Biotecnologia translacional: hemopressina e outros peptídeos intracelulares

Biotechnology has been used since ancient Egypt for the production of bread and beer. In the modern world, biotechnology has been used in several ways, including for the treatment of diseases. In academia, biotechnology has allowed a rapid advance of knowledge. In this article, we make a brief summary of what is biotechnology and its relation to the process of innovation and production of biopharmaceuticals. In academia, biotechnology has contributed decisively to the discovery of new bioactive molecules, such as in hemopressin and several other intracellular peptides.A biotecnologia é uma prática antiga, sendo utilizada desde o antigo Egito para a produção de pão e cerveja. No mundo contemporâneo, a biotecnologia tem sido utilizada de diversas formas, incluindo o tratamento de doenças. No universo acadêmico, a biotecnologia tem permitido um avanço rápido do conhecimento. Neste artigo, fazemos um breve resumo sobre o que é biotecnologia, sua relação com o processo de inovação e produção de biofármacos. No universo acadêmico, a biotecnologia tem contribuído de forma decisiva para a descoberta de novas moléculas bioativas, como no caso da hemopressina e de diversos outros peptídeos intracelulares

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K-M for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K-M of 2x10(5) M-1 s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K-M = 3x10(5) M-1 s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K-M of 2.5x10(4) M-1 s(-1) for this substrate. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.Univ Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, BrazilInst Butantan, CAT, Ctr Toxicol Aplicada, BR-05467010 São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Program Biol Celular, Dept Histol & Embriol, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Secreção e regulação da ENDO-S, uma enzima metabolizadora de mensageiros peptídicos semelhante a endo-oligopeptidase A, por linguagens celulares mantidas em cultura

BV UNIFESP: Teses e dissertaçõe

Endopeptidase 22.19 na retina de vertebrados: um estudo bioquímico e imuno-histoquímico

BV UNIFESP: Teses e dissertaçõe

Peptidase Activities In Rats Treated Chronically With N(omega)-nitro-l-arginine Methyl Ester (l-name).

The chronic treatment of rats with N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) biosynthesis, results in hypertension. This inhibition of NO production results in activation of the renin-angiotensin system, with increased activity of the carboxypeptidase angiotensin I-converting enzyme (ACE). Since chronic NO inhibition increases ACE activity, we hypothesized that this inhibition could also affect the activities of other peptidases involved in cardiovascular functions. To test this possibility, we examined the activities of aminopeptidase M (APM), dipeptidyl peptidase IV (DPP IV), metalloendopeptidase 24.15 (MEP 24.15) and neutral endopeptidase 24.11 (NEP 24.11) in rat brain, heart, kidney, liver, lung and thoracic aorta. Male Wistar rats were treated chronically with L-NAME (80mgkg(-1) per day) administered in the drinking water for 4 weeks and their organs then removed and processed for the determination of peptidase activities. Treatment with L-NAME did not significantly alter the activities of the four peptidases in brain, heart, kidney, liver and lung. In contrast, in aorta, the activity of APM was slightly but significantly reduced whereas those of DPP IV and MEP 24.15 were markedly enhanced; NEP 24.11 was not detected in this tissue. Immunoblotting for DPP IV and MEP 24.15 showed increased expression in aortic tissue. Neither L-NAME (1-100microM) nor the NO donors sodium nitroprusside and 3-morpholinosydnonimine (SIN-1; 1-100microM) had any consistent effect on the activity of recombinant MEP 24.15 or renal DPP IV. The importance of MEP 24.15 in peptide metabolism was confirmed in pentobartibal-anesthetized rats pretreated with the MEP 24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2), which significantly potentiated the hypotensive response to bradykinin. The altered peptidase activities seen in aorta may contribute to modulating vascular responses in this model of hypertension.68205-1

Peptidomic Analysis of HEK293T Cells: Effect of the Proteasome Inhibitor Epoxomicin on Intracellular Peptides

Peptides derived from cytosolic, mitochondrial, and nuclear proteins have been detected in extracts of animal tissues and cell lines. To test whether the proteasome is involved in their formation, HEK293T cells were treated with epoxomicin (0.2 or 2 mu M) for 1 h and quantitative peptidomics analysis was performed. Altogether, 147 unique peptides were identified by mass spectrometry sequence analysis. Epoxomicin treatment decreased the levels of the majority of intracellular peptides, consistent with inhibition of the proteasome beta-2 and beta-5 subunits. Treatment with the higher concentration of epoxomicin elevated the levels of some peptides. Most of the elevated peptides resulted from cleavages at acidic residues, suggesting that epoxomicin increased the processing of proteins through the beta-1 subunit. Interestingly, some of the peptides that were elevated by the epoxomicin treatment had hydrophobic residues in P1 cleavage sites. Taken together, these findings suggest that, while the proteasome is the major source of intracellular peptides, other peptide-generating mechanisms exist. Because intracellular peptides are likely to perform intracellular functions, studies using proteasome inhibitors need to be interpreted with caution, as it is possible that the effects of these inhibitors are due to a change in the peptide levels rather than inhibition of protein degradation.National Institutes of HealthNational Institutes of Health [DA-004494]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) through the Rede Genoprot [559698/2009-7]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) through the Rede GenoprotUniversity of Sao PauloUniversity of Sao Paulo [2011.1.9333.1.3]CNPqCNP

Structural features that make oligopeptides susceptible substrates for hydrolysis by recombinant thimet oligopeptidase

A systematic analysis of the peptide sequences and lengths of several homologues of bioactive peptides and of a number of quenched-fluorescence (qf) opioid- and bradykinin-related peptides was performed to determine the main features leading the oligopeptides to hydrolysis by the recombinant rat testis thimet oligopeptidase (EC 3.4.24.15). The results indicate that a minimum substrate length of six amino acids is required and that among the oligopeptides six to thirteen amino acid residues long, their susceptibility as substrates is highly variable. Thimet oligopeptidase was able to hydrolyse, with similar catalytic efficiency, peptide bonds having hydrophobic or hydrophilic amino acids as well as proline in the P1 position of peptides, ranging from a minimum of six to a maximum of approximately thirteen amino acid residues. An intriguing observation was the shift of the cleavage site, at a Leu-Arg bond in qf dynorphin (2-8) [qf-Dyn(2-8); Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl) ethylenediamine], to Arg-Arg in qf-Dyn(2-8)Q, in which Gln was substituted for Val at its C-terminus. Similarly, a cleavage site displacement was also observed with the hydrolysis of the internally quenched-fluorescence bradykinin analogues containing Gin at the C-terminal position, namely Abz-RPPGFSPFR-EDDnp and Abz-GFSPFR-EDDnp are cleaved at the Phe-Ser bond, but Abz-RPPGFSPFRQ-EDDnp and Abz-GFSPFRQ-EDDnp are cleaved at the Pro-Phe bond.UNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT BIOFIS,BR-04044020 SAO PAULO,SP,BRAZILINST BUTANTAN,BIOCHEM & BIOPHYS LAB,BR-05503900 SAO PAULO,BRAZILUNIV SAO PAULO,INST BIOMED SCI,SAO PAULO,BRAZILFDN ANTONIO PRUDENTE,BR-01509010 SAO PAULO,BRAZILUNIV FED SAO PAULO,ESCOLA PAULISTA MED,DEPT BIOFIS,BR-04044020 SAO PAULO,SP,BRAZILWeb of Scienc

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.Brazilian National Research Council (CNPq) [559698/2009-7]Brazilian National Research Council (CNPq)University of Sao PauloUniversity of Sao Paulo [2011.1.9333.1.3, NAPNA]CNPqCNPqFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paul