzhanskaya N, Nolin SL, Brown T, Currie JR and Denman RB

    Department of Anatomy and Cell Biology, Columbia University, New York, NY 10032, USA.

    Loss of the RNA-binding protein FMRP (fragile X mental retardation protein) leads to fragile X syndrome, the most common form of inherited mental retardation. Although some of the messenger RNA targets of this protein, including FMR1, have been ascertained, many have yet to be identified. We have found that Xenopus elongation factor 1A (EF-1A) mRNA binds tightly to recombinant human FMRP in vitro. Binding depended on protein determinants located primarily in the C-terminal end of hFMRP, but the hnRNP K homology domain influenced binding as well. When hFMRP was expressed in cultured cells, it dramatically reduced endogenous EF-1A protein expression but had no effect on EF-1A mRNA levels. In contrast, the translation of several other mRNAs, including those coding for dynamin and constitutive heat shock 70 protein, was not affected by the hFMRP expression. Most importantly, EF-1A mRNA and hFMR1 mRNA were coimmunoprecipitated with hFMRP. Finally, in fragile X lymphoblastoid cells in which hFMRP is absent, human EF-1A protein but not its corresponding mRNA is elevated compared with normal lymphoblastoid cells. These data suggest that hFMRP binds to EF-1A mRNA and also strongly argue that FMRP negatively regulates EF-1A expression in vivo.

    The Journal of biological chemistry 2003;278;18;15669-78

  • Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm.

    Bohnsack MT, Regener K, Schwappach B, Saffrich R, Paraskeva E, Hartmann E and Görlich D

    ZMBH, INF 282, D-69120 Heidelberg, Germany.

    Importin beta-type transport receptors mediate the vast majority of transport pathways between cell nucleus and cytoplasm. We identify here the translation elongation factor 1A (eEF1A) as the predominant nuclear export substrate of RanBP21/exportin 5 (Exp5). This cargo-exportin interaction is rather un usual in that eEF1A binds the exportin not directly, but instead via aminoacylated tRNAs. Exp5 thus represents the second directly RNA-binding exportin and mediates tRNA export in parallel with exportin-t. It was suggested recently that 10-15% of the cellu 1f40 lar translation would occur in the nucleus. Our data rule out such a scenario and instead suggest that nuclear translation is actively suppressed by the nuclear export machinery. We found that the vast majority of translation initiation factors (eIF2, eIF2B, eIF3, eIF4A1, eIF5 and eIF5B), all three elongation factors (eEF1A, eEF1B and eEF2) and the termination factor eRF1 are strictly excluded from nuclei. Besides Exp5 and importin 13, CRM1 and as yet unidentified exportins also contribute to the depletion of translation factors from nuclei.

    The EMBO journal 2002;21;22;6205-15

  • Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA.

    Calado A, Treichel N, Müller EC, Otto A and Kutay U

    Swiss Federal Institute of Technology (ETH) Zürich, Institute of Biochemistry, HPM F11.1, CH-8093 Zürich, Switzerland.

    Transport of proteins and RNA into and out of the cell nucleus is mediated largely by a family of RanGTP-binding transport receptors. Export receptors (exportins) need to bind RanGTP for efficient loading of their export cargo. We have identified eukaryotic elongation factor 1A (eEF1A) and tRNA as RanGTP-dependent binding partners of exportin-5 (Exp5). Exp5 stimulates nuclear export of eEF1A when microinjected into the nucleus of Xenopus laevis oocytes. Surprisingly, the interaction between eEF1A and Exp5 is dependent on tRNA that can interact directly with Exp5 and, if aminoacylated, recruits eEF1A into the export complex. These data suggested to us that Exp5 might support tRNA export. Indeed, not only the canonical tRNA export receptor, exportin-t, but also Exp5 can drive nuclear export of tRNA. Taken together, we show that there exists an alternative tRNA export pathway which can be exploited to keep eEF1A out of the cell nucleus.

    The EMBO journal 2002;21;22;6216-24

  • Novel complexes of mammalian translation elongation factor eEF1A.GDP with uncharged tRNA and aminoacyl-tRNA synthetase. Implications for tRNA channeling.

    Petrushenko ZM, Budkevich TV, Shalak VF, Negrutskii BS and El'skaya AV

    Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine.

    Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47-78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl-tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13-17]. The [eEF1A.GDP.tRNA] complex has been hypothesized to interact with aminoacyl-tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A.GDP.tRNA] complex with phenylalanyl-tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel-retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A.GDP.tRNA] and [eEF1A.GDP.tRNAPhe.PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A.GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A.GDP with tRNA and ARS in the channeled elongation cycle is discussed.

    European journal of biochemistry 2002;269;19;4811-8

  • Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.

    Chang JS, Seok H, Kwon TK, Min DS, Ahn BH, Lee YH, Suh JW, Kim JW, Iwashita S, Omori A, Ichinose S, Numata O, Seo JK, Oh YS and Suh PG

    Department of Life Science, College of Natural Science, Daejin University, Kyeonggido 487-711, Korea. jchang@road.daijin.ac.kr

    The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with va de4 rious domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.

    The Journal of biological chemistry 2002;277;22;19697-702

  • Interaction network of human aminoacyl-tRNA synthetases and subunits of elongation factor 1 complex.

    Sang Lee J, Gyu Park S, Park H, Seol W, Lee S and Kim S

    National Creative Research Initiatives Center for ARS Network, College of Pharmacy, Seoul National University, Shinlim-Dong, Kwanak-Ku, Seoul, 157-742, Korea.

    Aminoacyl-tRNA synthetases (ARSs) ligate amino acids to their cognate tRNAs. It has been suggested that mammalian ARSs are linked to the EF-1 complex for efficient channeling of aminoacyl tRNAs to ribosome. Here we systemically investigated possible interactions between human ARSs and the subunits of EF-1 (alpha, beta, gamma, and delta) using a yeast two-hybrid assay. Among the 80 tested pairs, leucyl- and histidyl-tRNA synthetases were found to make strong and specific interaction with the EF-1gamma and beta while glu-proly-, glutaminyl-, alanyl-, aspartyl-, lysyl-, phenylalanyl-, glycyl-, and tryptophanyl-tRNA synthetases showed moderate interactions with the different EF-1 subunits. The interactions of leucyl- and histidyl-tRNA synthetase with the EF-1 complex were confirmed by immunoprecipitation and in vitro pull-down experiments. Interestingly, the aminoacylation activities of these two enzymes, but not other ARSs, were stimulated by the cofactor of EF-1, GTP. These data suggest that a systematic interaction network may exist between mammalian ARSs and EF-1 subunits probably to enhance the efficiency of in vivo protein synthesis.

    Biochemical and biophysical research communications 2002;291;1;158-64

  • Interactions of elongation factor 1alpha with F-actin and beta-actin mRNA: implications for anchoring mRNA in cell protrusions.

    Liu G, Grant WM, Persky D, Latham VM, Singer RH and Condeelis J

    Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, New York 10461, USA. liug@mail.amc.edu

    The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/microtubule-binding protein, elongation factor 1alpha (EF1alpha) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1alpha colocalizes with beta-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and beta-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1alpha in mRNA targeting, we mapped the two actin-binding sites on EF1alpha at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1alpha and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1alpha-F-actin complex is the scaffold that is important for beta-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1alpha and the EF1alpha-binding site of yeast Bni1p, a protein that inhibits EF1alpha binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1alpha or the EF1alpha-binding site of Bni1p inhibits EF1alpha binding to beta-actin mRNA in vitro and causes delocalization of beta-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1alpha in the anchoring of beta-actin mRNA to the protrusion in crawling cells.

    Molecular biology of the cell 2002;13;2;579-92

  • Functional interactions of human immunodeficiency virus type 1 integrase with human and yeast HSP60.

    Parissi V, Calmels C, De Soultrait VR, Caumont A, Fournier M, Chaignepain S and Litvak S

    REGER, UMR-5097 Centre National de la Recherche Scientifique (CNRS)-Université Victor Segalen Bordeaux 2, Bordeaux, France. vincent.parissi@reger.u-bordeaux2.fr

    Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast Saccharomyces cerevisiae was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.

    Journal of virology 2001;75;23;11344-53

  • LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1.

    Weiskirchen R, Moser M, Weiskirchen S, Erdel M, Dahmen S, Buettner R and Gressner AM

    Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany. rweiskirchen@post.klinikum.rwth-aachen.de

    Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.

    The Biochemical journal 2001;359;Pt 3;485-96

  • Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

    Zhu J, Hayakawa A, Kakegawa T and Kaspar RL

    Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.

    Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.

    Funded by: NIGMS NIH HHS: GM57644

    Biochimica et biophysica acta 2001;1521;1-3;19-29

  • Elongation factor-1alpha as a homologous complement activator of Jurkat cells.

    Aoki H, Takizawa F, Tsuji S and Nagasawa S

    Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.

    Although increasing evidence exist suggesting that apoptotic cells activate homologous complement, the homologous complement activators are only poorly characterized. We found that cell lysate of Jurkat cells contained a homologous complement activator of 50 kDa. Digestion of the 50 kDa activator with lysylendopeptidase yielded peptide fragments, with sequences identical to those of EF-1alpha. The 50 kDa activator was removed by immunoadsorption with anti-EF-1alpha, suggesting that the 50 kDa activator is EF-1alpha. The homologous complement activation did not proceed with EGTA-serum. In addition, C4b, a fragment produced by activation of the classical or lectin pathways was found to bind with EF-1alpha. These results suggest that EF-1alpha activates the homologous complement through the classical or lectin pathway.

    International journal of molecular medicine 2000;6;1;87-92

  • Extensive gene duplications and a large inversion characterize the human leukocyte receptor cluster.

    Wende H, Volz A and Ziegler A

    Institut für Immungenetik, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Germany.

    The chromosomal region 19q13.4 harbors the human leukocyte receptor cluster (LRC) which has been demonstrated to contain 19 genes encoding leukocyte-expressed receptors of the immunoglobulin (Ig) superfamily. A spotted PAC library was used to construct a contig of 65 overlapping clones spanning the complete LRC. Within the 900 kb covered by the contig, we identified one cluster containing killer cell inhibitory receptor genes and two clusters containing Ig-like transcript (ILT) genes. Of these, the second ILT cluster, located at the centromeric end of the LRC, was previously unknown. Detailed analysis of the ILT receptor genes in this cluster revealed one novel (ILT11) and six already known ILT genes. The two ILT clusters are transcribed in opposite directions and are separated by about 200 kb, which contains two leukocyte-associated inhibitory receptor (LAIR) genes. The data suggest that the two ILT clusters, each including one LAIR locus, arose from a single ancestral ILT/LAIR cluster by inverse duplication of a c large genomic fragment. Furthermore, the NK cell-expressed NKp46 gene was localized 20 kb telomeric of FCAR; and 14 novel genes mapping within the LRC were identified by cDNA selection. Together with the gene for the ribosomal protein S9 (RPS9), which had previously been assigned to 19q13.4, the total number of LRC genes is now 44. Of these, 29 belong to the Ig superfamily.

    Immunogenetics 2000;51;8-9;703-13

  • Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein.

    Cimarelli A and Luban J

    Departments of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

    Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle. Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins. MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes. EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein. This interaction is mediated 18f4 not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein. EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin. The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions. The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells. Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.

    Funded by: NIAID NIH HHS: AI41857, P30 AI042848, P30 AI42848, R01 AI041857

    Journal of virology 1999;73;7;5388-401

  • The solution structure of the guanine nucleotide exchange domain of human elongation factor 1beta reveals a striking resemblance to that of EF-Ts from Escherichia coli.

    Pérez JM, Siegal G, Kriek J, Hård K, Dijk J, Canters GW and Möller W

    Department of Molecular Cell Biology, Sylvius Laboratory, University ofLeiden, Wassenaarseweg 72 NL-2333, AL Leiden, The Netherlands.

    Background: In eukaryotic protein synthesis, the multi-subunit elongation factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the rate of translation. EF-1alpha, which transports the aminoacyl tRNA to the ribosome, is a member of the G-protein superfamily. EF-1beta regulates the activity of EF-1alpha by catalyzing the exchange of GDP for GTP and thereby regenerating the active form of EF-1alpha. The structure of the bacterial analog of EF-1alpha, EF-Tu has been solved in complex with its GDP exchange factor, EF-Ts. These structures indicate a mechanism for GDP-GTP exchange in prokaryotes. Although there is good sequence conservation between EF-1alpha and EF-Tu, there is essentially no sequence similarity between EF-1beta and EF-Ts. We wished to explore whether the prokaryotic exchange mechanism could shed any light on the mechanism of eukaryotic translation elongation.

    Results: Here, we report the structure of the guanine-nucleotide exchange factor (GEF) domain of human EF-1beta (hEF-1beta, residues 135-224); hEF-1beta[135-224], determined by nuclear magnetic resonance spectroscopy. Sequence conservation analysis of the GEF domains of EF-1 subunits beta and delta from widely divergent organisms indicates that the most highly conserved residues are in two loop regions. Intriguingly, hEF-1beta[135-224] shares structural homology with the GEF domain of EF-Ts despite their different primary sequences.

    Conclusions: On the basis of both the structural homology between EF-Ts and hEF-1beta[135-224] and the sequence conservation analysis, we propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1beta[135-224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.

    Structure (London, England : 1993) 1999;7;2;217-26

  • A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII.

    Sheu GT and Traugh JA

    Department of Biochemistry and Genetics Graduate Group, University of California, Riverside 92521, USA.

    EF-1alpha binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the betagammadelta complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant beta, gamma, and delta subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the beta and delta subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the beta subunit and reduced the Km, while addition of alpha to beta or the betagammacomplex inhibited phosphorylation by CKII. However, alpha had little effect on phosphorylation of delta. Thus, the beta and delta subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of beta was altered by association with other subunits, while the site on delta was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of(alpha2betagamma2delta)2 is proposed and discussed.

    Molecular and cellular biochemistry 1999;191;1-2;181-6

  • Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

    Gangwani L, Mikrut M, Galcheva-Gargova Z and Davis RJ

    Howard Hughes Medical Institute and Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

    The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

    Funded by: NCI NIH HHS: CA 58396

    The Journal of cell biology 1998;143;6;1471-84

  • Antisense inhibition of the PTI-1 oncogene reverses cancer phenotypes.

    Su Z, Goldstein NI and Fisher PB

    Departments of Pathology and Urology, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.

    The genetic alterations and molecular events mediating human prostate cancer development and progression remain to be defined. Rapid expression cloning and differential RNA display detect a putative oncogene, prostate tumor-inducing gene 1 (PTI-1), that is differentially expressed in human prostate (as well as breast, colon, and small cell lung) cancer cell lines, patient-derived prostate carcinomas, and blood from patients with metastatic prostate cancer. PTI-1 consists of a unique 5' untranslated region (5' UTR) with significant sequence homology to Mycoplasma hyopneumoniae 23S ribosomal RNA juxtaposed to a sequence that encodes a truncated and mutated human elongation factor 1alpha (Trun-EF). Stable expression of a nearly full-length 1.9-kb PTI-1 gene, but not the separate PTI-1 5' UTR or Trun-EF region, in normal rat embryo fibroblast cells, CREF-Trans 6, induces an aggressive tumorigenic phenotype in athymic nude mice. Blocking PTI-1 expression with antisense PTI-1 results in reversion of transformed PTI-1-expressing cells to a more normal cellular morphology with suppression in both anchorage-independent growth and tumorigenic potential in athymic nude mice. These findings document that PTI-1 is indeed an oncogene, and directly blocking PTI-1 expression can nullify cancer phenotypes. In these contexts, PTI-1 not only represents a gene with discriminating diagnostic properties but also may serve as a target for the gene-based therapy of human prostate and other cancers.

    Funded by: NCI NIH HHS: CA35675, R01 CA035675

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;4;1764-9

  • Recombinant subunits of mammalian elongation factor 1 expressed in Escherichia coli. Subunit interactions, elongation activity, and phosphorylation by protein kinase CKII.

    Sheu GT and Traugh JA

    Department of Biochemistry and the Genetics Graduate Group, University of California, Riverside, California 92521-0129, USA.

    The first step in elongation requires two different activities; elongation factor (EF)-1alpha transfers aminoacyl-tRNA to the ribosome and is released upon hydrolysis of GTP, EF-1betagammadelta catalyzes exchange of GDP on EF-1alpha with GTP. To analyze the role of the individual subunits of EF-1 in elongation, the cDNAs for the beta, gamma, and delta subunits of EF-1 from rabbit were cloned, and proteins of 225, 437, and 280 amino acids, respectively, were expressed in Escherichia coli. The purified recombinant beta subunit migrates as a dimer and the gamma subunit as a trimer upon gel filtration, whereas the delta subunit forms a large aggregate. Complexes of betagamma, gammadelta and betagammadelta were formed by self-association and eluted with a molecular mass of approximately 160, 530, and 670 kDa, respectively; no interaction was observed between beta and delta. The activity of the recombinant subunits was determined with native EF-1alpha by measuring stimulation of the rate of elongation by poly(U)-directed polyphenylalanine synthesis. Recombinant beta and delta alone stimulated the rate of elongation by 10-fold, with a ratio of 5alpha:2beta or delta. The betagammadelta complex stimulated EF-1alpha activity up to 10-fold with a ratio of 20alpha to 1betagammadelta. Phosphorylation of the beta and delta subunits alone or in betagammadelta by protein kinase CKII had no effect on the rate of elongation.

    The Journal of biological chemistry 1997;272;52;33290-7

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Expression of elongation factor-1 alpha and S1 in young and old human skeletal muscle.

    Welle S, Thornton C, Bhatt K and Krym M

    University of Rochester, New York, USA.

    Previous research has indicated that reduced expression of elongation factor-1 alpha (EF-1 alpha) may be an important determinant of the reduced rate of protein synthesis in senescent animals and cultured cells. The present study examined whether expression of EF-1 alpha or S1, a homologous protein found exclusively in postmitotic tissues, is reduced in senescent human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis muscles of healthy young (22-31 yr old) and old (61-74 yr old) subjects. As reported previously, myofibrillar protein synthesis was approximately 40% slower in the older muscle (p < .001) as determined by incorporation of a stable isotope. Immunoblotting revealed no difference in the concentration of EF-1 alpha + S1 between younger and older muscle. RT-PCR assays indicated that S1 mRNA was much more abundant than EF-1 alpha mRNA in muscles of both age groups, with no reduction in either EF-1 alpha or S1 mRNA abundance in older muscles. We conclude that expression of EF-1 alpha and S1 is not diminished in older muscles and does not explain the age-related slowing of protein synthesis in human skeletal muscle. However, we cannot exclude the possibility that the activity of these proteins declines during senescence due to post-translational modifications.

    Funded by: NCRR NIH HHS: RR-00044; NIA NIH HHS: AG-10463, AG-13070

    The journals of gerontology. Series A, Biological sciences and medical sciences 1997;52;5;B235-9

  • Assignment of human elongation factor 1alpha genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3.

    Lund A, Knudsen SM, Vissing H, Clark B and Tommerup N

    Department of Biostructural Chemistry, Aarhus University, Aarhus, Denmark.

    The human elongation factor 1alpha gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed in a tissue-specific manner, whereas EEF1A is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21.

    Genomics 1996;36;2;359-61

  • Immunofluorescence studies of human fibroblasts demonstrate the presence of the complex of elongation factor-1 beta gamma delta in the endoplasmic reticulum.

    Sanders J, Brandsma M, Janssen GM, Dijk J and Möller W

    Department of Medical Biochemistry, Sylvius Laboratory, University of Leiden, The Netherlands.

    The eukaryotic elongation factor-1 (EF-1) consists of four subunits, EF-1 alpha, EF-1 beta, EF-1 gamma and EF-1 delta which induce efficient transfer of aminoacyl-tRNA to the ribosome. In this process EF-1 alpha.GTP acts as the carrier of the aminoacyl-tRNA on its way to the ribosome. After release of aminoacyl-tRNA to the ribosome under concomitant hydrolysis of GTP, the inactive EF-1 alpha.GDP form is recycled to EF-1 alpha.GTP by EF-1 beta gamma delta. In eukaryotic cells the concentration of EF-1 alpha exceeds that of the complex beta gamma delta by a factor of 5-10. In order to delineate the intracellular localization of the different subunits of EF-1, antibodies against the EF-1 subunits have been elicited and indirect immunofluorescence microscopy experiments were performed. In human fibroblasts, the guanine nucleotide exchange part of EF-1, EF-1 beta gamma delta, was found to co-localize with the endoplasmic reticulum (ER), displaying a distinct fine-structure in its staining pattern. The guanine nucleotide-binding subunit of EF-1, EF-1 alpha, shows a more diffuse distribution throughout the cytoplasm and is, in addition, associated with the nucleus.

    Journal of cell science 1996;109 ( Pt 5);1113-7

  • Identification of a group of cellular cofactors that stimulate the binding of RNA polymerase II and TRP-185 to human immunodeficiency virus 1 TAR RNA.

    Wu-Baer F, Lane WS and Gaynor RB

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, 75235-8594, USA.

    A double-stranded RNA structure transcribed from the HIV-1 long terminal repeat known as TAR is critical for increasing gene expression in response to the transactivator protein Tat. Two cellular factors, RNA polymerase II and TRP-185, bind specifically to TAR RNA, but require the presence of cellular proteins known as cofactors which by themselves are unable to bind to TAR RNA. In an attempt to determine the mechanism by which these cofactors stimulate binding to TAR RNA, we purified these factors from HeLa nuclear extract and amino acid microsequence analysis performed. Three proteins were identified in the cofactor fraction including two previously described proteins, elongation factor 1alpha (EF-1alpha) and the polypyrimidine tract-binding protein (PTB), and a novel protein designated the stimulator of TAR RNA-binding proteins (SRB). SRB has a high degree of homology with a variety of cellular proteins known as chaperonins. Recombinant EF-1alpha, PTB, and SRB produced from vaccinia expression vectors stimulated the binding of RNA polymerase II and TRP-185 to TAR RNA in gel retardation analysis. These studies define a group of cellular factors that function in concert to stimulate the binding of TRP-185 and RNA polymerase II to HIV-1 TAR RNA.

    The Journal of biological chemistry 1996;271;8;4201-8

  • Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.

    Shen R, Su ZZ, Olsson CA and Fisher PB

    Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.

    Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.

    Funded by: NCI NIH HHS: CA35675

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;15;6778-82

  • Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431.

    Kielbassa K, Müller HJ, Meyer HE, Marks F and Gschwendt M

    German Cancer Research Center, Heidelberg.

    Two cytosolic proteins of murine epidermis or porcine spleen with molecular masses of 37 kDa (p37) and 50 kDa (p50) are differentially phosphorylated in vitro by the purified protein kinase C (PKC) isoenzymes alpha, beta, gamma (cPKC) and PKC delta. p37, identified as annexin I, is preferentially phosphorylated by cPKC, whereas p50, identified as elongation factor eEF-1 alpha, is phosphorylated with much greater efficacy by PKC delta than by cPKC. Using the recombinant PKC isoenzymes alpha, beta, gamma, delta, epsilon, eta, and zeta, we could show that purified eEF-1 alpha is indeed a specific substrate of PKC delta. It is not significantly phosphorylated by PKC epsilon, -eta, and -zeta and only slightly by PKC alpha, -beta, and -gamma. PKC delta phosphorylates eEF-1 alpha at Thr-431 (based on the murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a sequence corresponding to that of 422-443 from murine eEF-1 alpha and containing Thr-431 is an absolutely specific substrate for the delta-type of PKC. The single basic amino acid close to Thr-431 (Arg-429) is essential for recognition of the peptide as a substrate by PKC delta and for the selectivity of this recognition. Substitution of Arg-429 by alanine abolishes the ability of PKC delta to phosphorylate the peptide, and insertion of additional basic amino acids in the vicinity of Thr-431 causes a complete loss of selectivity.

    The Journal of biological chemistry 1995;270;11;6156-62

  • Mechanisms of the transfer of aminoacyl-tRNA from aminoacyl-tRNA synthetase to the elongation factor 1 alpha.

    Reed VS, Wastney ME and Yang DC

    Department of Chemistry, Georgetown University, Washington, D.C. 20057.

    Aspartylation of mammalian tRNAAsp by bacteria-expressed human aspartyl-tRNA synthetase (hDRS) was examined. The kinetics of the aspartylation of tRNA was consistent with the following reaction pathway, [formula: see text] where E, represents aspartyl-tRNA synthetase. A set of rate constants was obtained which fit single turnover time courses at varying concentrations of the enzyme, tRNA, and AMP using the SAAM program. The dissociation of Asp-tRNA (k3) was found to be rate limiting. The elongation factor 1 alpha (EF1 alpha) and GTP stimulated the hDRS aspartylation. The stimulation depended on the presence of both EF1 alpha and GTP. Kinetic analysis indicated that EF1 alpha formed a complex with the hDRS-Asp-tRNA complex and stimulated the dissociation of Asp-tRNA. In the presence of 0.5 M NH4Cl, which enhances the binding of Asp-tRNA by EF1 alpha, hDRS-bound Asp-tRNA can be transferred directly to EF1 alpha. The implications of these results on the function of the multi-tRNA synthetase complex will be discussed.

    Funded by: NIGMS NIH HHS: GM-25848

    The Journal of biological chemistry 1994;269;52;32932-6

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Retropseudogenes constitute the major part of the human elongation factor 1 alpha gene family.

    Madsen HO, Poulsen K, Dahl O, Clark BF and Hjorth JP

    Department of Molecular Biology and Plant Physiology, Aarhus University, Denmark.

    The elongation factor 1 alpha (EF-1 alpha) is a protein which promotes the GTP-dependent binding of aminoacyl-tRNA to ribosomes in the protein synthesis process. A human gene coding for EF-1 alpha has previously been cloned and sequenced along with a pseudo-gene. Here, we have further analyzed the family of human EF-1 alpha genes. Using an EF-1 alpha cDNA as probe twelve genomic EF-1 alpha-like clones were isolated and analyzed. Four of these were sequenced and found to contain EF-1 alpha retropseudogenes. A Southern blot analysis indicated that the remaining eight clones also contained retropseudogenes. Genomic Southern blot analysis revealed at least twenty loci in the human genome with sequence homology to the EF-1 alpha cDNA. Besides the already described active gene only one potentially active locus was found. The others appeared to be retropseudogenes. EF-1 alpha retropseudogenes were also found to be abundant in the mammalian species mouse and pig, while the chicken contained only one presumably active EF-1 alpha gene.

    Nucleic acids research 1990;18;6;1513-6

  • Valyl-tRNA synthetase from rabbit liver. I. Purification as a heterotypic complex in association with elongation factor 1.

    Bec G, Kerjan P, Zha XD and Waller JP

    Laboratoire d'Enzymologie, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.

    Valyl-tRNA synthetase occurs as a high molecular mass entity of approximately equal to 700 kDa in the crude extract from rabbit liver. The enzyme was purified as a heterotypic complex comprising four polypeptides of 140, 50, 35, and 27 kDa in the molar proportions of 1:2:1:1, respectively, as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Co-purification of these components at each step of the purification supports the conclusion that they are physically associated within the same complex. In addition to valyl-tRNA synthetase activity, which was assigned to the 140-kDa component, the purified complex exhibits a potent Elongation Factor 1 activity, determined by its ability to sustain poly(U)-dependent polyphenylalanine synthesis in the presence of Elongation Factor 2. Our results are essentially in agreement with those from a recent report (Motorin, Y., Wolfson, A., Orlovsky, A., and Gladilin, K. (1988) FEBS Lett. 238, 262-264), according to which the polypeptides other than that assigned to valyl-tRNA synthetase correspond to the subunits of Elongation Factor 1H.

    The Journal of biological chemistry 1989;264;35;21131-7

  • Murine elongation factor 1 alpha (EF-1 alpha) is posttranslationally modified by novel amide-linked ethanolamine-phosphoglycerol moieties. Addition of ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1 alpha.

    Whiteheart SW, Shenbagamurthi P, Chen L, Cotter RJ and Hart GW

    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    Elongation Factor 1 alpha (EF-1 alpha), an important eukaryotic translation factor, transports charged aminoacyl-tRNA from the cytosol to the ribosomes during poly-peptide synthesis. Metabolic radiolabeling with [3H] ethanolamine shows that, in all cells examined, EF-1 alpha is the major radiolabeled protein. Radiolabeled EF-1 alpha has an apparent Mr = 53,000 and a basic isoelectric point. It is cytosolic and does not contain N-linked oligosaccharides. Trypsin digestion of murine EF-1 alpha generated two major [3H]ethanolamine-labeled peptides. Three peptides were sequenced and were identical to two distinct regions of the human EF-1 alpha protein. Blank sequencing cycles coinciding with glutamic acid in the human cDNA-derived sequence were also found to release [3H]ethanolamine, and compositional analysis of these peptides confirmed the presence of glutamic acid. Dansylation analysis demonstrates that the amine group of the ethanolamine is blocked. These results indicate that EF-1 alpha is posttranslationally modified by the covalent attachment of ethanolamine via an amide bond to at least two specific glutamic acid residues (Glu-301 and Glu-374). The hydroxyl group of the attached ethanolamine was shown by mass spectrometry and compositional analysis, to be further modified by the addition of a phosphoglycerol unit. This novel posttranslational modification may represent an important alteration of EF-1 alpha, comparable to the regulatory effects of posttranslational methylation of EF-1 alpha lysine residues.

    Funded by: NICHD NIH HHS: HD-13563; NIGMS NIH HHS: GM-07445

    The Journal of biological chemistry 1989;264;24;14334-41

  • Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor-1 10a4 alpha.

    Uetsuki T, Naito A, Nagata S and Kaziro Y

    Institute of Medical Science, University of Tokyo, Japan.

    The cDNA for human elongation factor-1 alpha was isolated from a cDNA library of human fibroblast cells. Using the cDNA as a probe, a number of chromosomal genes encoding the human elongation factor-1 alpha were isolated. Characterization of the clones by restriction enzyme mapping and nucleotide sequence analysis has revealed that only one of them is an active gene, whereas all of the other genes are processed pseudogenes. The active gene consists of 8 exons and 7 introns spanning about 3.5 kilobases, and the sequence of its exons is completely identical to that of the human elongation factor-1 alpha cDNA. The first non-coding exon of 33 base pairs is separated by a 943-base pair intron from the coding exons. The primer extension of human elongation factor-1 alpha mRNA has indicated that the transcription of human elongation factor-1 alpha gene starts from a C residue, and a "TATA" box was found 24 base pairs upstream of the initiation site. Three and five Sp1 binding sites are present on the 5'-flanking region and the 1st intron, respectively. Furthermore, one Ap-1 binding site is located in the 1st intron. By using nuclear extracts from HeLa cells, the promoter of human elongation factor-1 alpha gene could stimulate in vitro transcription better than the adenovirus major late promoter.

    The Journal of biological chemistry 1989;264;10;5791-8

  • Mammalian valyl-tRNA synthetase forms a complex with the first elongation factor.

    Motorin YuA, Wolfson AD, Orlovsky AF and Gladilin KL

    A.N. Bakh Institute of Biochemistry, Academy of Sciences of the USSR, Moscow.

    The high-molecular-mass form of valyl-tRNA synthetase is associated with the first elongation factor activity. It includes two polypeptides of about 50 kDa and two others of 40 and 30 kDa, identified as alpha, beta, gamma and delta subunits of eEF-1H. The complex of valyl-tRNA synthetase with eEF-1H is suggested to be a novel form of the first elongation factor.

    FEBS letters 1988;238;2;262-4

  • Retinol-regulated gene expression in human tracheobronchial epithelial cells. Enhanced expression of elongation factor EF-1 alpha.

    Ann DK, Wu MM, Huang T, Carlson DM and Wu R

    Department of Biochemistry and Biophysics, California Primate Research Center, Davis.

    Conducting airway epithelial cells requires vitamin A or its synthetic chemicals (retinoids) for their survival and for the expression of normal mucociliary functions. By using molecular cloning, we have shown that one of the effects of retinol on cultured human tracheobronchial epithelial (HTBE) cells is the enhancement (from 2- to 4-fold) of the mRNA encoding the elongation factor EF-1 alpha. Sequence analysis has shown that clone HT7, which was identified by differential hybridization procedures, contained a cDNA insert which encoded a protein closely resembling (81%) elongation factor EF-1 alpha from brine shrimp and completely identical to the published sequence of human elongation factor EF-1 alpha (Brands, H.H.G.M., Maassen, J.A., Van Hemert, F.J., Amons, R., and Moller, W. (1986) Eur. J. Biochem. 155, 167-171). Regions of homology of HT7 to EF-Tu from yeast mitochondria, plant chloroplasts, and Escherichia coli are also evident. A single RNA band at 1700 bases was observed for both untreated and retinol-treated HTBE cells, and for mouse liver and parotid glands when Northern transfer from denaturing agarose gel was probed with a 32P-labeled HT7 insert. An enhanced amino acid incorporation and increased protein content per cell for HTBE cells grown in the presence of retinol were observed. Results presented by these studies indicate that retinol may regulate the transcription of a factor required for translation.

    Funded by: NHLBI NIH HHS: HL 35635, HL 36031; NIDDK NIH HHS: DK 36812; ...

    The Journal of biological chemistry 1988;263;8;3546-9

  • Human elongation factor 1 alpha: a polymorphic and conserved multigene family with multiple chromosomal localizations.

    Opdenakker G, Cabeza-Arvelaiz Y, Fiten P, Dijkmans R, Van Damme J, Volckaert G, Billiau A, Van Elsen A, Van der Schueren B, Van den Berghe H et al.

    One of the genes activated in human melanoma cells by the tumor-promoting phorbol ester is that of the elongation factor 1 alpha. A cDNA clone containing the complete 3'-end untranslated region and the nucleotide sequences coding for 227 carboxyterminal amino acids was isolated. Computer-assisted comparison with known sequences of elongation factors from other species revealed homologies up to 73% and 63% on amino acid and nucleotide sequences, respectively. Northern blot analysis of mRNA from unstimulated and phorbol ester-treated cells showed a 3- to 5-fold increase in cytoplasmic elongation factor 1 alpha mRNA after phorbol ester induction. When compared with the phorbol ester-inducible single-copy gene transcripts coding for the tissue-type plasminogen activator, the cellular mRNA content of elongation factor 1 alpha is 30 times higher. By Southern blot analysis experiments on human genomic DNA, a multi-gene family was found showing polymorphisms in restriction endonuclease fragment lengths (RFLP). Several polymorphisms were studied more extensively in the population on more than 100 DNA samples from normal individuals and in three-generation families. In situ hybridization of the cDNA probe to normal human metaphase chromosomes showed multiple chromosomal localizations of the elongation factor gene(s), with peak hybridization on the chromosomes 1, 2, 4, 5, 6, 7, and 15. The estimate of the gene copy number in humans is more than ten copies per (haploid) genome.

    Human genetics 1987;75;4;339-44

  • Structure of the amino-terminal end of mammalian elongation factor Tu.

    Rao TR and Slobin LI

    Nucleic acids research 1986;14;5;2409

  • The primary structure of the alpha subunit of human elongation factor 1. Structural aspects of guanine-nucleotide-binding sites.

    Brands JH, Maassen JA, van Hemert FJ, Amons R and Möller W

    The primary structure of the alpha subunit of elongation factor 1 (EF-1 alpha) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF-1 alpha. An inventory of amino acid sequences around the guanine-nucleotide-binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved sequence elements. Region I has the sequence apolar-Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Yaa-Xaa-Gly-LYs-Thr(Ser)- -Xaa-Xaa-Xaa-Xaa-X-apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar-apolar-Xaa-Xaa-Asn-Lys-Xaa-Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.

    European journal of biochemistry 1986;155;1;167-71

  • Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes.

    Carvalho MD, Carvalho JF and Merrick WC

    Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (Mr greater than 500,000) containing four distinct polypeptides (alpha, beta, gamma, delta). The other form, EF-1 alpha, consists of a single polypeptide which is the same as the alpha subunit of EF-1H. Both EF-1 alpha and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1 alpha is enhanced in polyphenylalanine synthesis by a complementary component, EF-1 beta delta. It is also shown that EF-1 beta delta can facilitate an exchange of EF-1 alpha-bound GDP for GTP. The EF-1 alpha dissociation constants for GDP and GTP were 0.47 and 0.55 microM respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 microM, respectively. Thus, while EF-1 alpha and EF-1H had approximately the same affinities for GDP and GTP, the EF-1 alpha dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1 alpha or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along wit 1f40 h studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.

    Funded by: NIGMS NIH HHS: GM-26796

    Archives of biochemistry and biophysics 1984;234;2;603-11

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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