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/ kummari-2019-1 ecoli Escherichia coli (strain K12) molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Kummari R., Dutta S., Chaganti LK., Bose K. Beta Casein has been assumed to refer to P02666 (Bovine Casein -from Milk) throughout this publication, although this is only explicitly stated in regard to Enzyme activity assays (FITC conjugated). It has been assumed to have been purified. kbose@actrec.gov.in The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation kummari-2019-2 in vitro In vitro protease assay protease assay weight by comassie molecular weight estimation by coomasie staining weight by comassie Kummari R., Dutta S., Chaganti LK., Bose K. Beta Casein has been assumed to refer to P02666 (Bovine Casein -from Milk) throughout this publication, although this is only explicitly stated in regard to Enzyme activity assays (FITC conjugated). It has been assumed to have been purified. kbose@actrec.gov.in The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation kummari-2019-3 in vitro In vitro protease assay protease assay tag fluorescence tag visualisation by fluorescence tag fluorescence Kummari R., Dutta S., Chaganti LK., Bose K. Casein was labeled using a large molar excess of fluorescein isothiocyanate (FITC). Fluorescence properties of this heavily-labeled, intact protein substrate change dramatically upon digestion by proteases, resulting in a measurable indication of proteolysis. Beta Casein has been assumed to refer to P02666 (Bovine Casein -from Milk) throughout this publication, although this is only explicitly stated in regard to Enzyme activity assays (FITC conjugated). It has been assumed to have been purified. kbose@actrec.gov.in The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation kummari-2019-4 in vitro In vitro protease assay protease assay edman degradation edman degradation Kummari R., Dutta S., Chaganti LK., Bose K. Beta Casein has been assumed to refer to P02666 (Bovine Casein -from Milk) throughout this publication, although this is only explicitly stated in regard to Enzyme activity assays (FITC conjugated). It has been assumed to have been purified. kbose@actrec.gov.in The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation kummari-2019-5 in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Kummari R., Dutta S., Chaganti LK., Bose K. Beta Casein has been assumed to refer to P02666 (Bovine Casein -from Milk) throughout this publication, although this is only explicitly stated in regard to Enzyme activity assays (FITC conjugated). It has been assumed to have been purified. kbose@actrec.gov.in The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation htra4_human Serine protease HTRA4 High-temperature requirement factor A4 HTRA4 protein protein human Homo sapiens Human MIRPQLRTAGLGRCLLPGLLLLLVPVLWAGAEKLHTQPSCPAVCQPTRCPALPTCALGTTPVFDLCRCCRVCPAAEREVCGGAQGQPCAPGLQCLQPLRPGFPSTCGCPTLGGAVCGSDRRTYPSMCALRAENRAARRLGKVPAVPVQWGNCGDTGTRSAGPLRRNYNFIAAVVEKVAPSVVHVQLWGRLLHGSRLVPVYSGSGFIVSEDGLIITNAHVVRNQQWIEVVLQNGARYEAVVKDIDLKLDLAVIKIESNAELPVLMLGRSSDLRAGEFVVALGSPFSLQNTATAGIVSTKQRGGKELGMKDSDMDYVQIDATINYGNSGGPLVNLDGDVIGVNSLRVTDGISFAIPSDRVRQFLAEYHEHQMKGKAFSNKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVYVCKVVEGTAAQSSGLRDHDVIVNINGKPITTTTDVVKALDSDSLSMAVLRGKDNLLLTVIPETIN casb_bovin Beta-casein CSN2 protein protein bovin Bos taurus (Bovine) Bovine MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV 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 htra4-6 3676639 N/A 3676644 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 P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false N-SPD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 physical association physical association false false false Table 1 HtrA4 and N-SPD are trimers;some amount (20%) of monomeric population is also observed in HtrA4. htra4-7 3676639 N/A 3676645 unspecified role unspecified role neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3676644 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 P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 physical association physical association false false false Table 1 Only the trimeric peak was observed with complete disappearance of the monomeric population. csn2-htra4-1 3676640 N/A 3676645 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk N/A 3676644 enzyme enzyme neutral component neutral component purified purified P83105:p.Ser326Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain false N-SPD 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) Stoichiometry: 3.0 protein cleavage protein cleavage false false false Figure 2 A/B Both the trimeric protein HtrA4 and N-SPD cleaved beta-casein; although the activity of N-SPD found to be less when compared with HtrA4, particularly at higher temperatures. xiap-htra4-1 3676640 N/A 3676646 enzyme target enzyme target substrate substrate neutral component neutral component purified purified n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag 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 3676644 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 motif required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false P83105:p.Ser326Ala 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) Stoichiometry: 3.0 protein cleavage protein cleavage false false false Figure 4B csn2-htra4-2 3676641 N/A 3676645 enzyme target enzyme target substrate substrate neutral component neutral component purified purified region fitc labelled fluorescein isothiocyanate label fluorescein isothiocyanate labbeled FITC labelled FITC labelled fluorescein isothiocyanate labbeled fitc labelled undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false bovin-milk Bos taurus milk milk N/A 3676644 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) Stoichiometry: 3.0 protein cleavage protein cleavage false false false 3676641 3676641 Figure 2 C Vmax was 1.9 x 10E-12 M/Sec csn2-htra4-3 3676642 N/A 3676645 enzyme target enzyme target substrate substrate neutral component neutral component purified purified Ala15-Arg16 biological feature biological feature certain certain sequence position certain certain certain sequence position certain false Met108-Gly109 biological feature biological feature certain certain sequence position certain certain certain sequence position certain false Val170-Met171 biological feature biological feature certain certain sequence position certain certain certain sequence position certain false bovin-milk Bos taurus milk milk N/A 3676644 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) Stoichiometry: 3.0 protein cleavage protein cleavage false false false Figure 3 htra4-xiap-1 3676643 N/A 3676646 unspecified role unspecified role bait bait purified purified n-terminus gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag 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 3676644 unspecified role unspecified role prey prey 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 motif required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain false P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain false ecoli Escherichia coli (strain K12) Stoichiometry: 3.0 protein cleavage protein cleavage false false false Figure 4A