Inhibition of cathepsin B by its propeptide: Use of overlapping peptides to identify a critical segment

AbstractTen overlapping 15-mer peptides (peptidyl amides) spanning the proregion of rat cathepsin B (residues 1p–60p) were constructed to identify minimal segments having inhibitory activity towards the mature enzyme, that could be used to develop a new generation of peptide-derived inhibitors specifically targeting the active site of the corresponding proteinase. Three synthetic peptides, containing the pentapeptide Leu-Cys-Gly-Thr-Val (residues 41p–45p) in their sequence, inhibited cathepsin B with Ki values in the micromolar range. Alkylation of the thiol group of Cys-42p of peptide PB8 (36p–50p) resulted in its rapid proteolytic degradation, suggesting that this residue is essential for inhibition. The inhibition constant was slightly improved (Ki = 2 μM) using a longer peptide (26p–50p) which was completely resistant to cleavage even after a prolonged incubation. Alkylation of its cysteinyl residue also resulted in rapid cleavage of the peptide chain. Peptides derived from the rat cathepsin B prosequence also inhibited human cathepsin B with similar Ki values. Unlike rat cathepsin B, which cleaves peptide PB8 at the G47p–G48p bond after prolonged incubation, the human enzyme cleaved both PB8 and PB11 at the Lys-40p-Leu41p bond, in agreement with the different kinetic properties of these two proteinases. New probes with improved specificity for cysteine proteinases may therefore be designed based on the sequences of their propeptides

Purificação e caracterização de um inibidor de trombina de saliva de Boophilus microplus

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Purificação e caracterização de um inibidor de trombina de saliva de Boophilus microplus

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Serum Amyloid A Production Is Triggered by Sleep Deprivation in Mice and Humans: Is That the Link between Sleep Loss and Associated Comorbidities?

Serum amyloid A (SAA) was recently associated with metabolic endotoxemia, obesity and insulin resistance. Concurrently, insufficient sleep adversely affects metabolic health and is an independent predisposing factor for obesity and insulin resistance. In this study we investigated whether sleep loss modulates SAA production. The serum SAA concentration increased in C57BL/6 mice subjected to sleep restriction (SR) for 15 days or to paradoxical sleep deprivation (PSD) for 72 h. Sleep restriction also induced the upregulation of Saa1.1/Saa2.1 mRNA levels in the liver and Saa3 mRNA levels in adipose tissue. SAA levels returned to the basal range after 24 h in paradoxical sleep rebound (PSR). Metabolic endotoxemia was also a finding in SR. Increased plasma levels of SAA were also observed in healthy human volunteers subjected to two nights of total sleep deprivation (Total SD), returning to basal levels after one night of recovery. The observed increase in SAA levels may be part of the initial biochemical alterations caused by sleep deprivation, with potential to drive deleterious conditions such as metabolic endotoxemia and weight gain

Corneal angiogenesis modulation by cysteine cathepsins: in vitro and in vivo studies

Corneal avascularization is essential for normal vision. Several antiangiogenic factors were identified in cornea such as endostatin and angiostatin. Cathepsin V, which is highly expressed in the cornea, can hydrolyze human plasminogen to release angiostatin fragments. Herein, we describe a detailed investigation of the expression profile of cathepsins B, L, S and V in the human cornea and the role of cysteine peptidases in modulating angiogenesis both in vitro and in vivo. We used various methodological tools for this purpose, including real-time PCR, SDS-PAGE, western blotting, catalytic activity assays, cellular assays and induction of corneal neovascularity in rabbit eyes. Human corneal enzymatic activity assays revealed the presence of cysteine proteases that were capable of processing endogenous corneal plasminogen to produce angiostatin-like fragments. Comparative real-time analysis of cathepsin B, L, S and V expression revealed that cathepsin V was the most highly expressed, followed by cathepsins L, B and S. However, cathepsin V depletion revealed that this enzyme is not the major cysteine protease responsible for plasminogen degradation under non-pathological conditions. Furthermore, western blotting analysis indicated that only cathepsins B and S were present in their enzymatically active forms. in vivo analysis of angiogenesis demonstrated that treatment with the cysteine peptidase inhibitor E64 caused a reduction in neovascularization. Taken together, our results show that human corneal cysteine proteases are critically involved in angiogenesis. (C) 2015 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)Universidade Federal de São Paulo, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Psicobiol, São Paulo, SP, BrazilUniv Fed Maranhao, Dept Med 1, Sao Luis, MA, BrazilUniversidade Federal de São Paulo, Inst Visao IPEPO, Dept Oftalmol & Ciencias Visuals, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Saude, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Clencias Exatas & Terra, Diadema, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Psicobiol, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Inst Visao IPEPO, Dept Oftalmol & Ciencias Visuals, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Saude, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Clencias Exatas & Terra, Diadema, SP, BrazilCNPq: 482400/2013-7Web of Scienc

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

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, 558924/2008-5, 481104/2004-6, 306587/2010-6]Associacao de Fundo e Incentivo a Pesquisa (AFIP)Associacao de Fundo e Incentivo a Pesquisa (AFIP