Studies on the peptidase activity of transthyretin (TTR)

Transthyretin (TTR) is a plasma protein transporter of thyroxine (T-4) and retinol and also has peptidase activity. in order to characterize TTR peptidase activity we used fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLRSSK-Q-EDDnp and from two portion-mixing libraries as substrates. Most of the susceptible FRET peptides were cleaved at more than one peptide bond, without particular substrate specificity. the more relevant observation was that the peptides containing E or D were cleaved at only one peptide bond and TTR was competitively inhibited by glutathione analog peptide gamma-E-A-G-OH that contains two free carboxyl groups. the dependence on ionic interactions of TTR hydrolytic activity was confirmed by the large inhibitory effects of salt and ionic surfactants. TTR was not inhibited by any usual peptidase inhibitors, except by ortho-phenanthroline and EDTA. the mechanism of TTR catalysis was explored by the pH-profile of TTR hydrolytic activity in different temperatures and by proton inventory. the obtained pK and heat of ionization values suggest that a carboxylate and an ammonium group, possibly from a lysine side chain are involved. These results support the recently proposed inducible metalloprotease mechanism for TTR based on its 3D structure in presence of Zn2+ and a series of point mutations [Liz et al., Biochem. J 443 (2012) 769-778]. (c) 2012 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundacao para a Ciencia e Tecnologia under the program FEDER in PortugalFundacao para a Ciencia e Tecnologia under the program COMPETE in PortugalUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilInst Biol Mol & Celular, P-4150180 Oporto, PortugalUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilFundacao para a Ciencia e Tecnologia under the program FEDER in Portugal: PTDC/SAU-GMG/111761/2009Fundacao para a Ciencia e Tecnologia under the program FEDER in Portugal: PTDC/SAU-ORG/118863/2010Fundacao para a Ciencia e Tecnologia under the program COMPETE in Portugal: PTDC/SAU-GMG/111761/2009Fundacao para a Ciencia e Tecnologia under the program COMPETE in Portugal: PTDC/SAU-ORG/118863/2010Web of Scienc