Improvement of cathepsin S detection using a designed FRET peptide based on putative natural substrates

Improvement of cathepsin S detection using a designed FRET peptide based on putative natural substrates

Autor Oliveira, Marcela Autor UNIFESP Google Scholar
Torquato, Ricardo J. S. Autor UNIFESP Google Scholar
Alves, Marcio F. M. Autor UNIFESP Google Scholar
Juliano, Maria A. Autor UNIFESP Google Scholar
Bromme, Dieter Google Scholar
Barros, Nilana M. T. Autor UNIFESP Google Scholar
Carmona, Adriana K. Autor UNIFESP Google Scholar
Instituição Universidade Federal de São Paulo (UNIFESP)
Univ British Columbia
Resumo Cathepsin S is a lysosomal cysteine peptidase of the papain superfamily which is implicated in physiological and pathological states. the enzyme is highly expressed in antigen presenting cells and is thought to play an important role in the processing of the major histocompatibility complex (MHC) class II-associated invariant chain. in pathological processes, cathepsin S is associated with Alzheimer's disease, atherosclerosis and obesity and can be regarded as a potential target in related disorders. However, due to the broad substrate specificities of the lysosomal cathepsins, the specific detection of cathepsin S is difficult when other cathepsins are present. in an attempt to distinguish cathepsin S from other cathepsins we synthesized and tested fluorescence resonance energy transfer (FRET) peptides derived from two of its putative natural substrates, namely insulin beta-chain and class II-associated invariant chain (CLIP). the influence of ionic strength on the catalytic activity and the enzyme stability in neutral pH was also analyzed. Using data gathered from our study we developed a selective substrate for cathepsin S and establish the assay conditions to differentiate the enzyme from cathepsins L, B, V and K. the peptide Abz-LEQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl]ethylenediamine]) in 50 mM sodium phosphate buffer, pH 7.4, containing 1 M NaCl was hydrolyzed by cathepsin S with k(cat)/K(m) value of 3585 mM(-1) s(-1), and was resistant to hydrolysis by cathepsins L, V, K and B. Thus, we developed a sensitive and selective cathepsins S substrate that permits continuous measurement of the enzymatic activity even in crude tissue extracts. (C) 2009 Elsevier Inc. All rights reserved.
Palavra-chave Lysosomal cathepsins
Cathepsin S
FRET peptides
Continuous assay
Selective substrate
Idioma Inglês
Financiador Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Canada Research Chair
Data de publicação 2010-04-01
Publicado em Peptides. New York: Elsevier B.V., v. 31, n. 4, p. 562-567, 2010.
ISSN 0196-9781 (Sherpa/Romeo, fator de impacto)
Publicador Elsevier B.V.
Extensão 562-567
Fonte http://dx.doi.org/10.1016/j.peptides.2009.12.027
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:000276887600006
Endereço permanente http://repositorio.unifesp.br/handle/11600/32431

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