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/ RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell (0092-8674) 2007 imex curation Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar 22-Jan-2008: Contacted by JYOTI. DAVET Only protein-protein interactions 11 in vitro In vitro pull down pull down western blot western blot Immuno blot Immuno blot Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar Accepted 2007-Not a month!-6 by DAVET. Cell (0092-8674) 2007 DAVET Only protein-protein interactions 11 imex curation RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell (0092-8674) 2007 imex curation Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar 22-Jan-2008: Contacted by JYOTI. DAVET Only protein-protein interactions 11 in vitro In vitro anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar Accepted 2007-Not a month!-6 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 DAVET Only protein-protein interactions 11 imex curation RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell (0092-8674) 2007 imex curation Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar 22-Jan-2008: Contacted by JYOTI. DAVET Only protein-protein interactions 11 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar Accepted 2007-Not a month!-6 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 DAVET Only protein-protein interactions 11 imex curation RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell (0092-8674) 2007 imex curation Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar 22-Jan-2008: Contacted by JYOTI. DAVET Only protein-protein interactions 11 chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar Accepted 2007-Not a month!-6 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 DAVET Only protein-protein interactions 11 imex curation RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. RSUME, a Small RWD-Containing Protein, Enhances SUMO Conjugation and Stabilizes HIF-1alpha during Hypoxia. Cell (0092-8674) 2007 imex curation Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar 22-Jan-2008: Contacted by JYOTI. DAVET Only protein-protein interactions 11 human-hela Homo sapiens epitheloid cervix carcinoma cells Human hela Human epitheloid cervix carcinoma HeLa cells derivation: cervix; epithelial; adenocarcinoma ATCC no:CCL-2 confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Carbia-Nagashima A., Gerez J., Perez-Castro C., Paez-Pereda M., Silberstein S., Stalla GK., Holsboer F., Arzt E. Source of SUMO-1 and Ubc9 taken from PMID: 9409737 earzt@fbmc.fcen.uba.ar Accepted 2007-Not a month!-6 by DAVET. 22-Jan-2008: Contacted by JYOTI. Cell (0092-8674) 2007 DAVET Only protein-protein interactions 11 imex curation sumo1_human Small ubiquitin-related modifier 1 SUMO1 Sentrin Ubiquitin-like protein SMT3C Ubiquitin-homology domain protein PIC1 Ubiquitin-like protein UBL1 GAP-modifying protein 1 SMT3 homolog 3 SMT3C SMT3H3 UBL1 OK/SW-cl.43 protein protein human Homo sapiens Human MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV DIP protein P63165 original sequence version: 36 DIP protein P63165 original sequence version: 40 DIP protein P63165 original sequence version: 124 DIP protein P63165 original sequence version: 125 DIP protein P63165 original sequence version: 83 DIP protein P63165 original sequence version: 85 DIP protein P63165 original sequence version: 81 DIP protein P63165 original sequence version: 84 DIP protein P63165 original sequence version: 103 DIP protein P63165 original sequence version: 100 DIP protein P63165 original sequence version: 95 DIP protein P63165 original sequence version: 98 DIP protein P63165 original sequence version: 101 DIP protein P63165-1 original sequence version: 131 DIP protein P63165 original sequence version: 102 DIP protein P63165 original sequence version: 122 DIP protein P63165 original sequence version: 62 DIP protein P63165 original sequence version: 119 DIP protein P63165 original sequence version: 82 89BE97D2D054FB33 uba1_human Ubiquitin-like modifier-activating enzyme 1 Ubiquitin-activating enzyme E1 Protein A1S9 UBA1 A1S9T UBE1 protein protein human Homo sapiens Human MSSSPLSKKRRVSGPDPKPGSNCSPAQSVLSEVPSVPTNGMAKNGSEADIDEGLYSRQLYVLGHEAMKRLQTSSVLVSGLRGLGVEIAKNIILGGVKAVTLHDQGTAQWADLSSQFYLREEDIGKNRAEVSQPRLAELNSYVPVTAYTGPLVEDFLSGFQVVVLTNTPLEDQLRVGEFCHNRGIKLVVADTRGLFGQLFCDFGEEMILTDSNGEQPLSAMVSMVTKDNPGVVTCLDEARHGFESGDFVSFSEVQGMVELNGNQPMEIKVLGPYTFSICDTSNFSDYIRGGIVSQVKVPKKISFKSLVASLAEPDFVVTDFAKFSRPAQLHIGFQALHQFCAQHGRPPRPRNEEDAAELVALAQAVNARALPAVQQNNLDEDLIRKLAYVAAGDLAPINAFIGGLAAQEVMKACSGKFMPIMQWLYFDALECLPEDKEVLTEDKCLQRQNRYDGQVAVFGSDLQEKLGKQKYFLVGAGAIGCELLKNFAMIGLGCGEGGEIIVTDMDTIEKSNLNRQFLFRPWDVTKLKSDTAAAAVRQMNPHIRVTSHQNRVGPDTERIYDDDFFQNLDGVANALDNVDARMYMDRRCVYYRKPLLESGTLGTKGNVQVVIPFLTESYSSSQDPPEKSIPICTLKNFPNAIEHTLQWARDEFEGLFKQPAENVNQYLTDPKFVERTLRLAGTQPLEVLEAVQRSLVLQRPQTWADCVTWACHHWHTQYSNNIRQLLHNFPPDQLTSSGAPFWSGPKRCPHPLTFDVNNPLHLDYVMAAANLFAQTYGLTGSQDRAAVATFLQSVQVPEFTPKSGVKIHVSDQELQSANASVDDSRLEELKATLPSPDKLPGFKMYPIDFEKDDDSNFHMDFIVAASNLRAENYDIPSADRHKSKLIAGKIIPAIATTTAAVVGLVCLELYKVVQGHRQLDSYKNGFLNLALPFFGFSEPLAAPRHQYYNQEWTLWDRFEVQGLQPNGEEMTLKQFLDYFKTEHKLEITMLSQGVSMLYSFFMPAAKLKERLDQPMTEIVSRVSKRKLGRHVRALVLELCCNDESGEDVEVPYVRYTIR DIP protein P22314 original sequence version: 157 DIP protein P22314 original sequence version: 152 4B413AAA75FAA562 rwdd3_human RWD domain-containing protein 3 RWDD3 RSUME RWD domain-containing sumoylation enhancer protein protein human Homo sapiens Human MAEPVQEELSVLAAIFCRPHEWEVLSRSETDGTVFRIHTKAEGFMDVDIPLELVFHLPVNYPSCLPGISINSEQLTRAQCVTVKENLLEQAESLLSEPMVHELVLWIQQNLRHILSQPETGSGSEKCTFSTSTTMDDGLWITLLHLDHMRAKTKYVKIVEKWASDLRLTGRLMFMGKIILILLQGDRNNLKEYLILQKTSKVDVDSSGKKCKEKMISVLFETKVQTEHKRFLAFEVKEYSALDELQKEFETAGLKKLFSEFVLALVK 00F6892F29A57CA7 hif1a_human Hypoxia-inducible factor 1-alpha ARNT-interacting protein Member of PAS protein 1 Basic-helix-loop-helix-PAS protein MOP1 HIF1A MOP1 BHLHE78 PASD8 PAS domain-containing protein 8 Class E basic helix-loop-helix protein 78 protein protein human Homo sapiens Human MEGAGGANDKKKISSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHNVSSHLDKASVMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDGFVMVLTDDGDMIYISDNVNKYMGLTQFELTGHSVFDFTHPCDHEEMREMLTHRNGLVKKGKEQNTQRSFFLRMKCTLTSRGRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCLVLICEPIPHPSNIEIPLDSKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNSQPQCIVCVNYVVSGIIQHDLIFSLQQTECVLKPVESSDMKMTQLFTKVESEDTSSLFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQQTQIQEPTANATTTTATTDELKTVTKDRMEDIKILIASPSPTHIHKETTSATSSPYRDTQSRTASPNRAGKGVIEQTEKSHPRSPNVLSVALSQRTTVPEEELNPKILALQNAQRKRKMEHDGSLFQAVGIGTLLQQPDDHAATTSLSWKRVKGCKSSEQNGMEQKTIILIPSDLACRLLGQSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN DIP protein Q16665 original sequence version: 94 DIP protein Q16665 original sequence version: 98 DIP protein Q16665-1 original sequence version: 102 DIP protein Q16665 original sequence version: 102 DIP protein Q16665 original sequence version: 127 DIP protein Q16665 original sequence version: 120 DIP protein Q16665 original sequence version: 169 DIP protein Q16665 original sequence version: 174 DIP protein Q16665-1 original sequence version: 174 DIP protein Q16665-1 original sequence version: 191 DIP protein Q16665 original sequence version: 191 DIP protein Q16665 original sequence version: 179 DIP protein Q16665 original sequence version: 150 DIP protein Q16665 original sequence version: 156 DIP protein Q16665 original sequence version: 177 ABD4F7DAA135BE2D ikba_human NF-kappa-B inhibitor alpha NFKBIA IKBA MAD3 NFKBI I-kappa-B-alpha Major histocompatibility complex enhancer-binding protein MAD3 protein protein human Homo sapiens Human MFQAAERPQEWAMEGPRDGLKKERLLDDRHDSGLDSMKDEEYEQMVKELQEIRLEPQEVPRGSEPWKQQLTEDGDSFLHLAIIHEEKALTMEVIRQVKGDLAFLNFQNNLQQTPLHLAVITNQPEIAEALLGAGCDPELRDFRGNTPLHLACEQGCLASVGVLTQSCTTPHLHSILKATNYNGHTCLHLASIHGYLGIVELLVSLGADVNAQEPCNGRTALHLAVDLQNPDLVSLLLKCGADVNRVTYQGYSPYQLTWGRPSTRIQQQLGQLTLENLQMLPESEDEESYDTESEFTEFTEDELPYDDCVFGGQRLTL DIP protein P25963 original sequence version: 160 DIP protein P25963 original sequence version: 157 DIP protein P25963 original sequence version: 145 DIP protein P25963 original sequence version: 170 DIP protein P25963 original sequence version: 122 DIP protein P25963 original sequence version: 128 DIP protein P25963 original sequence version: 78 DIP protein P25963 original sequence version: 121 DIP protein P25963 original sequence version: 124 088B313226786395 ubc9_human SUMO-conjugating enzyme UBC9 UBE2I p18 SUMO-protein ligase UBC9 UBCE9 Ubiquitin carrier protein 9 Ubiquitin carrier protein I Ubiquitin-conjugating enzyme E2 I Ubiquitin-protein ligase I RING-type E3 SUMO transferase UBC9 protein protein human Homo sapiens Human MSGIALSRLAQERKAWRKDHPFGFVAVPTKNPDGTMNLMNWECAIPGKKGTPWEGGLFKLRMLFKDDYPSSPPKCKFEPPLFHPNVYPSGTVCLSILEEDKDWRPAITIKQILLGIQELLNEPNIQDPAQAEAYTIYCQNRVEYEKRVRAQAKKFAPS DIP protein P63279 original sequence version: 33 DIP protein P63279 original sequence version: 57 DIP protein P63279 original sequence version: 80 DIP protein P63279 original sequence version: 137 DIP protein P63279 original sequence version: 99 DIP protein P63279 original sequence version: 140 DIP protein P63279 original sequence version: 120 DIP protein P63279 original sequence version: 117 DIP protein P63279 original sequence version: 93 E2C826E9C8D0683D rwdd3-ube2i-1 1 8 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role bait bait region 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) direct interaction direct interaction To address this hypothesis, we performed an in vitro pull-down experiment using recombinant GST-Ubc9 and RSUME, showing that RSUME interacts with Ubc9 (Figure 4A). Figure 4A rwdd3-sumo1-1 2 6 unspecified role unspecified role bait bait 8 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Further immunoprecipitation experiments showed that RSUME could also interact with SUMO-1 (Figure 4B). Figure 4B rwdd3-sumo1-2 2 6 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Further immunoprecipitation experiments showed that RSUME could also interact with SUMO-1 (Figure 4B). Figure 4B rwdd3-ube2i-2 1 11 unspecified role unspecified role bait bait region 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) 6 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) association association In competition experiments,RSUME enhanced SUMO-1 binding to Ubc9 (lower concentrations of SUMO-1 only interacted with Ubc9 when RSUME was present) (Figure 4C) Figure 4A, 4C, 4D RSUME appeared to enhance binding of SUMO-1 to UBE2I. High concentrations of SUMO-1 could compete with RSUME for binding to UBE2I but could not displace protein which was already bound. rwdd3-ube2i-3 1 8 unspecified role unspecified role prey prey region v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 11 unspecified role unspecified role bait bait region 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) physical association physical association The interaction of RSUME to Ubc9 was confirmed in COS-7 cell extracts using GST-Ubc9 Figure 4F rwdd3-ube2i-4 4 11 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role neutral component neutral component region egfp tag enhanced green fluorescent protein tag enhanced green fluorescent protein EGFP enhanced green fluorescent protein EGFP egfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization To explore whether this interaction was supported by the intracellular localization of RSUME and Ubc9, COS-7 and HeLa cells were cotransfected with EGFP-RSUME and RFP-Ubc9 constructs. Figure 4G rwdd3-ube2i-5 5 8 unspecified role unspecified role neutral component neutral component region egfp tag enhanced green fluorescent protein tag enhanced green fluorescent protein EGFP enhanced green fluorescent protein EGFP egfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 11 unspecified role unspecified role neutral component neutral component region rfp tag red fluorescent protein tag red fluorescent protein RFP red fluorescent protein RFP rfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization To explore whether this interaction was supported by the intracellular localization of RSUME and Ubc9, COS-7 and HeLa cells were cotransfected with EGFP-RSUME and RFP-Ubc9 constructs. Figure 4G rwdd3-hif1a-1 3 9 unspecified role unspecified role prey prey region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role bait bait region v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association Immunoprecipitation experiments detected HIF-1a in an extract where RSUME was immunoprecipitated (Figure 6F), indicating an interaction between RSUME and HIF-1a during hypoxia that could account for the observed effects Figure 6F ube1-ube2i-1 1 7 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) 11 unspecified role unspecified role bait bait region 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) physical association physical association To further understand RSUME's action, we studied its effect on the interaction of Ubc9 with E1 and found that RSUME increased the binding of Ubc9 and E1 Figure 4E ube1-ube2i-2 1 7 unspecified role unspecified role bait bait region 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) 11 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) physical association physical association To further understand RSUME's action, we studied its effect on the interaction of Ubc9 with E1 and found that RSUME increased the binding of Ubc9 and E1 Figure 4E rwdd3-nkfbia-1 2 10 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait ecoli Escherichia coli (strain K12) direct interaction direct interaction Immunoprecipitation experiments showed a direct interaction of RSUME with IκB (Figure 5B). Figure 5B