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/ bekhouche-2010-3 in vitro In vitro cleavage cleavage assay cleavage predetermined predetermined participant predetermined Bekhouche M., Kronenberg D., Vadon-Le Goff S., Bijakowski C., Lim NH., Font B., Kessler E., Colige A., Nagase H., Murphy G., Hulmes DJ., Moali C. Enzymatic assay from Fig. 3, Fig. 5 and Fig. 6 were not captured because it was not possible to identify an unambiguous UniprotAC for collagen proteins. d.hulmes@ibcp.fr The Journal of biological chemistry 2010 Only protein-protein interactions imex curation imex curation bekhouche-2010-1 in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Bekhouche M., Kronenberg D., Vadon-Le Goff S., Bijakowski C., Lim NH., Font B., Kessler E., Colige A., Nagase H., Murphy G., Hulmes DJ., Moali C. To further investigate the mechanism of superstimulation, we studied the binding of heparin to each of the molecules involved: PCPE-1, mini-procollagen III, and BMP-1. By surface plasmon resonance using biotinylated heparin stably immobilized to a streptavidin surface, we observed binding of both BMP-1 and mini-procollagen III when injected at a concentration of 50 nm (Fig. 7). This was also the case for PCPE-1, which has previously been reported to bind to heparin, via the NTR domain, with nanomolar affinity (23, 41). Interestingly, whereas mini-procollagen III bound heparin in a rather stable manner with a slow dissociation phase, BMP-1 displayed fast association and dissociation phases and was washed off from the surface very rapidly. Dissociation constants were found to be in the range of 10–150 nm for PCPE-1 and mini-procollagen III and 0.5–2 μm for BMP-1. In contrast, the C-propeptide trimer of procollagen III did not bind at all to the immobilized heparin, suggesting that the binding site for heparin is located in the triple-helical or telopeptide regions of mini-procollagen III. Enzymatic assay from Fig. 3, Fig. 5 and Fig. 6 were not captured because it was not possible to identify an unambiguous UniprotAC for collagen proteins. d.hulmes@ibcp.fr The Journal of biological chemistry 2010 Only protein-protein interactions imex curation imex curation bekhouche-2010-4 in vitro In vitro cleavage cleavage assay cleavage protein sequence protein sequence identification protein sequence Bekhouche M., Kronenberg D., Vadon-Le Goff S., Bijakowski C., Lim NH., Font B., Kessler E., Colige A., Nagase H., Murphy G., Hulmes DJ., Moali C. Enzymatic assay from Fig. 3, Fig. 5 and Fig. 6 were not captured because it was not possible to identify an unambiguous UniprotAC for collagen proteins. d.hulmes@ibcp.fr The Journal of biological chemistry 2010 Only protein-protein interactions imex curation imex curation peptide_yvadap peptide_YVADAP peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro YVADAP bmp1_human Bone morphogenetic protein 1 Procollagen C-proteinase Mammalian tolloid protein BMP1 PCOLC protein protein human Homo sapiens Human MPGVARLPLLLGLLLLPRPGRPLDLADYTYDLAEEDDSEPLNYKDPCKAAAFLGDIALDEEDLRAFQVQQAVDLRRHTARKSSIKAAVPGNTSTPSCQSTNGQPQRGACGRWRGRSRSRRAATSRPERVWPDGVIPFVIGGNFTGSQRAVFRQAMRHWEKHTCVTFLERTDEDSYIVFTYRPCGCCSYVGRRGGGPQAISIGKNCDKFGIVVHELGHVVGFWHEHTRPDRDRHVSIVRENIQPGQEYNFLKMEPQEVESLGETYDFDSIMHYARNTFSRGIFLDTIVPKYEVNGVKPPIGQRTRLSKGDIAQARKLYKCPACGETLQDSTGNFSSPEYPNGYSAHMHCVWRISVTPGEKIILNFTSLDLYRSRLCWYDYVEVRDGFWRKAPLRGRFCGSKLPEPIVSTDSRLWVEFRSSSNWVGKGFFAVYEAICGGDVKKDYGHIQSPNYPDDYRPSKVCIWRIQVSEGFHVGLTFQSFEIERHDSCAYDYLEVRDGHSESSTLIGRYCGYEKPDDIKSTSSRLWLKFVSDGSINKAGFAVNFFKEVDECSRPNRGGCEQRCLNTLGSYKCSCDPGYELAPDKRRCEAACGGFLTKLNGSITSPGWPKEYPPNKNCIWQLVAPTQYRISLQFDFFETEGNDVCKYDFVEVRSGLTADSKLHGKFCGSEKPEVITSQYNNMRVEFKSDNTVSKKGFKAHFFSDKDECSKDNGGCQQDCVNTFGSYECQCRSGFVLHDNKHDCKEAGCDHKVTSTSGTITSPNWPDKYPSKKECTWAISSTPGHRVKLTFMEMDIESQPECAYDHLEVFDGRDAKAPVLGRFCGSKKPEPVLATGSRMFLRFYSDNSVQRKGFQASHATECGGQVRADVKTKDLYSHAQFGDNNYPGGVDCEWVIVAEEGYGVELVFQTFEVEEETDCGYDYMELFDGYDSTAPRLGRYCGSGPPEEVYSAGDSVLVKFHSDDTITKKGFHLRYTSTKFQDTLHSRK peptide_plgl peptide_PLGL peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro PLGL mmp2_human 72 kDa type IV collagenase MMP2 CLG4A 72 kDa gelatinase Matrix metalloproteinase-2 Gelatinase A TBE-1 protein protein human Homo sapiens Human MEALMARGALTGPLRALCLLGCLLSHAAAAPSPIIKFPGDVAPKTDKELAVQYLNTFYGCPKESCNLFVLKDTLKKMQKFFGLPQTGDLDQNTIETMRKPRCGNPDVANYNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDPGFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSVKFGSIKSDWLGC peptide_aelqgrpisiak peptide_AELQGRPISIAK peptide peptide polypeptide oligopeptide oligopeptide polypeptide in vitro In vitro AELQGRPISIAK ats4_human A disintegrin and metalloproteinase with thrombospondin motifs 4 ADAMTS4 KIAA0688 ADMP-1 Aggrecanase-1 UNQ769/PRO1563 protein protein human Homo sapiens Human MSQTGSHPGRGLAGRWLWGAQPCLLLPIVPLSWLVWLLLLLLASLLPSARLASPLPREEEIVFPEKLNGSVLPGSGAPARLLCRLQAFGETLLLELEQDSGVQVEGLTVQYLGQAPELLGGAEPGTYLTGTINGDPESVASLHWDGGALLGVLQYRGAELHLQPLEGGTPNSAGGPGAHILRRKSPASGQGPMCNVKAPLGSPSPRPRRAKRFASLSRFVETLVVADDKMAAFHGAGLKRYLLTVMAAAAKAFKHPSIRNPVSLVVTRLVILGSGEEGPQVGPSAAQTLRSFCAWQRGLNTPEDSDPDHFDTAILFTRQDLCGVSTCDTLGMADVGTVCDPARSCAIVEDDGLQSAFTAAHELGHVFNMLHDNSKPCISLNGPLSTSRHVMAPVMAHVDPEEPWSPCSARFITDFLDNGYGHCLLDKPEAPLHLPVTFPGKDYDADRQCQLTFGPDSRHCPQLPPPCAALWCSGHLNGHAMCQTKHSPWADGTPCGPAQACMGGRCLHMDQLQDFNIPQAGGWGPWGPWGDCSRTCGGGVQFSSRDCTRPVPRNGGKYCEGRRTRFRSCNTEDCPTGSALTFREEQCAAYNHRTDLFKSFPGPMDWVPRYTGVAPQDQCKLTCQAQALGYYYVLEPRVVDGTPCSPDSSSVCVQGRCIHAGCDRIIGSKKKFDKCMVCGGDGSGCSKQSGSFRKFRYGYNNVVTIPAGATHILVRQQGNPGHRSIYLALKLPDGSYALNGEYTLMPSPTDVVLPGAVSLRYSGATAASETLSGHGPLAQPLTLQVLVAGNPQDTRLRYSFFVPRPTPSTPRPTPQDWLHRRAQILEILRRRPWAGRK ats5_human A disintegrin and metalloproteinase with thrombospondin motifs 5 ADAMTS5 ADAMTS11 ADMP2 A disintegrin and metalloproteinase with thrombospondin motifs 11 ADMP-2 Aggrecanase-2 protein protein human Homo sapiens Human MLLGWASLLLCAFRLPLAAVGPAATPAQDKAGQPPTAAAAAQPRRRQGEEVQERAEPPGHPHPLAQRRRSKGLVQNIDQLYSGGGKVGYLVYAGGRRFLLDLERDGSVGIAGFVPAGGGTSAPWRHRSHCFYRGTVDGSPRSLAVFDLCGGLDGFFAVKHARYTLKPLLRGPWAEEEKGRVYGDGSARILHVYTREGFSFEALPPRASCETPASTPEAHEHAPAHSNPSGRAALASQLLDQSALSPAGGSGPQTWWRRRRRSISRARQVELLLVADASMARLYGRGLQHYLLTLASIANRLYSHASIENHIRLAVVKVVVLGDKDKSLEVSKNAATTLKNFCKWQHQHNQLGDDHEEHYDAAILFTREDLCGHHSCDTLGMADVGTICSPERSCAVIEDDGLHAAFTVAHEIGHLLGLSHDDSKFCEETFGSTEDKRLMSSILTSIDASKPWSKCTSATITEFLDDGHGNCLLDLPRKQILGPEELPGQTYDATQQCNLTFGPEYSVCPGMDVCARLWCAVVRQGQMVCLTKKLPAVEGTPCGKGRICLQGKCVDKTKKKYYSTSSHGNWGSWGSWGQCSRSCGGGVQFAYRHCNNPAPRNNGRYCTGKRAIYRSCSLMPCPPNGKSFRHEQCEAKNGYQSDAKGVKTFVEWVPKYAGVLPADVCKLTCRAKGTGYYVVFSPKVTDGTECRLYSNSVCVRGKCVRTGCDGIIGSKLQYDKCGVCGGDNSSCTKIVGTFNKKSKGYTDVVRIPEGATHIKVRQFKAKDQTRFTAYLALKKKNGEYLINGKYMISTSETIIDINGTVMNYSGWSHRDDFLHGMGYSATKEILIVQILATDPTKPLDVRYSFFVPKKSTPKVNSVTSHGSNKVGSHTSQPQWVTGPWLACSRTCDTGWHTRTVQCQDGNRKLAKGCPLSQRPSAFKQCLLKKC pcoc1_human Procollagen C-endopeptidase enhancer 1 PCOLCE PCPE1 Procollagen COOH-terminal proteinase enhancer 1 Type 1 procollagen C-proteinase enhancer protein Type I procollagen COOH-terminal proteinase enhancer protein protein human Homo sapiens Human MLPAATASLLGPLLTACALLPFAQGQTPNYTRPVFLCGGDVKGESGYVASEGFPNLYPPNKECIWTITVPEGQTVSLSFRVFDLELHPACRYDALEVFAGSGTSGQRLGRFCGTFRPAPLVAPGNQVTLRMTTDEGTGGRGFLLWYSGRATSGTEHQFCGGRLEKAQGTLTTPNWPESDYPPGISCSWHIIAPPDQVIALTFEKFDLEPDTYCRYDSVSVFNGAVSDDSRRLGKFCGDAVPGSISSEGNELLVQFVSDLSVTADGFSASYKTLPRGTAKEGQGPGPKRGTEPKVKLPPKSQPPEKTEESPSAPDAPTCPKQCRRTGTLQSNFCASSLVVTATVKSMVREPGEGLAVTVSLIGAYKTGGLDLPSPPTGASLKFYVPCKQCPPMKKGVSYLLMGQVEENRGPVLPPESFVVLHRPNQDQILTNLSKRKCPSQPVRAAASQD heparin Heparin small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) bmp1-peptide_yvadap-1 3451238 N/A 3451241 enzyme target enzyme target substrate substrate neutral component neutral component purified purified Dnp fluorophore fluorophore c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Mca fluorophore fluorophore n-term range n-terminal range n-term range n-term range n-terminal range n-term range false chemical synthesis Chemical synthesis (Chemical synthesis) N/A 3451242 enzyme enzyme neutral component neutral component purified purified flag tag flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false human-293 ebna Homo sapiens 293 ebna 293 ebna cleavage cleavage reaction cleavage false false false Fig. S2 We conclude that the NTR domain of PCPE-1 is not an inhibitor of BMP-1 or ADAMTS-2, -4, or -5. mmp2-peptide_plgl-1 3451238 N/A 3451243 enzyme target enzyme target substrate substrate neutral component neutral component purified purified Dnp fluorophore fluorophore c-term range C-terminal range c-term range c-term range C-terminal range c-term range false Mca fluorophore fluorophore n-term range n-terminal range n-term range n-term range n-terminal range n-term range false chemical synthesis Chemical synthesis (Chemical synthesis) N/A 3451244 enzyme enzyme neutral component neutral component purified purified mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc cleavage cleavage reaction cleavage false false false Fig. 3 We also tested the effects of both forms of NTR on MMP-2 cleavage of denatured collagen I but found no evidence of inhibition at a NTR concentration of 2 μm (supplemental Fig. S1). We conclude that the NTR domain of PCPE-1 is not an inhibitor of MMP-2. adamts4-peptide_aelqgrpisiak-1 3451238 N/A 3451245 enzyme target enzyme target substrate substrate neutral component neutral component purified purified TAMRA fluorophore fluorophore c-term range C-terminal range c-term range c-term range C-terminal range c-term range false FAM fluorophore fluorophore n-term range n-terminal range n-term range n-term range n-terminal range n-term range false chemical synthesis Chemical synthesis (Chemical synthesis) N/A 3451246 enzyme enzyme neutral component neutral component purified purified human-293 ebna Homo sapiens 293 ebna 293 ebna cleavage cleavage reaction cleavage false false false Fig. S2 We conclude that the NTR domain of PCPE-1 is not an inhibitor of BMP-1 or ADAMTS-2, -4, or -5. adamts5-peptide_aelqgrpisiak-1 3451238 N/A 3451245 enzyme target enzyme target substrate substrate neutral component neutral component purified purified TAMRA fluorophore fluorophore c-term range C-terminal range c-term range c-term range C-terminal range c-term range false FAM fluorophore fluorophore n-term range n-terminal range n-term range n-term range n-terminal range n-term range false chemical synthesis Chemical synthesis (Chemical synthesis) N/A 3451247 enzyme enzyme neutral component neutral component purified purified human-293 ebna Homo sapiens 293 ebna 293 ebna cleavage cleavage reaction cleavage false false false Fig. S2 We conclude that the NTR domain of PCPE-1 is not an inhibitor of BMP-1 or ADAMTS-2, -4, or -5. pcpe1-bmp1-1 3451239 N/A 3451242 unspecified role unspecified role bait bait purified purified human-293 ebna Homo sapiens 293 ebna 293 ebna N/A 3451248 unspecified role unspecified role prey prey purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain range range false human-293 ebna Homo sapiens 293 ebna 293 ebna direct interaction direct interaction false false false 3451239 Fig. 4 heparin-bmp1-1 3451239 N/A 3451249 unspecified role unspecified role bait bait purified purified biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false pig Sus scrofa (Pig) Pig N/A 3451242 unspecified role unspecified role prey prey purified purified human-293 ebna Homo sapiens 293 ebna 293 ebna direct interaction direct interaction false false false 3451239 Fig. 7 heparin-pcolce-3 3451239 N/A 3451249 unspecified role unspecified role bait bait purified purified biotin tag biotin tag biotin tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false pig Sus scrofa (Pig) Pig N/A 3451248 unspecified role unspecified role prey prey purified purified human-293 ebna Homo sapiens 293 ebna 293 ebna direct interaction direct interaction false false false 3451239 Fig. 7 mmp2-pcolce-1 3451240 N/A 3451248 enzyme target enzyme target substrate substrate neutral component neutral component purified purified His tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false cleaved residue uncategorized protein modification uncategorized protein modification partial id prot seq partial identification of protein sequence certain certain sequence position certain certain certain sequence position certain false human-293 ebna Homo sapiens 293 ebna 293 ebna N/A 3451244 enzyme enzyme neutral component neutral component purified purified mouse-nso NSO-mouse myeloma subclone of NS-1 ns0 NS0 Mouse Myeloma subclone of NS-1 http://www.hpacultures.org.uk/products/celllines/generalcell/detail.jsp?refId=85110503&collection=ecacc_gc from ecacc cleavage cleavage reaction cleavage false false false Fig. 1