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/ Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. The Biochemical journal 2019 imex curation 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 Only protein-protein interactions imex curation 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 Accepted 2020-JUN-24 AT 04:51 BST AT 04:51 BST by KALPANAP The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. The Biochemical journal 2019 imex curation 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 Only protein-protein interactions imex curation 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 Accepted 2020-JUN-24 AT 04:51 BST AT 04:51 BST by KALPANAP The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. The Biochemical journal 2019 imex curation 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 Only protein-protein interactions imex curation 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 Accepted 2020-JUN-24 AT 04:51 BST AT 04:51 BST by KALPANAP The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. The Biochemical journal 2019 imex curation 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 Only protein-protein interactions imex curation 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 Accepted 2020-JUN-24 AT 04:51 BST AT 04:51 BST by KALPANAP The Biochemical journal 2019 Only protein-protein interactions imex curation imex curation I have moved it to Expt-2 where I think it should be, most likely as no method is stated for detection. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. Discerning the mechanism of action of HtrA4: a serine protease implicated in the cell death pathway. The Biochemical journal 2019 imex curation 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 Only protein-protein interactions imex curation 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 Accepted 2020-JUN-24 AT 04:51 BST AT 04:51 BST by KALPANAP 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 DIP protein P02666 original sequence version: 88 DIP protein P02666 original sequence version: 101 F0BBDD8148A238AE 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 DIP protein P98170 original sequence version: 168 DIP protein P98170 original sequence version: 118 DIP protein P98170 original sequence version: 153 9D394C16D45EB635 htra4-6 5 6 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 P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain S A Tue Jun 30 17:50:54 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser326Ala' N-SPD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) physical association physical association Table 1 HtrA4 and N-SPD are trimers;some amount (20%) of monomeric population is also observed in HtrA4. htra4-7 5 7 unspecified role unspecified role neutral component neutral component purified purified bovin-milk Bos taurus milk milk 6 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 P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain S A Tue Jun 30 16:26:23 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser326Ala' ecoli Escherichia coli (strain K12) physical association physical association Table 1 Only the trimeric peak was observed with complete disappearance of the monomeric population. csn2-htra4-1 4 7 enzyme target enzyme target substrate substrate neutral component neutral component purified purified bovin-milk Bos taurus milk milk 6 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 S A Tue Jun 30 15:41:09 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser326Ala' N-SPD sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 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 ecoli Escherichia coli (strain K12) protein cleavage protein cleavage 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. csn2-htra4-2 2 7 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 bovin-milk Bos taurus milk milk 6 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 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 ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Figure 2 C Vmax was 1.9 x 10E-12 M/Sec the Km is from THIS publication, not Ref. 38, as Htra4 is not mentioned there. csn2-htra4-3 1 7 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 resulting-cleavage resulting-cleavage Met108-Gly109 biological feature biological feature certain certain sequence position certain certain certain sequence position certain resulting-cleavage resulting-cleavage Val170-Met171 biological feature biological feature certain certain sequence position certain certain certain sequence position certain resulting-cleavage resulting-cleavage bovin-milk Bos taurus milk milk 6 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 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 ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Figure 3 htra4-xiap-1 3 8 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 ecoli Escherichia coli (strain K12) 6 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 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 motif required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain P83105:p.Ser326Ala mutation mutation certain certain sequence position certain certain certain sequence position certain S A Tue Jun 30 18:34:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser326Ala' ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Figure 4A xiap-htra4-1 4 8 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 ecoli Escherichia coli (strain K12) 6 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 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 motif required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain P83105:p.Ser326Ala mutation disrupting rate mutation disrupting interaction rate mutation disrupting rate certain certain sequence position certain certain certain sequence position certain S A Tue Jun 30 17:43:25 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser326Ala' ecoli Escherichia coli (strain K12) protein cleavage protein cleavage Figure 4B