Navegando por Palavras-chave "Thimet oligopeptidase"

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    Acute cocaine treatment increases thimet oligopeptidase in the striatum of rat brain
    (Elsevier B.V., 2012-03-23) Dalio, Fernanda Montiel [UNIFESP]; Visniauskas, Bruna [UNIFESP]; Bicocchi, Eliane S.; Perry, Juliana Cini [UNIFESP]; Freua, Rodrigo [UNIFESP]; Gesteira, Tarsis Ferreira [UNIFESP]; Nader, Helena Bonciani [UNIFESP]; Machado, Mauricio Ferreira Marcondes [UNIFESP]; Tufik, Sergio [UNIFESP]; Ferro, Emer Suavinho [UNIFESP]; Andersen, Monica Levy [UNIFESP]; Toledo, Claudio A. B.; Chagas, Jair Ribeiro [UNIFESP]; Oliveira, Vitor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Cidade São Paulo UNICID; Universidade de São Paulo (USP)
    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.
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    Avaliação das alterações das atividades de metalopeptidases no sistema nervoso central em modelos de privação de sono
    (Universidade Federal de São Paulo (UNIFESP), 2010-04-28) Visniauskas, Bruna [UNIFESP]; Chagas, Jair Ribeiro [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Neuropeptides have a fundamental role on sleep-wake cycle control and their actions are regulated by proteolytic processing. Proteolytic enzymes are essential for the metabolism of neuropeptides, either by release from active or inactive precursor protein, or the inactivation of active neuropeptides. Using fluorescence resonance energy transfer substrates, Western blot, specific inhibitors and real time PCR, we have demonstrated changes in the expression and activity of angiotensin I-converting enzyme (ACE - EC 3.4.15.1) and thimet oligopeptidase (EP24.15 – EC 3.4.24.15) in the central nervous system of rats submitted to paradoxical sleep deprivation. Male rats were distributed in 5 groups: control, paradoxical sleep deprivation during 96h (PSD96), and sleep recovery (recovery after sleep deprivation 24, 48 and 96h after PSD96). Alterations in the activities, expression and mRNA levels of ACE and EP24.15 were found in extracts of the hypothalamus, hippocampus, brainstem, cortex and striatum. Significant changes in these parameters (activity, mRNA levels, protein expression) were observed in groups PSD96 and recovery. Modifications on ACE and EP24.15 activities can result in alterations in the metabolism of angiotensins, bradykinin, opioid peptides (dynorphins and enkephalins), substance P and GnRH and be related to some of the physiological changes (stress, memory and cognition, nociception and endocrine changes) observed during and after sleep deprivation.
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    Catalytic properties of thimet oligopeptidase H600A mutant
    (Elsevier B.V., 2010-04-02) Machado, Mauricio F. M. [UNIFESP]; Marcondes, Marcelo F. [UNIFESP]; Rioli, Vanessa; Ferro, Emer S.; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Oliveira, Vitor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Inst Butantan; Universidade de São Paulo (USP)
    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.
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    Expression and activity of thimet oligopeptidase (TOP) are modified in the hippocampus of subjects with temporal lobe epilepsy (TLE)
    (Wiley-Blackwell, 2014-05-01) Rodrigues Simoes, Priscila Santos [UNIFESP]; Visniauskas, Bruna [UNIFESP]; Perosa, Sandra Regina [UNIFESP]; Yacubian, Elza Márcia Targas [UNIFESP]; Centeno, Ricardo [UNIFESP]; Canzian, Mauro; Lopes-Cendes, Iscia; Maurer Morelli, Claudia Vianna; Carrete, Henrique [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Tufik, Sergio [UNIFESP]; Chagas, Jair Ribeiro [UNIFESP]; Naffah-Mazzacoratti, Maria da Graca [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Universidade Estadual de Campinas (UNICAMP)
    ObjectiveThimet oligopeptidase (TOP) is a metalloprotease that has been associated with peptide processing in several nervous system structures, and its substrates include several peptides such as bradykinin, amyloid beta (A), and major histocompatibility complex (MHC) class I molecules. As shown previously by our research group, patients with temporal lobe epilepsy (TLE) have a high level of kinin receptors as well as kallikrein, a kinin-releasing enzyme, in the hippocampus.MethodsIn this study, we evaluated the expression, distribution, and activity of TOP in the hippocampus of patients with TLE and autopsy-control tissues, through reverse-transcription polymerase chain reaction (RT-PCR), enzymatic activity, Western blot, and immunohistochemistry. in addition, hippocampi of rats were analyzed using the pilocarpine-induced epilepsy model. Animals were grouped according to the epilepsy phases defined in the model as acute, silent, and chronic.ResultsIncreased TOP mRNA expression, decreased protein levels and enzymatic activity were observed in tissues of patients, compared to control samples. in addition, decreased TOP distribution was also visualized by immunohistochemistry. Similar results were observed in tissues of rats during the acute phase of epilepsy model. However, increased TOP mRNA expression and no changes in immunoreactivity were found in the silent phase, whereas increased TOP mRNA expression and increased enzymatic activity were observed in the chronic phase.SignificanceThe results show that these alterations could be related to a failure in the mechanisms involved in clearance of inflammatory peptides in the hippocampus, suggesting an accumulation of potentially harmful substances in nervous tissue such as A, bradykinin, and antigenic peptides. These accumulations could be related to hippocampal inflammation observed in TLE subjects.
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    Identificação de atividade metalo-oligopeptidásica Thimet-like em Paracoccidioides brasiliensis: um novo fator de patogenicidade fúngica?
    (Universidade Federal de São Paulo (UNIFESP), 2010-10-28) Gravi, Ellen Tihe [UNIFESP]; Rodrigues, Elaine Guadelupe [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Paracoccidioidomycosis (PCM), caused by the pathogenic fungus Paracoccidioides brasiliensis (Pb) is a systemic mycosis with severe acute and chronic forms. Proteases or peptidases are proteolytic enzymes that occur in all organisms and constitute 1-5% of their genetic contents. These enzymes are involved in biological processes such as blood clotting, cell death and tissue differentiation. Several important proteolytic steps occur during the invasion of metastatic tumors, as well as in the infection of a large number of viruses and pathogens. To date, a small number of Pb proteases were isolated and characterized, also, their activities during the development of the disease was not determined, and an oligopeptidase activity was not detected in this fungus. In the present work, we demonstrated a metallopeptidase thimet oligopeptidase (TOP)-like activity in the cytosolic extract of Pb18 yeasts. Our results shown a major hydrolysis of the fluorescence resonance energy transfer (FRET) peptide Abz-GFSPFRQ-EDDnp, preferentially cleaved by TOP from mammals, and the inhibition of the hydrolysis of this peptide by orthophenantrolin and JA-2, selective inhibitors of metalloproteases and TOP, respectively. The presence of neurolysin- like and neprilysin-like, serinepeptidases, cysteine-peptidases and angiotensin converting enzyme I was discarded by analyzing selective FRET peptides and inhibitors. The higher peptidase activity of cytosolic extracts over the membrane/cell wall and total yeast lysate preparations may indicate that this enzyme is localized in the yeast cytosol. The metallo-oligopeptidase activity was not detected on in vitro culture supernatants, even after addition of fetal calf serum. However, the peptidase with TOP-like activity of P. brasiliensis seems to be secreted in vivo, or released after fungal lysis by immune factors, since antibodies that can inhibit this enzymatic activity were found in sera from paracoccidioidomycosis patients, and serum with highest titer in immunodiffusion contains higher concentrations of enzymespecific antibodies. Bradykinin, an important inflammatory mediator in vivo, is cleaved by several enzymes from the M3 family. The same fragments observed after hydrolysis by TOP were observed after cytosolic extract hydrolysis of bradykinin and the substrate Abz-GFSPFRQ-EDDnp. MIP and bacterial OpdA hydrolysis of these peptides generate different fragments, and this is an additional indicator of a major TOP-like activity in P. brasiliensis yeast cells Bradykinin hydrolysis by the TOP-like metallopeptidase of P. brasiliensis may occur in inflammatory processes and this suggests that the enzyme may be involved in the inhibition of a protective anti-fungal response induction, limiting fungal elimination. We also observed that the expression of the TOP homologous gene in P. brasiliensis has almost a two-fold increased in the virulent isolate 18 compared to the non-virulent isolate. Increased hydrolysis of the substrate Abz-GFSPFRQ-EDDnp was also observed in the most virulent isolate compared to the non-virulent. The possible correlation between TOP-like peptidase expression and fungal virulence suggests that this peptidase could be classified as a fungal virulence factor, however, additional experiments are needed to confirm this hypothesis. Gp43 expression was also analyzed in both isolates, and it was observed a thirteen-fold increase in the expression on the virulent isolate. In order to better characterize the P. brasiliensis TOP-like activity, we attempted to obtain the purified recombinant or the native protein, isolated from fungal lysate. However, we were not successful in the expression of recombinant proteins and neither on the isolation of the native protein using chromatographic methods. Our results suggest the presence of a TOP-like activity in the cytosolic fraction of P. brasiliensis yeasts. In vivo release of this enzyme after fungal lysis, or host factors-stimulated secretion, may have a role in inflammation and development of the disease.