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/ http://www.ebi.ac.uk/ falkevall-2006-1 Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP. in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Falkevall A., Alikhani N., Bhushan S., Pavlov PF., Busch K., Johnson KA., Eneqvist T., Tjernberg L., Ankarcrona M., Glaser E. Apoptosis - Interactions involving proteins with a function related to apoptosis Degradadation assay: Recombinant hPreP incubated with substrate in degradation buffer for 1 hour at room temperature. Reaction was stopped by addition of laemmli sample buffer and analysed on 10-20% Tris-Tricine gels, stained with coomassie blue to detect degradation of substrates. The degradation pattern of selected substrates was studied using liquid chromatography nanospray tandem mass spectrometry. e_glaser@dbb.su.se J. Biol. Chem. (0021-9258) 2006 Only protein-protein interactions imex curation Neurodegeneration - Publications depicting interactions involved in neurodegenerative diseases. Alzheimers - Interactions investigated in the context of Alzheimers disease prep_human Presequence protease, mitochondrial PITRM1 KIAA1104 MP1 Pitrilysin metalloproteinase 1 PREP protein protein human Homo sapiens Human MWRCGGRQGLCVLRRLSGGHAHHRAWRWNSNRACERALQYKLGDKIHGFTVNQVTSVPELFLTAVKLTHDDTGARYLHLAREDTNNLFSVQFRTTPMDSTGVPHILEHTVLCGSQKYPCRDPFFKMLNRSLSTFMNAFTASDYTLYPFSTQNPKDFQNLLSVYLDATFFPCLRELDFWQEGWRLEHENPSDPQTPLVFKGVVFNEMKGAFTDNERIFSQHLQNRLLPDHTYSVVSGGDPLCIPELTWEQLKQFHATHYHPSNARFFTYGNFPLEQHLKQIHEEALSKFQKIEPSTVVPAQTPWDKPREFQITCGPDSFATDPSKQTTISVSFLLPDITDTFEAFTLSLLSSLLTSGPNSPFYKALIESGLGTDFSPDVGYNGYTREAYFSVGLQGIAEKDIETVRSLIDRTIDEVVEKGFEDDRIEALLHKIEIQMKHQSTSFGLMLTSYIASCWNHDGDPVELLKLGNQLAKFRQCLQENPKFLQEKVKQYFKNNQHKLTLSMRPDDKYHEKQAQVEATKLKQKVEALSPGDRQQIYEKGLELRSQQSKPQDASCLPALKVSDIEPTIPVTELDVVLTAGDIPVQYCAQPTNGMVYFRAFSSLNTLPEELRPYVPLFCSVLTKLGCGLLDYREQAQQIELKTGGMSASPHVLPDDSHMDTYEQGVLFSSLCLDRNLPDMMQLWSEIFNNPCFEEEEHFKVLVKMTAQELANGIPDSGHLYASIRAGRTLTPAGDLQETFSGMDQVRLMKRIAEMTDIKPILRKLPRIKKHLLNGDNMRCSVNATPQQMPQTEKAVEDFLRSIGRSKKERRPVRPHTVEKPVPSSSGGDAHVPHGSQVIRKLVMEPTFKPWQMKTHFLMPFPVNYVGECIRTVPYTDPDHASLKILARLMTAKFLHTEIREKGGAYGGGAKLSHNGIFTLYSYRDPNTIETLQSFGKAVDWAKSGKFTQQDIDEAKLSVFSTVDAPVAPSDKGMDHFLYGLSDEMKQAHREQLFAVSHDKLLAVSDRYLGTGKSTHGLAILGPENPKIAKDPSWIIQ p05067-pro_0000000093 Amyloid-beta protein 40 APP A4 AD1 ABPP Alzheimer disease amyloid protein Cerebral vascular amyloid peptide PreA4 Protease nexin-II APPI Amyloid precursor protein Amyloid-beta A4 protein Alzheimer disease amyloid A4 protein homolog Amyloid-beta (A4) precursor protein protein protein human Homo sapiens Human DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV 672 711 p05067-pro_0000000092 Amyloid-beta protein 42 Alzheimer disease amyloid protein Cerebral vascular amyloid peptide APP A4 AD1 ABPP PreA4 Protease nexin-II APPI Amyloid precursor protein Amyloid-beta A4 protein Alzheimer disease amyloid A4 protein homolog Amyloid-beta (A4) precursor protein protein protein human Homo sapiens Human DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 672 713 p01317-pro_0000015764 Insulin B chain INS protein protein bovin Bos taurus (Bovine) Bovine FVNQHLCGSHLVEALYLVCGERGFFYTPKA 25 54 p05067pro-pitrm1 Abeta(1-40) is degraded by presequence protease, mitochondrial precursor (PREP) 854448 n/a 854449 enzyme enzyme neutral component neutral component 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 false ecoli Escherichia coli (strain K12) n/a 854450 enzyme target enzyme target substrate substrate neutral component neutral component in vitro In vitro protein cleavage protein cleavage false false false Figure 1B Prep cloned into pGEX-6P-2 vector and expressed in E. coli overexpression strain BL21(DE3). The supernatent from whole cell lysate we loaded onto a a GSTrap column and Prep was eluted with PreScission Protease. Thus the GST tag is presumed to be removed prior to degradation studies. Investigation of different types of protease inhibitors showed that neither PMSF, nor bestatin affected proteolysis, whereas NEM showed a small inhibitory effect of 10% and the metalloprotease inhibitor oPh completed inhibited degradation. The author state that this demonstrates that hPreP is a thiol-sensitive metalloprotease. Email confirmation from author Elzbieta Glaser confirmed source of Abeta 1-40 to be chemically synthesised human sequence obtained from Sigma-Aldrich. p05067pro-pitrm1-1 Abeta(1-42) is degraded by presequence protease, mitochondrial precursor (PREP) 854448 n/a 854449 enzyme enzyme neutral component neutral component 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 false ecoli Escherichia coli (strain K12) n/a 854451 enzyme target enzyme target substrate substrate neutral component neutral component ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 1B The GST tag may be removed prior to degradation studies. Email confirmation from author Elzbieta Glaser confirmed source of Abeta 1-42 was human sequence and gift of Prof. Lars Lannfelt. APP Arctic protein (1-42 E22G), a protein that causes an AD-like pathology, was also degraded by PITRM1 p01317pro0000-pitrm1 Insulin B-chain is degraded by presequence protease, mitochondrial precursor (PREP) 854448 n/a 854452 enzyme target enzyme target substrate substrate neutral component neutral component n/a 854449 enzyme enzyme neutral component neutral component 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 false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 1C Prep cloned into pGEX-6P-2 vector and expressed in E. coli overexpression strain BL21(DE3). The supernatent from whole cell lysate we loaded onto a a GSTrap column and Prep was eluted with PreScission Protease. Thus the GST tag is presumed to be removed prior to degradation studies. The authors also showed that Prep was unable to degrade Bovine insulin. Email confirmation from author Elzbieta Glaser confirmed source of insulin B chain was bovine sequence and obtained from Sigma-Aldrich.