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‣ Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

BERTI, Denise A.; MORANO, Cain; RUSSO, Lilian C.; CASTRO, Leandro M.; CUNHA, Fernanda M.; ZHANG, Xin; SIRONI, Juan; KLITZKE, Clecio F.; FERRO, Emer S.; FRICKER, Lloyd D.
Fonte: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC Publicador: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Tipo: Artigo de Revista Científica
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Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However...

‣ Catalytic properties of thimet oligopeptidase H600A mutant

MACHADO, Mauricio F. M.; MARCONDES, Marcelo F.; RIOLI, Vanessa; FERRO, Emer S.; JULIANO, Maria A.; JULIANO, Luiz; OLIVEIRA, Vitor
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
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Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His(600). In the present work, the role of His(600) of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S(1) and S(1)`, specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His(600) residue makes important interactions with the substrate, supporting the prediction that His(600) moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K(m) and k(cat), showing the importance of His(600) for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His(600) in TOP catalysis, transferring a proton to the newly generated NH(2)-terminus or helping Tyr(605) and/or Tyr(612) in the intermediate oxyanion stabilization. (C) 2010 Elsevier Inc. All rights reserved.; Sao Paulo State Research Foundation (FAPESP) MFMM[08/57336-2]; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); CNPq

‣ Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

RUSSO, Lilian C.; GONI, Camila N.; CASTRO, Leandro M.; ASEGA, Amanda F.; CAMARGO, Antonio C. M.; TRUJILLO, Cleber A.; ULRICH, Henning; GLUCKSMAN, Marc J.; SCAVONE, Cristoforo; FERRO, Emer S.
Fonte: WILEY-BLACKWELL PUBLISHING, INC Publicador: WILEY-BLACKWELL PUBLISHING, INC
Tipo: Artigo de Revista Científica
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Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together...

‣ A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15

RIOLI, Vanessa; PREZOTO, Benedito C.; KONNO, Katsuhiro; MELO, Robson L.; KLITZKE, Clecio F.; FERRO, Emer S.; FERREIRA-LOPES, Monica; CAMARGO, Antonio C. M.; PORTARO, Fernanda C. V.
Fonte: BLACKWELL PUBLISHING Publicador: BLACKWELL PUBLISHING
Tipo: Artigo de Revista Científica
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Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP), < ENWPHPQIPP (Xc), < EGGWPRPGPEIPP (XIIIa) and < EARPPHPPIPP (XIe) (where < E is a pyroglutamyl residue). In addition, we identified a novel BPP peptide containing additional AP amino acids in the C-terminus (< EARPPHPPIPPAP); this novel peptide was named BPP-AP. Next, dermal and muscle microcirculations were visualized using intravital microscopy to establish the roles of peptides BPP-XIe and BPP-AP in this process. After local administration of peptide BPP-XIe (0.5 mu g.mu L-1), leukocyte rolling flux and adhesion were increased by fivefold in post-capillary venules, without any increments in vasodilatation of arterioles compared to control experiments. In contrast, local administration of BPP-AP (0.5 mu g.mu L-1) potently induced vasodilatation of arterioles (nearly 100% increase compared with the vehicle saline control)...

‣ Oligomerization of the cysteinyl-rich oligopeptidase EP24.15 is triggered by S-glutathionylation

DEMASI, Marilene; PIASSA FILHO, Gilberto M.; CASTRO, Leandro M.; FERREIRA, Juliana C.; RIOLI, Vanessa; FERRO, Erner S.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
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Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is a thiol-rich metallopeptidase ubiquitously distributed in mammalian tissues and involved in oligopeptide metabolism both within and outside cells. Fifteen Cys residues are present in the rat EP24.15 protein, seven are solvent accessible, and two are found inside the catalytic site cleft; no intraprotein disulfide is described. In the present investigation, we show that mammalian immunoprecipitated EP24.15 is S-glutathionylated. In vitro EP24.15 S-glutathionylation was demonstrated by the incubation of bacterial recombinant EP24.15 with oxidized glutathione concentration as low as 10 mu M. The in vitro S-glutathionylation of EP24.15 was responsible for its oxidative oligomerization to dimer and trimer complexes. EP24.15 immunoprecipitated from cells submitted to oxidative challenge showed increased trimeric forms and decreased S-glutathionylation compared to immunoprecipitated protein from control cells. Our present data also show that EP24.15 maximal enzymatic activity is maintained by partial S-glutathionylation, a mechanism that apparently regulates the protein oligomerization. Present results raise the possibility of an unconventional property of protein S-glutathionylation, inducing oligomerization by interprotein thiol-disulfide exchange. (c) 2007 Elsevier Inc. All rights reserved.

‣ Intracellular peptides as natural regulators of cell signaling

CUNHA, Fernanda M.; BERTI, Denise A.; FERREIRA, Zulma S.; KLITZKE, Clecio F.; MARKUS, Regina P.; FERRO, Emer S.
Fonte: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC Publicador: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Tipo: Artigo de Revista Científica
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Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns...

‣ Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Russo, Lilian C.; Castro, Leandro M.; Gozzo, Fabio C.; Ferro, Emer S.
Fonte: ELSEVIER SCIENCE BV; AMSTERDAM Publicador: ELSEVIER SCIENCE BV; AMSTERDAM
Tipo: Artigo de Revista Científica
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Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 mu M). The protein kinase A inhibitor KT5720 (1 mu M) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.; Brazilian National Research Council (CNPq) [559698/2009-7]; Brazilian National Research Council (CNPq); University of Sao Paulo; University of Sao Paulo [2011.1.9333.1.3, NAPNA]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

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

Vieira, Lilian Cristina Russo; Asega, Amanda F.; Castro, Leandro Mantovani de; Negraes, Priscilla Davidson; Cruz, Lilian; Gozzo, Fabio C.; Ulrich, Henning; Camargo, Antonio C. M.; Rioli, Vanessa; Ferro, Emer Suavinho
Fonte: WILEY-BLACKWELL; HOBOKEN Publicador: WILEY-BLACKWELL; HOBOKEN
Tipo: Artigo de Revista Científica
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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 Paulo; University of Sao Paulo [2011.1.9333.1.3...

‣ The Cysteine-Rich Protein Thimet Oligopeptidase as a Model of the Structural Requirements for S-glutathiolation and Oxidative Oligomerization

Malvezzi, Alberto; Higa, Patricia M.; Amaral, Antonia Tavares do; Silva, Gustavo M.; Gozzo, Fabio C.; Ferro, Emer Suavinho; Castro, Leandro Mantovani de; Rezende, Leandro de; Monteiro, Gisele; Demasi, Marilene
Fonte: PUBLIC LIBRARY SCIENCE; SAN FRANCISCO Publicador: PUBLIC LIBRARY SCIENCE; SAN FRANCISCO
Tipo: Artigo de Revista Científica
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Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 mu M) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [08/06731-9...

‣ Acute cocaine treatment increases thimet oligopeptidase in the striatum of rat brain

Dalio, Fernanda M.; Visniauskas, Bruna; Bicocchi, Eliane S.; Perry, Juliana C.; Freua, Rodrigo; Gesteira, Tarsis F.; Nader, Helena B.; Machado, Mauricio F. M.; Tufik, Sergio; Ferro, Emer Suavinho; Andersen, Monica L.; Toledo, Claudio A. B.; Chagas, Jair R
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE; SAN DIEGO Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE; SAN DIEGO
Tipo: Artigo de Revista Científica
Português
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Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, alpha-neoendorphin, beta-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects. (C) 2012 Elsevier Inc. All rights reserved.; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (CEPID) [98/14303-3]; Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) (CEPID); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [553645/2009-9...

‣ Busca de novos substratos e/ou inibidores das enzimas timet oligopeptidase (E.C.3.4.24.15) e neurolisina (E.C.3.4.24.16) nas frações de baixa massa molecular do veneno do escorpião Tityus serrulatus.; Search for new substrates and/or inhibitors of thimet oligopeptidase (EC3.4.24.15) and neurolysin (EC3.4.24.16) enzymes in low molecular weight fractions of Tityus serrulatus scorpion venom.

Duzzi, Bruno
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 27/05/2014 Português
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O escorpião Tityus serrulatus é o responsável pelos acidentes mais graves no Brasil. Dentre os componentes já estudados de sua peçonha estão neurotoxinas capazes de interagir com canais iônicos, além de peptídeos biologicamente ativos também estarem presentes. Neste estudo foram isolados peptídeos da fração de baixa massa molecular da peçonha que interagiram com as oligopeptidases timet oligopeptidase (EP24.15) e neurolisina (EP24.16) através do emprego de substratos fluorescentes específicos como ferramentas. Usando espectrometria de massas, as sequências KEILG, FTR, YLPT e do análogo KELLG foram determinadas e posteriormente sintetizadas. In vitro, os peptídeos não foram substratos para enzimas já citadas, além de testes com a neprelisina e ECA. Em relação à inibição, os destaques ficam por conta de KELLG e KEILG, capazes de inibirem a EP 24.15 e de não inibirem a EP 24.16. Outro destaque foi o YLPT, apresentando um Ki de 0,94 mM perante a neprilisina. In vivo os peptídeos foram testados em relação à nocicepção, rolamento de leucócitos e reatividade vascular, onde se destacou o FTR, apresentando efeito antinociceptivo e o KEILG, capaz de aumentar o número de leucócitos, ressaltando a importância de pequenas moléculas na composição da peçonha.; The scorpion Tityus serrulatus is responsible for the most serious accidents in Brazil. Among the components already studied in its venom...

‣ Differential subcellular distribution of neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.3.24.15) in the rat brain

Massarelli, E. E.; Casatti, C. A.; Kato, A.; Camargo, ACM; Bauer, J. A.; Glucksman, M. J.; Roberts, J. L.; Hirose, S.; Ferro, E. S.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 261-265
Português
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Immunohistochemistry was used to analyze the rat brain distribution of thimet oligopeptidase and neurolysin. Both enzymes appear ubiquitously distributed within the entire rat brain. However, neuronal perikarya and processes stained for neurolysin, while intense nuclear labeling was only observed for thimet oligopeptidase. These data suggest that neurolysin and thimet oligopeptidase, endopeptidases sharing several functional and structural similarities, are present in distinctive intracellular compartments in neuronal cells. (C) 1999 Elsevier B.V. B.V. All rights reserved.

‣ Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Russo, Lilian C.; Castro, Leandro M.; Gozzo, Fabio C.; Ferro, Emer S.
Fonte: Elsevier; Amsterdam Publicador: Elsevier; Amsterdam
Tipo: Artigo de Revista Científica
Português
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Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 mu M). The protein kinase A inhibitor KT5720 (1 mu M) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

‣ Thimet oligopeptidase specificity: evidence of preferential cleavage near the C-terminus and product inhibition from kinetic analysis of peptide hydrolysis.

Knight, C G; Dando, P M; Barrett, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/05/1995 Português
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The substrate-size specificity of human thimet oligopeptidase (EC 3.4.24.15) was investigated with oligomers of glycyl-prolyl-leucine (GPL)n where n = 2, 3, 4 and 5. These peptides were cleaved only at Leu-Gly bonds to give GPL as the single final product. Hydrolysis was most rapid with (GPL)3 and slowest with (GPL)5. The more water-soluble oligomers of Gly-Hyp-Leu showed the same trend. (Gly-Hyp-Leu)6 was not hydrolysed, consistent with the previous finding that substrates larger than 17 amino acids are not cleaved by thimet oligopeptidase. The cleavage of (GPL)3 to GPL fitted a sequential first-order model. First-order kinetics were unexpected as the initial substrate concentration was greater than Km. The anomaly was also seen during the cleavage of bradykinin and neurotensin, and in these cases first-order behaviour was due to potent competitive inhibition by the C-terminal product. The sequential mechanism for (GPL)3 breakdown by thimet oligopeptidase does not discriminate between initial cleavages towards the N- or C-terminus. As isoleucine is an unfavourable residue in P1, substrates were made in which selected leucine residues were replaced by isoleucine. GPL--GPI--GPL (where--represents the bond between the tripeptide units) was resistant to hydrolysis and GPI--GPL--GPL was cleaved only at the -Leu-Gly- bond. Experiments with isoleucine-containing analogues of (Gly-Hyp-Leu)4 showed that thimet oligopeptidase preferred to cleave these peptides near the C-terminus.

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

Camargo, A C; Gomes, M D; Reichl, A P; Ferro, E S; Jacchieri, S; Hirata, I Y; Juliano, L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/06/1997 Português
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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-Dyn2-8; Abz-GGFLRRV-EDDnp, where Abz stands for o-aminobenzoyl and EDDnp for N-(2,4-dinitrophenyl)ethylenediamine], to Arg-Arg in qf-Dyn2-8Q, 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 Gln at the C-terminal position...

‣ Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition

Ray, Kallol; Hines, Christina S.; Rodgers, David W.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /09/2002 Português
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The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall...

‣ Thimet oligopeptidase expression is differentially regulated in neuroendocrine and spermatid cell lines by transcription factor binding to SRY (sex-determining region Y), CAAT and CREB (cAMP-response-element-binding protein) promoter consensus sequences.

Morrison, Lesley S; Pierotti, Adrian R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/11/2003 Português
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The zinc metalloprotease thimet oligopeptidase (EP24.15) is found predominantly in the neuroendocrine-gonadal axis where it is implicated in the processing of bioactive peptides, including GnRH (gonadotropin-releasing hormone), beta-neoendorphin, alpha-neoendorphin and dynorphin(1-8), the progression of spermatogenesis and the normal clearance of beta-amyloid in brain cells. Regulation of the enzyme's activity may occur in part by phosphorylation and redox disruption of intermolecular disulphide bridges. The elevated levels of both EP24.15 activity and mRNA within testicular and neuroendocrine tissues indicate that EP24.15 gene expression is differentially regulated. In the present paper, we present a detailed analysis of the rat EP24.15 promoter region previously isolated and partially characterized in this laboratory. Employing site-directed mutagenesis to create a series of promoter deletions and full-length promoter mutants, and measuring their activity in luciferase reporter gene and electrophoretic mobility-shift assays, we have shown that the transcription of the EP24.15 gene is differentially regulated in neuroendocrine and spermatid cell lines by transcription factor binding to SRY (sex-determining region Y), CAAT and CREB (cAMP-response-element-binding protein) promoter consensus sequences. The key to identifying the in vivo role of thimet oligopeptidase is likely to be found within the mechanisms by which it is regulated...

‣ Rat thimet oligopeptidase: large-scale expression in Escherichia coli and characterization of the recombinant enzyme.

McKie, N; Dando, P M; Brown, M A; Barrett, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/1995 Português
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The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system. Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams. The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP. The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate 'Pz-peptidase' or 'endo-oligopeptidase A' enzymes.

‣ Human thimet oligopeptidase.

Dando, P M; Brown, M A; Barrett, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/09/1993 Português
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We have purified human thimet oligopeptidase to homogeneity from erythrocytes, and compared it with the enzyme from rat testis and chicken liver. An antiserum raised against rat thimet oligopeptidase also recognized the human and chicken enzymes, suggesting that the structure of the enzyme has been strongly conserved in evolution. Consistent with this, the properties of the human enzyme were very similar to those for the other species. Thus human thimet oligopeptidase also is a thiol-dependent metallo-oligopeptidase with M(r) about 75,000. Specificity for cleavage of a number of peptides was indistinguishable from that of the rat enzyme, but Ki values for the four potent reversible inhibitors tested were lower. In discussing the results, we consider the determinants of the complex substrate specificity of thimet oligopeptidase. We question whether substrates containing more than 17 amino acid residues are cleaved, as has been suggested. We also point out that the favourable location of a proline residue and a free C-terminus in the substrate may be as important as the hydrophobic residues in the P2, P1 and P3' positions that have been emphasized in the past.

‣ Thimet oligopeptidase: similarity to 'soluble angiotensin II-binding protein' and some corrections to the published amino acid sequence of the rat testis enzyme.

McKie, N; Dando, P M; Rawlings, N D; Barrett, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1993 Português
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The deduced amino acid sequence of pig liver soluble angiotensin II-binding protein [Sugiura, Hagiwara and Hirose (1992) J. Biol. Chem. 267, 18067-18072] is similar over most of its length to that reported for rat testis thimet oligopeptidase (EC 3.4.24.15) by Pierotti, Dong, Glucksman, Orlowski and Roberts [(1990) (Biochemistry 29, 10323-10329]. We have found that homogeneous rat testis thimet oligopeptidase binds angiotensin II with the same distinctive characteristics as the pig liver protein. Analysis of the nucleotide sequences reported for the two proteins pointed to the likelihood that sequencing errors had caused two segments of the amino acid sequence of the rat protein to be translated out of frame, and re-sequencing of selected parts of the clone (kindly provided by the previous authors) confirmed this. The revised deduced amino acid sequence of rat thimet oligopeptidase contains 687 residues, representing a protein of 78,308 Da, and is more closely related to those of the pig liver protein and other known homologues of thimet oligopeptidase than that described previously.