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dc.contributor.authorGarcía-Pardo, Javier
dc.contributor.authorTanco, Sebastian
dc.contributor.authorDiaz, Lucia
dc.contributor.authorDasgupta, Sayani
dc.contributor.authorFernandez-Recio, Juan
dc.contributor.authorLorenzo, Julia
dc.contributor.authorAviles, Francesc X.
dc.contributor.authorFricker, Lloyd D.
dc.contributor.otherBarcelona Supercomputing Center
dc.date.accessioned2018-01-03T16:23:38Z
dc.date.available2018-01-03T16:23:38Z
dc.date.issued2017-11-13
dc.identifier.citationGarcía-Pardo, J. [et al.]. Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains. "PLoS ONE", 13 Novembre 2017, vol. 12, núm. 11.
dc.identifier.issn1932-6203
dc.identifier.urihttp://hdl.handle.net/2117/112434
dc.description.abstractMetallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell.
dc.description.sponsorshipThis work was funded by the Spanish Ministry of Innovation and Competitiveness grants BIO2013-44973-R and BIO2016-78057-R (to FXA), by Plan Estatal grant number BIO2016-79960-R from the Spanish Ministry of Economy and Competitiveness (to JFR), and by grant R01-DA004494 from the United States’ National Institute of Health (to LDF).
dc.format.extent29 p.
dc.language.isoeng
dc.publisherPublic Library of Science
dc.rightsAttribution-NonCommercial-NoDerivs 4.0 Spain
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/es/
dc.subjectÀrees temàtiques de la UPC::Enginyeria biomèdica
dc.subject.lcshProtein engineering
dc.subject.otherMetallocarboxypeptidase D (CPD)
dc.subject.otherCarboxypeptidase domains
dc.titleSubstrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains
dc.typeArticle
dc.subject.lemacEnginyeria de proteïnes
dc.identifier.doi10.1371/journal.pone.0187778
dc.description.peerreviewedPeer Reviewed
dc.relation.publisherversionhttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187778
dc.rights.accessOpen Access
dc.description.versionPostprint (published version)
dc.relation.projectidinfo:eu-repo/grantAgreement/MINECO/PE2013-2016/BIO2013-44973-R
dc.relation.projectidinfo:eu-repo/grantAgreement/MINECO/PE2013-2016/BIO2016-78057-R
dc.relation.projectidinfo:eu-repo/grantAgreement/MINECO/PE2013-2016/BIO2016-79960-R
upcommons.citation.publishedtrue
upcommons.citation.publicationNamePLoS ONE
upcommons.citation.volume12
upcommons.citation.number11


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