S3 to S3 ' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates

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dc.contributor.authorAlves, Marcio FM
dc.contributor.authorPuzer, Luciano
dc.contributor.authorCotrin, Simone Silva [UNIFESP]
dc.contributor.authorJuliano, Maria Aparecida [UNIFESP]
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorBromme, Dieter
dc.contributor.authorCarmona, Adriana Karaoglanovic [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionCUNY Mt Sinai Sch Med
dc.date.accessioned2016-01-24T12:33:57Z
dc.date.available2016-01-24T12:33:57Z
dc.date.issued2003-08-01
dc.description.abstractWe have systematically examined the S3 to S3' subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. the results indicated that the S3-S1 subsite requirements are more restricted than those of S1'-S3'. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. the substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg-Gly and Gly-Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.en
dc.description.affiliationUNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.affiliationCUNY Mt Sinai Sch Med, Dept Human Genet, New York, NY 10029 USA
dc.description.affiliationUnifespUNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, Brazil
dc.description.sourceWeb of Science
dc.format.extent981-986
dc.identifierhttp://dx.doi.org/10.1042/BJ20030438
dc.identifier.citationBiochemical Journal. London: Portland Press, v. 373, p. 981-986, 2003.
dc.identifier.doi10.1042/BJ20030438
dc.identifier.issn0264-6021
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/27333
dc.identifier.wosWOS:000184800300037
dc.language.isoeng
dc.publisherPortland Press
dc.relation.ispartofBiochemical Journal
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectlysosomal proteinaseen
dc.subjectcysteine proteaseen
dc.subjectfluorogenic substrateen
dc.titleS3 to S3 ' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substratesen
dc.typeinfo:eu-repo/semantics/article
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