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/ wenta-2017-2 in vitro In vitro protease assay protease assay predetermined predetermined participant predetermined Wenta T., Glaza P., Jarząb M., Zarzecka U., Żurawa-Janicka D., Lesner A., Skórko-Glonek J., Lipińska B. Beta Casein has been assumed to be of Bovine (milk) origin as this protein is used in multiple publications by this group. barbara.lipinska@biol.ug.edu.pl Biochimica et biophysica acta. Proteins and proteomics 2017 Only protein-protein interactions imex curation imex curation wenta-2017-1 ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column experimental particp experimental participant identification experimental particp Wenta T., Glaza P., Jarząb M., Zarzecka U., Żurawa-Janicka D., Lesner A., Skórko-Glonek J., Lipińska B. barbara.lipinska@biol.ug.edu.pl Biochimica et biophysica acta. Proteins and proteomics 2017 Only protein-protein interactions imex curation imex curation casb_bovin Beta-casein CSN2 protein protein bovin Bos taurus (Bovine) Bovine MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV p83110-1 Serine protease HTRA3 HTRA3 PRSP High-temperature requirement factor A3 Pregnancy-related serine protease Long pL protein protein human Homo sapiens Human MQARALLLAALAALALAREPPAAPCPARCDVSRCPSPRCPGGYVPDLCNCCLVCAASEGEPCGGPLDSPCGESLECVRGLCRCRWSHAVCGTDGHTYANVCALQAASRRALQLSGTPVRQLQKGACPLGLHQLSSPRYKFNFIADVVEKIAPAVVHIELFLRHPLFGRNVPLSSGSGFIMSEAGLIITNAHVVSSNSAAPGRQQLKVQLQNGDSYEATIKDIDKKSDIATIKIHPKKKLPVLLLGHSADLRPGEFVVAIGSPFALQNTVTTGIVSTAQREGRELGLRDSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDRITRFLTEFQDKQIKDWKKRFIGIRMRTITPSLVDELKASNPDFPEVSSGIYVQEVAPNSPSQRGGIQDGDIIVKVNGRPLVDSSELQEAVLTESPLLLEVRRGNDDLLFSIAPEVVM xiap_human E3 ubiquitin-protein ligase XIAP Inhibitor of apoptosis protein 3 X-linked inhibitor of apoptosis protein IAP-like protein XIAP API3 IAP3 BIRC4 Baculoviral IAP repeat-containing protein 4 RING-type E3 ubiquitin transferase XIAP protein protein human Homo sapiens Human MTFNSFEGSKTCVPADINKEEEFVEEFNRLKTFANFPSGSPVSASTLARAGFLYTGEGDTVRCFSCHAAVDRWQYGDSAVGRHRKVSPNCRFINGFYLENSATQSTNSGIQNGQYKVENYLGSRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNWPDYAHLTPRELASAGLYYTGIGDQVQCFCCGGKLKNWEPCDRAWSEHRRHFPNCFFVLGRNLNIRSESDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGFYALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTHSLEECLVRTTEKTPSLTRRIDDTIFQNPMVQEAIRMGFSFKDIKKIMEEKIQISGSNYKSLEVLVADLVNAQKDSMQDESSQTSLQKEISTEEQLRRLQEEKLCKICMDRNIAIVFVPCGHLVTCKQCAEAVDKCPMCYTVITFKQKIFMS ala(mca)irrvsysf–anb–nh2 peptide peptide polypeptide oligopeptide oligopeptide polypeptide chemical synthesis Chemical synthesis (Chemical synthesis) IRRVSYSF htra1_human Serine protease HTRA1 HTRA1 HTRA PRSS11 High-temperature requirement A serine peptidase 1 L56 Serine protease 11 protein protein human Homo sapiens Human MQIPRAALLPLLLLLLAAPASAQLSRAGRSAPLAAGCPDRCEPARCPPQPEHCEGGRARDACGCCEVCGAPEGAACGLQEGPCGEGLQCVVPFGVPASATVRRRAQAGLCVCASSEPVCGSDANTYANLCQLRAASRRSERLHRPPVIVLQRGACGQGQEDPNSLRHKYNFIADVVEKIAPAVVHIELFRKLPFSKREVPVASGSGFIVSEDGLIVTNAHVVTNKHRVKVELKNGATYEAKIKDVDEKADIALIKIDHQGKLPVLLLGRSSELRPGEFVVAIGSPFSLQNTVTTGIVSTTQRGGKELGLRNSDMDYIQTDAIINYGNSGGPLVNLDGEVIGINTLKVTAGISFAIPSDKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAKELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITVIPEEIDP htra2_human Serine protease HTRA2, mitochondrial High temperature requirement protein A2 Omi stress-regulated endoprotease Serine proteinase OMI Serine protease 25 HTRA2 PRSS25 OMI protein protein human Homo sapiens Human MAAPRAGRGAGWSLRAWRALGGIRWGRRPRLTPDLRALLTSGTSDPRARVTYGTPSLWARLSVGVTEPRACLTSGTPGPRAQLTAVTPDTRTREASENSGTRSRAWLAVALGAGGAVLLLLWGGGRGPPAVLAAVPSPPPASPRSQYNFIADVVEKTAPAVVYIEILDRHPFLGREVPISNGSGFVVAADGLIVTNAHVVADRRRVRVRLLSGDTYEAVVTAVDPVADIATLRIQTKEPLPTLPLGRSADVRQGEFVVAMGSPFALQNTITSGIVSSAQRPARDLGLPQTNVEYIQTDAAIDFGNSGGPLVNLDGEVIGVNTMKVTAGISFAIPSDRLREFLHRGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQLREPSFPDVQHGVLIHKVILGSPAHRAGLRPGDVILAIGEQMVQNAEDVYEAVRTQSQLAVQIRRGRETLTLYVTPEVTE degp_ecoli Periplasmic serine endoprotease DegP degP htrA ptd b0161 JW0157 Heat shock protein DegP Protease Do protein protein ecoli Escherichia coli (strain K12) MKKTTLALSALALSLGLALSPLSATAAETSSATTAQQMPSLAPMLEKVMPSVVSINVEGSTTVNTPRMPRNFQQFFGDDSPFCQEGSPFQSSPFCQGGQGGNGGGQQQKFMALGSGVIIDADKGYVVTNNHVVDNATVIKVQLSDGRKFDAKMVGKDPRSDIALIQIQNPKNLTAIKMADSDALRVGDYTVAIGNPFGLGETVTSGIVSALGRSGLNAENYENFIQTDAAINRGNSGGALVNLNGELIGINTAILAPDGGNIGIGFAIPSNMVKNLTSQMVEYGQVKRGELGIMGTELNSELAKAMKVDAQRGAFVSQVLPNSSAAKAGIKAGDVITSLNGKPISSFAALRAQVGTMPVGSKLTLGLLRDGKQVNVNLELQQSSQNQVDSSSIFNGIEGAEMSNKGKDQGVVVNNVKTGTPAAQIGLKKGDVIIGANQQAVKNIAELRKVLDSKPSVLALNIQRGDSTIYLLMQ htra3-20 3672634 N/A 3672636 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3672637 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified P83110-1:p.Phe142Asp mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false p.Asn196_Gly201del mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P83110-1:p.[Arg362Ala;Glu371Ala;Gln389Ala] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P83110-1:p.Ser305Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 4, 7B/C, 8,Supp. fig 2. Cleavage rate values for Delta-N-HtrA3L was: 3.7 nM b-casein/uM enzyme/min. htra3-xiap-17 3672634 N/A 3672638 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) N/A 3672637 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Ser305Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false p.Asn196_Gly201del mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P83110-1:p.[Arg362Ala;Glu371Ala;Gln389Ala] mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 4, 8, Supp. fig 2. Cleavage rate values for Delta-N-HtrA3L was: 60.1nM XIAP/uM enzyme/min. ala(mca)irrvsysfanbnh2-htra3-8 3672634 N/A 3672639 enzyme target enzyme target substrate substrate neutral component neutral component purified purified N-terminus fluorophore fluorophore n-term range n-terminal range n-term range n-term range n-terminal range n-term range false ecoli Escherichia coli (strain K12) N/A 3672637 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified P83110-1:p.Arg362Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Glu371Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Gln389Ala mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false p.Asn196_Gly201del mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false P83110-1:p.[Arg362Ala;Glu371Ala;Gln389Ala] mutation decreasing rate mutation decreasing interaction rate mutation decreasing rate certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Table 5, Figure 5, Supp. fig 3 k cat /Km (units /M/sec) (catalytic efficiency) for Wt =38,160. Mutant values range between ~10,000 to ~16, 000. At temperatures 25–40 deg.C all mutations had a negative effect on the peptide cleavage rate but as the temperature increased, the effect decreased. However, the effect of the DeltaLB mutation could not be alleviated. htra3-xiap-18 3672634 N/A 3672638 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) N/A 3672640 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 8 htra1-xiap-1 3672634 N/A 3672638 enzyme target enzyme target substrate substrate neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) N/A 3672641 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 8 htra1-xiap-2 3672634 N/A 3672636 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3672640 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 8 csn2-htra1-1 3672634 N/A 3672636 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3672641 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 8 htra2-xiap-6 3672634 N/A 3672636 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3672642 weight silver stain molecular weight estimation by silver staining weight silver stain enzyme enzyme neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false ecoli Escherichia coli (strain K12) protein cleavage protein cleavage false false false Figure 8 The bacterial HtrA(DegP) very efficiently digested beta-casein but was practically unable to cut XIAP. htra3-17 3672635 N/A 3672637 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P83110-1:p.Phe142Asp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Glu371Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Gln389Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P83110-1:p.Arg362Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 3.0 physical association physical association false false false 3A/C/D,7A, Table 4. Supp. fig. 1. htra3-18 3672635 N/A 3672637 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region_minus_LB sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P83110-1:p.[Arg362Ala;Glu371Ala;Gln389Ala] mutation mutation certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 6.0 physical association physical association false false false 3A/B, Table 4. Supp. fig. 1 htra3-19 3672635 N/A 3672640 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 3.0 physical association physical association false false false Supp. fig. 1, Table 4. htra2-1 3672635 N/A 3672641 weight silver stain molecular weight estimation by silver staining weight silver stain predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified C-terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false Stoichiometry: 3.0 physical association physical association false false false Supp. fig. 1, Table 4.