Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate

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dc.citation.volume76
dc.contributor.authorKondo, Marcia Y. [UNIFESP]
dc.contributor.authorGouvea, Iuri E. [UNIFESP]
dc.contributor.authorOkamoto, Debora N. [UNIFESP]
dc.contributor.authorSantos, Jorge A. N. [UNIFESP]
dc.contributor.authorSouccar, Caden [UNIFESP]
dc.contributor.authorOda, Kohei
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorJuliano, Maria A. [UNIFESP]
dc.coverageNew York
dc.date.accessioned2020-11-03T14:40:35Z
dc.date.available2020-11-03T14:40:35Z
dc.date.issued2016
dc.description.abstractTripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endopeptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S-4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca2+ and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K-m compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys. (C) 2016 Elsevier Inc. All rights reserved.en
dc.description.affiliationUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Tres de Maio 100, BR-0404420 Sao Paulo, Brazil
dc.description.affiliationUniv Fed Sao Paulo, Escola Paulista Med, Dept Pharmacol, Rua Tres de Maio 100, BR-0404420 Sao Paulo, Brazil
dc.description.affiliationKyoto Inst Technol, Dept Appl Biol, Kyoto 606, Japan
dc.description.affiliationUnifespUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Tres de Maio 100, BR-0404420 Sao Paulo, Brazil
dc.description.affiliationUnifespUniv Fed Sao Paulo, Escola Paulista Med, Dept Pharmacol, Rua Tres de Maio 100, BR-0404420 Sao Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-Projects)
dc.description.sponsorshipConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Projects)
dc.description.sponsorshipIDFAPESP: 12/50191-4R
dc.description.sponsorshipIDFAPESP: 2013/12106-8
dc.description.sponsorshipIDCNPq: 471340/2011-1
dc.description.sponsorshipIDCNPq: 470388/2010-2
dc.format.extent80-86
dc.identifierhttps://www.sciencedirect.com/science/article/pii/S0196978116300092
dc.identifier.citationPeptides. New York, v. 76, p. 80-86, 2016.
dc.identifier.doi10.1016/j.peptides.2016.01.000
dc.identifier.issn0196-9781
dc.identifier.urihttps://repositorio.unifesp.br/handle/11600/58632
dc.identifier.wosWOS:000369597900010
dc.language.isoeng
dc.publisherElsevier Science Inc
dc.relation.ispartofPeptides
dc.rightsAcesso restrito
dc.subjectTPP-Ien
dc.subjectTripeptidyl amino peptidaseen
dc.subjectEndopeptidaseen
dc.subjectFRET peptidesen
dc.titleAnalysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrateen
dc.typeArtigo
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