Analysis of catalytic properties of tripeptidyl peptidase I (TTP-I), a serine carboxyl lysosomal protease, and its detection in tissue extracts using selective FRET peptide substrate

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endopeptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S-4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca2+ and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K-m compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys. (C) 2016 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP-Projects)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Projects)Univ Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Tres de Maio 100, BR-0404420 Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Pharmacol, Rua Tres de Maio 100, BR-0404420 Sao Paulo, BrazilKyoto Inst Technol, Dept Appl Biol, Kyoto 606, JapanUniv Fed Sao Paulo, Escola Paulista Med, Dept Biophys, Rua Tres de Maio 100, BR-0404420 Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Pharmacol, Rua Tres de Maio 100, BR-0404420 Sao Paulo, BrazilFAPESP: 12/50191-4RFAPESP: 2013/12106-8CNPq: 471340/2011-1CNPq: 470388/2010-2Web of Scienc

Controlled peptide solvation in portion-mixing libraries of FRET peptides: Improved specificity determination for dengue 2 virus NS2B-NS3 protease and human cathepsin S

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. in principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. the influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P(1) position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P(2)-P(1) positions). in conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilCtr Gamle Carlsberg, SPOCC, Carlsberg Lab, DK-2500 Copenhagen, DenmarkUniv British Columbia, Dept Dent, Vancouver, BC V6T 1Z3, CanadaUniv British Columbia, UBC Ctr Blood Res, Vancouver, BC V6T 1Z3, CanadaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. the peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO42- > HPO42- > acetate(-) > HCO3- > Cl-. the activation appeared to be independent of substrate sequence and arose primarily from an increase in k(cat). A shift to higher pH was also observed for the pK(1) of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilHungarian Acad Sci, Biol Res Ctr, Inst Enzymol, H-1518 Budapest 112, HungaryUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Poliovirus 3C proteinase inhibition by organotelluranes

The 3C proteinase, essential for human poliovirus (PV) replication, has unique characteristics as its three-dimensional structure resembles chymotrypsin, but its catalytic nucleophile is a cysteine SH group rather than the OH group of serine. Here, we describe the use of tellurium compounds as inhibitors of PV3C proteinase. A rapid, stoichiometric and covalent inactivation of PV3C was observed with both a chloro-telluroxetane and a bis-vinylic organotellurane. These compounds also inhibit human cathepsins B, L, S, and K with second order rate constants higher than those obtained for PV3C. Chloro-telluroxetane inhibits replication of PV in human embryonic rhabdomyosarcoma cells in the low micromolar range and below the toxic level for the host cells. Bis-vinylic organotellurane is more effective as antiviral agent but reduces the cell viability by 20% at 10 mM, a concentration almost completely inhibiting virus growth. This is the first description of inhibition of viral 3C proteinase with antiviral property by this class of compounds.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilInst Oswaldo Cruz, Fundacao Oswaldo Cruz, Dept Virol, BR-21045060 Rio de Janeiro, BrazilUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08780911 Mogi das Cruzes, SP, BrazilUniv Fed ABC, Ctr Cieencias Nat & Humanas, BR-09210580 Santo Andre, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc