Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Autor Korkmaz, B. Google Scholar
Attucci, S. Google Scholar
Hazouard, E. Google Scholar
Ferrandiere, M. Google Scholar
Jourdan, M. L. Google Scholar
Brillard-Bourdet, M. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Gauthier, F. Google Scholar
Instituição Univ Tours
Universidade Federal de São Paulo (UNIFESP)
Resumo Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. the hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from a-l-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.
Idioma Inglês
Data 2002-10-18
Publicado em Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 277, n. 42, p. 39074-39081, 2002.
ISSN 0021-9258 (Sherpa/Romeo, fator de impacto)
Editor Amer Soc Biochemistry Molecular Biology Inc
Extensão 39074-39081
Fonte http://dx.doi.org/10.1074/jbc.M202918200
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000178662500005
URI http://repositorio.unifesp.br/handle/11600/27004

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