title: Studies on the peptidase activity of transthyretin (TTR).
abstract: 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 γ-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 Zn(2+) and a series of point mutations [Liz et al., Biochem. J 443 (2012) 769-778].
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-020 São Paulo, Brazil.
authors.forename: Iuri Estrada
authors.initials: IE
authors.lastname: Gouvea
authors.forename: Marcia Yuri
authors.initials: MY
authors.lastname: Kondo
authors.forename: Diego M
authors.initials: DM
authors.lastname: Assis
authors.forename: Fabiana Madureira
authors.initials: FM
authors.lastname: Alves
authors.forename: Márcia Almeida
authors.initials: MA
authors.lastname: Liz
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques; D004492:Edetic Acid / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010618:Phenanthrolines / Q000737:chemistry; D011228:Prealbumin / Q000737:chemistry*; D011480:Protease Inhibitors / Q000737:chemistry; D011522:Protons; D060327:Solid-Phase Synthesis Techniques; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
chemical_list: D019151:Peptide Library; D010618:Phenanthrolines; D011228:Prealbumin; D011480:Protease Inhibitors; D011522:Protons; D013501:Surface-Active Agents; C001635:ferroin; D004492:Edetic Acid; D008239:Lysine