IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions in vitro In vitro comigration in sds comigration in sds page comigration in sds weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions in vitro In vitro elisa enzyme linked immunosorbent assay ELISA ELISA elisa antibody detection identification by antibody antibody detection Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid predetermined predetermined participant predetermined Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner. Biochem. Biophys. Res. Commun. (0006-291X) 2009 imex curation Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Only protein-protein interactions human-lncap Human caucasian prostate carcinoma lncap Human caucasian prostate adenocarcinoma fluorescence imaging fluorescence microscopy fluorescence imaging anti tag immunost anti tag immunostaining anti tag immunost Jeong JY., Wang Y., Sytkowski AJ. asytkows@bidmc.harvard.edu Accepted 2010-FEB-4 by ORCHARD. 23-Feb-2010: Contacted by JYOTI. Human prostate cDNA library. Biochem. Biophys. Res. Commun. (0006-291X) 2009 Only protein-protein interactions imex curation ubp33_human Ubiquitin carboxyl-terminal hydrolase 33 Ubiquitin-specific-processing protease 33 Deubiquitinating enzyme 33 VHL-interacting deubiquitinating enzyme 1 USP33 KIAA1097 VDU1 Ubiquitin thioesterase 33 protein protein human Homo sapiens Human MTGSNSHITILTLKVLPHFESLGKQEKIPNKMSAFRNHCPHLDSVGEITKEDLIQKSLGTCQDCKVQGPNLWACLENRCSYVGCGESQVDHSTIHSQETKHYLTVNLTTLRVWCYACSKEVFLDRKLGTQPSLPHVRQPHQIQENSVQDFKIPSNTTLKTPLVAVFDDLDIEADEEDELRARGLTGLKNIGNTCYMNAALQALSNCPPLTQFFLDCGGLARTDKKPAICKSYLKLMTELWHKSRPGSVVPTTLFQGIKTVNPTFRGYSQQDAQEFLRCLMDLLHEELKEQVMEVEEDPQTITTEETMEEDKSQSDVDFQSCESCSNSDRAENENGSRCFSEDNNETTMLIQDDENNSEMSKDWQKEKMCNKINKVNSEGEFDKDRDSISETVDLNNQETVKVQIHSRASEYITDVHSNDLSTPQILPSNEGVNPRLSASPPKSGNLWPGLAPPHKKAQSASPKRKKQHKKYRSVISDIFDGTIISSVQCLTCDRVSVTLETFQDLSLPIPGKEDLAKLHSSSHPTSIVKAGSCGEAYAPQGWIAFFMEYVKRFVVSCVPSWFWGPVVTLQDCLAAFFARDELKGDNMYSCEKCKKLRNGVKFCKVQNFPEILCIHLKRFRHELMFSTKISTHVSFPLEGLDLQPFLAKDSPAQIVTYDLLSVICHHGTASSGHYIAYCRNNLNNLWYEFDDQSVTEVSESTVQNAEAYVLFYRKSSEEAQKERRRISNLLNIMEPSLLQFYISRQWLNKFKTFAEPGPISNNDFLCIHGGVPPRKAGYIEDLVLMLPQNIWDNLYSRYGGGPAVNHLYICHTCQIEAEKIEKRRKTELEIFIRLNRAFQKEDSPATFYCISMQWFREWESFVKGKDGDPPGPIDNTKIAVTKCGNVMLRQGADSGQISEETWNFLQSIYGGGPEVILRPPVVHVDPDILQAEEKIEVETRSL DIP protein Q8TEY7 original sequence version: 78 10A3AD632B901A74 se Selenium small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) q8tey7-2 Ubiquitin carboxyl-terminal hydrolase 33 USP33 KIAA1097 VDU1 Deubiquitinating enzyme 33 Ubiquitin-specific-processing protease 33 VHL-interacting deubiquitinating enzyme 1 Ubiquitin thioesterase 33 protein protein human Homo sapiens Human MSAFRNHCPHLDSVGEITKEDLIQKSLGTCQDCKVQGPNLWACLENRCSYVGCGESQVDHSTIHSQETKHYLTVNLTTLRVWCYACSKEVFLDRKLGTQPSLPHVRQPHQIQENSVQDFKIPSNTTLKTPLVAVFDDLDIEADEEDELRARGLTGLKNIGNTCYMNAALQALSNCPPLTQFFLDCGGLARTDKKPAICKSYLKLMTELWHKSRPGSVVPTTLFQGIKTVNPTFRGYSQQDAQEFLRCLMDLLHEELKEQVMEVEEDPQTITTEETMEEDKSQSDVDFQSCESCSNSDRAENENGSRCFSEDNNETTMLIQDDENNSEMSKDWQKEKMCNKINKVNSEGEFDKDRDSISETVDLNNQETVKVQIHSRASEYITDVHSNDLSTPQILPSNEGVNPRLSASPPKSGNLWPGLAPPHKKAQSASPKRKKQHKKYRSVISDIFDGTIISSVQCLTCDRVSVTLETFQDLSLPIPGKEDLAKLHSSSHPTSIVKAGSCGEAYAPQGWIAFFMEYVKRFVVSCVPSWFWGPVVTLQDCLAAFFARDELKGDNMYSCEKCKKLRNGVKFCKVQNFPEILCIHLKRFRHELMFSTKISTHVSFPLEGLDLQPFLAKDSPAQIVTYDLLSVICHHGTASSGHYIAYCRNNLNNLWYEFDDQSVTEVSESTVQNAEAYVLFYRKSSEEAQKERRRISNLLNIMEPSLLQFYISRQWLNKFKTFAEPGPISNNDFLCIHGGVPPRKAGYIEDLVLMLPQNIWDNLYSRYGGGPAVNHLYICHTCQIEAEKIEKRRKTELEIFIRLNRAFQKEDSPATFYCISMQWFREWESFVKGKDGDPPGPIDNTKIAVTKCGNVMLRQGADSGQISEETWNFLQSIYGGGPEVILRPPVVHVDPDILQAEEKIEVETRSL 7F3F40A75F764624 sbp1_human Methanethiol oxidase SELENBP1 SBP 56 kDa selenium-binding protein Selenium-binding protein 1 protein protein human Homo sapiens Human MATKCGNCGPGYSTPLEAMKGPREEIVYLPCIYRNTGTEAPDYLATVDVDPKSPQYCQVIHRLPMPNLKDELHHSGWNTCSSCFGDSTKSRTKLVLPSLISSRIYVVDVGSEPRAPKLHKVIEPKDIHAKCELAFLHTSHCLASGEVMISSLGDVKGNGKGGFVLLDGETFEVKGTWERPGGAAPLGYDFWYQPRHNVMISTEWAAPNVLRDGFNPADVEAGLYGSHLYVWDWQRHEIVQTLSLKDGLIPLEIRFLHNPDAAQGFVGCALSSTIQRFYKNEGGTWSVEKVIQVPPKKVKGWLLPEMPGLITDILLSLDDRFLYFSNWLHGDLRQYDISDPQRPRLTGQLFLGGSIVKGGPVQVLEDEELKSQPEPLVVKGKRVAGGPQMIQLSLDGKRLYITTSLYSAWDKQFYPDLIREGSVMLQVDVDTVKGGLKLNPNFLVDFGKEPLGPALAHELRYPGGDCSSDIWI 6DC68F9B45FEC1BC hsbp1-vdu1 2 7 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 10 unspecified role unspecified role bait bait physical association physical association In the present study, we identified von Hippel-Lindau protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein partner of hSP56 using a yeast two-hybrid screen. hsbp1-vdu1-2 1 9 unspecified role unspecified role bait bait c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role prey prey direct interaction direct interaction Fig 1B. hsbp1-vdu1-3 4 9 unspecified role unspecified role prey prey c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain n-terminal gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) 10 unspecified role unspecified role bait bait direct interaction direct interaction Fig 1C. We carried out another type of in vitro binding experiment using an ELISA format. BSA (negative control) and hSP56 were each used to coat wells of a 96-well plate and GST-VDU1f was added and allowed to incubate hsbp1-vdu1-1 6 9 unspecified role unspecified role neutral component neutral component c-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 10 unspecified role unspecified role neutral component neutral component c-terminal egfp tag enhanced green fluorescent protein tag enhanced green fluorescent protein EGFP enhanced green fluorescent protein EGFP egfp tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range colocalization colocalization Fig 1D. Co-localization of hSP56 with VDU1 in the cytoplasm of LNCaP cells. hsbp1-vdu1-se 4 9 unspecified role unspecified role prey prey c-terminal ha-tag ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range 10 unspecified role unspecified role bait bait chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 physical association physical association This interaction only occurred if hSBP1 was pre-incubated with sodium selenite for 30 mins at RT. Similarly, a reaction only took place under non-reducing conditions i.e. the absence of 2-ME. Fig 2C. Selenium selenite sbp1-vdu1 5 10 unspecified role unspecified role bait bait 9 unspecified role unspecified role prey prey c-terminal region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Fig. 1A We isolated the library plasmid encoding the VDU1 C-terminal fragment (AD-VDU1f) and examined its interaction with hSP56 using yeast two-hybrid analysis (Fig. 1A) selenbp1-se 3 8 unspecified role unspecified role neutral component neutral component 10 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Selenium binding to hSP56 was only maintained under non-reducing conditions. Radiolabelled sodium selenite (75-Selenium) was used in SDS-PAGE.