Navegando por Palavras-chave "Proteolysis"
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- ItemSomente Metadadados20S proteasome activity is modified via S-glutathionylation based on intracellular redox status of the yeast Saccharomyces cerevisiae: Implications for the degradation of oxidized proteins(Elsevier B.V., 2014-09-01) Demasi, Marilene; Hand, Adrian; Ohara, Erina; Oliveira, Cristiano L. P.; Bicev, Renata N.; Bertoncini, Clelia A. [UNIFESP]; Netto, Luis E. S.; Inst Butantan; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Protein S-glutathionylation is a post-translational modification that controls many cellular pathways. Recently, we demonstrated that the alpha 5-subunit of the 20S proteasome is S-glutathionylated in yeast cells grown to the stationary phase in rich medium containing glucose, stimulating 20S core gate opening and increasing the degradation of oxidized proteins. in the present study, we evaluated the correlation between proteasomal S-glutathionylation and the intracellular redox status. the redox status was controlled by growing yeast cells in distinct carbon sources which induced respiratory (glycerol/ethanol) or fermentative (glucose) metabolism. Cells grown under glycerol/ethanol displayed higher reductive power when compared to cells grown under glucose. When purified from cells grown in glucose, 20S proteasome alpha 5-subunit exhibited an intense anti-glutathione labeling. A higher frequency of the open catalytic chamber gate was observed in the S-glutathionylated preparations as demonstrated by transmission electron microscopy. Therefore, cells that had been grown in glucose displayed an increased ability to degrade oxidized proteins. the results of the present study suggest that 20S proteasomal S-glutathionylation is a relevant adaptive response to oxidative stress that is capable to sense the intracellular redox environment, leading to the removal of oxidized proteins via a process that is not dependent upon ubiquitylation and ATP consumption. (C) 2014 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosChronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3 beta and p70S6K levels in rats(Springer, 2009-06-01) Zanchi, Nelo Eidy; Siqueira Filho, Mario Alves de; Lira, Fabio Santos [UNIFESP]; Rosa, Jose Cesar [UNIFESP]; Yamashita, Alex Shimura; Oliveira Carvalho, Carla Roberta de; Seelaender, Marilia; Lancha, Antonio Herbert; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Long-term adaptation to resistance training is probably due to the cumulative molecular effects of each exercise session. Therefore, we studied in female Wistar rats the molecular effects of a chronic resistance training regimen (3 months) leading to skeletal muscle hypertrophy in the plantaris muscle. Our results demonstrated that muscle proteolytic genes MuRF-1 and Atrogin-1 were significantly decreased in the exercised group measured 24 h after the last resistance exercise session (41.64 and 61.19%, respectively; P < 0.05). Nonetheless, when measured at the same time point, 4EBP-1, GSK-3 beta and eIF2B epsilon mRNA levels and Akt, GSK-3 beta and p70S6K protein levels (regulators of translation initiation) were not modified. Such data suggests that if gene transcription constitutes a control point in the protein synthesis pathway this regulation probably occurs in early adaptation periods or during extreme situations leading to skeletal muscle remodeling. However, proteolytic gene expression is modified even after a prolonged resistance training regimen leading to moderate skeletal muscle hypertrophy.
- ItemSomente MetadadadosCytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp(Springer, 2015-01-01) Sant'ana, Viviane Peracini [UNIFESP]; Carrijo-Carvalho, Linda Christian [UNIFESP]; Foronda, Annette Silva [UNIFESP]; Chudzinski-Tavassi, Ana Marisa; Freitas, Denise de [UNIFESP]; Souza de Carvalho, Fabio Ramos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Butantan InstProteolytic enzymes secreted by trophozoites (amoebic secretome) are suggested as the main virulence factor involved in the severity of Acanthamoeba keratitis. the degradation profile of the main glycoprotein components of anterior and posterior portions of the cornea and the cytopathic effect of secretomes on endothelial cells by contact-independent mechanism were evaluated.Trophozoites were isolated primarily from corneal tissue samples (n = 11) and extracellular proteins were collected from axenic cell culture supernatants. the molecular weights of proteolytic enzymes were estimated by zymography. Enzymatic cleavage of laminin and fibronectin substrates by amoebic secretome was investigated and cluster analysis was applied to the proteolysis profiles. Primary cultures of endothelial cells were used in both qualitative and quantitative assays of cytophatogenicity.Differential patterns of proteolysis were observed among the Acanthamoeba secretomes that were analysed. the uniformity of laminin degradation contrasted with the diversity of the proteolysis profiles observed in the fibronectin substrate. Acanthamoeba secretome extracted from four clinical isolates was shown to be toxic when in contact with the endothelial cell monolayer (p < 0.01). Induction of apoptosis and membrane permeability, at different percentual values, were suggested as the main mechanisms that could induce endothelial cell death when in contact with amoebic secretome.Our results provide evidence that virulence factors secreted by Acanthamoeba trophozoites can be related to an increased pathogenicity pattern by an independent contact-trophozoite mechanism, through induction of endothelial cell death by apoptosis at a higher percentage than providing the lack of cell viability by the membrane-associated pore-forming toxin activity.
- ItemAcesso aberto (Open Access)Prospecção de compostos químicos terapêuticos com potencial inibitório do exoproteoma de Acanthamoeba spp(Universidade Federal de São Paulo (UNIFESP), 2016-12-21) Marujo, Fabio Iglesias [UNIFESP]; Carvalho, Fabio Ramos de Souza [UNIFESP]; http://lattes.cnpq.br/1910912718767159; http://lattes.cnpq.br/4914252375893578; Universidade Federal de São Paulo (UNIFESP)Introduction: Acanthamoeba keratitis has presented an increased incidence and ocular morbidity indexes. During pathogenesis, corneal invasion of the amoeba is obtained by the secretion of an exoproteome, which include enzymes believed to be its main virulence factors. Treatment should be started as soon as possible and last for a long period, but the specific therapeutic regimen still lacks a consensus. The use of low doses of antiamoebic medications as proteolysis inhibitors may be a new and promising form of treatment, in which the focus is stopping the pathological process, rather than just attacking the microorganism itself. Materials and Methods: A sterile 96 well assay black plate was used to dilute one antiamoebic drug, exoproteome, EnzChek® Gelatinase/Collagenase Assay Kit substrate and reaction buffer in each well. Four different concentrations of each drug (Polyhexamethylene biguanide, Chlorhexidine gluconate, Hexamidine di-isethionate and Propamidine Isethionate) were tested in exoproteome from four different strains of Acanthamoeba. A second assay consisted in evaluating toxicity in commercially available certified standard cell lines of corneal keratocytes (SIRC) exposed to different dosages of PHMB, chlorhexidine, hexamidine and propamidine in the presence and absence of exoproteom. For this, PrestoBlue® Cell Viability Reagent was used. All assays used saline solution (NaCl 0.9%) as solvent and were conducted in triplicate. Fluorescence readings were performed by SpectraMax® Paradigm® Multi-Mode Detection Platform. Results: All compounds reduced proteolysis and there was a clear correlation between the concentration of the drug and its inhibitory effect, which was more pronounced in higher concentrations of hexamidine, propamidine and chlorhexidine. However the opposite occurred with PHMB, with concentrations as low as 0.0025% having a powerful inhibitory effect compared to the null effect of 0.02%. Regarding the SIRC keratocytes assay all drugs tested caused a decrease in cell viability, which was directly proportional to its concentration, both in the presence or absence of exoproteome. Discussion and conclusion: The formation of micelles of PHMB may be responsible for the decrease in its inhibitory effect in high concentrations. Therefore, a high concentration of antiamoebic drugs does not necessarily improve the clinical outcome, not only by the increase of citotoxicity, but also by lowering the inhibitory effect it would have on proteolisis. Studies using low-concentration PHMB in Acanthamoeba keratitis should be conducted to establish the most effective dosage in those cases.