Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). the effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS-PAGE. Optimum pH and temperature were 9.0 and 65 degrees C, respectively. the enzyme was stable after incubation for 30 min in a wide pH range (6.0-11.5) and at 55 degrees C. the kinetic parameters K-m, k(cat) and k(cat)/K-m were 0.47 +/- 0.042 mM, 1.33 s(-1) and 2.82 s(-1) mM(-1), respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe2+, Hg2+, Zn2+, Al3+, Pb2+, and was highly inhibited by trypsin inhibitors. the trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. the features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries). (C) 2012 Elsevier B.V. All rights reserved.Financiadora de Estudos e Projetos (FINEP/RECAR-CINE)Ministerio da Aquicultura e Pesca (MAP)Empresa brasileira de pesquisa agropecuaria (Embrapa)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundacao de Apoio a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE)Petroleo do Brasil S/A (PETROBRAS)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Pernambuco, Lab Enzimol LABENZ, Dept Bioquim, BR-50670420 Recife, PE, BrazilUniv Fed Pernambuco, LIKA, BR-50670420 Recife, PE, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Dopamine induces the accumulation of insoluble prion protein and affects autophagic flux

Accumulation of protein aggregates is a histopathological hallmark of several neurodegenerative diseases, but in most cases the aggregation occurs without defined mutations or clinical histories, suggesting that certain endogenous metabolites can promote aggregation of specific proteins. One example that supports this hypothesis is dopamine and its metabolites. Dopamine metabolism generates several oxidative metabolites that induce aggregation of alpha-synuclein, and represents the main etiology of Parkinson's diseases. Because dopamine and its metabolites are unstable and can be highly reactive, we investigated whether these molecules can also affect other proteins that are prone to aggregate, such as cellular prion protein (PrPC). in this study, we showed that dopamine treatment of neuronal cells reduced the number of viable cells and increased the production of reactive oxygen species (ROS) as demonstrated in previous studies. Overall PrPC expression level was not altered by dopamine treatment, but its unglycosylated form was consistently reduced at 100 mu M of dopamine. At the same concentration, the level of phosphorylated mTOR and 4EBP1 was also reduced. Moreover, dopamine treatment decreased the solubility of PrPC, and increased its accumulation in autophagosomal compartments with concomitant induction of LC3-II and p62/SQSTM1 levels. in vitro oxidation of dopamine promoted formation of high-order oligomers of recombinant prion protein. These results suggest that dopamine metabolites alter the conformation of PrPC, which in turn is sorted to degradation pathway, causing autophagosome overload and attenuation of protein synthesis. Accumulation of PrPC aggregates is an important feature of prion diseases. Thus, this study brings new insight into the dopamine metabolism as a source of endogenous metabolites capable of altering PrPC solubility and its subcellular localization.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)EMU (programa de equipamentos multiusuarios)Universidade Federal de São Paulo, Dept Biochem Mol & Cell Biol, BR-04039032 São Paulo, BrazilUniv Metodista São Paulo, São Paulo, BrazilAC Camargo Canc Ctr, Int Res Ctr, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem Mol & Cell Biol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04039032 São Paulo, BrazilFAPESP: 2008/06152-9FAPESP: 2009/14027-2FAPESP: 2013/22413-5FAPESP: 2012/18093-2Web of Scienc

Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 degrees C to 45 degrees C, and increases of NaCl concentration up to 1 M led to activity decreases. the preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity. (C) 2012 Elsevier Masson SAS. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Ciencias Mar UFC, BR-60165081 Fortaleza, Ceara, BrazilUniv Estado Amazonas, Escola Super Ciencias Saude INCT, BR-69065001 Manaus, Amazonas, BrazilUniversidade Federal de São Paulo, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Tonin Overexpression in Mice Diminishes Sympathetic Autonomic Modulation and Alters Angiotensin Type 1 Receptor Response

Background: Tonin, a serine-protease that forms Angiotensin II (AngII) from angiotensinogen, is increased in failing human heart samples. Increased blood pressure (BP) and decreased heart rate (HR) variabilities are associated with higher risk of cardiovascular morbidity. Losartan has been used to reduce hypertension and, therefore, lowers the risk of fatal and non-fatal cardiovascular events. Determination of tonin's impact on BP and HR variabilities as well as the impact of losartan remain questions to be elucidated.Aim: Evaluation of cardiovascular autonomic profile in transgenic mice overexpressing the rat tonin enzyme TGM'(rton) and the impact of AT1 receptor blocker, losartan.Methods: Male C57BL/6 (WT) and TGM'(rTon) mice were cannulated for recording BP (Windaq, 4 MHz) for 30 min at baseline and 30 min after losartan injection (20 mg/kg). BP and HR variabilities were analyzed in time and frequency domain method. Low-frequency (LF) and high-frequency (HF) components were identified for sympathetic and parasympathetic modulations analysis. Ang I, AngII, and Ang1-7 were quantified by high performance liquid chromatography method. The total enzymatic activity for AngI, AngII, and Ang1-7 formation was evaluated in the heart and plasma by Liquid chromatography mass spectrometry (LC-MS/MS).Results: At the baseline TGM'(rTon) exhibited higher BP, lower cardiac LF, higher cardiac HF, lower LF/HF, and lower alpha index than wild type (WT). After losartan injection, TGM'(rTon) mice presented an additional decrease in cardiac LF and increase in HF in relation to baseline and WT. In the vasculature, losartan caused decreased in BP and LF of systolic BP in WT mice in relation to its baseline. A similar effect was observed in the BP of TGM'(rTon) mice; however, LF of systolic BP increased compared to baseline. Our data also indicates that AT1R receptor signaling has been altered in TGM’(rTon)mice. Interestingly, the dynamics of the renin-angiotensin system kinetics change, favoring production of Ang1-7.Conclusion: Autonomic evaluation of TGM’(rTon) mice indicates an unclear prognosis for diseases that affect the heart. HR variability in TGM’(rTon) mice indicates high risk of morbidity, and sympathetic and parasympathetic modulation indicate low risk of morbidity. The low risk of morbidity could be the biased production of Ang1-7 in the heart and circulation; however, the altered response of AT1R in the TGM’(rTon) remains to be elucidated, as well aswhether that signaling is pro-protection or pro-pathology

Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles

The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been conducted to develop biotechnological tools to make possible the use of both proteases to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases

Characterization and role of the 3-methylglutaconyl coenzyme A hidratase in Trypanosoma brucei

Trypanosoma brucei, the agent of African Trypanosomiasis, is a flagellated protozoan parasite that develops in tsetse flies and in the blood of various mammals. The parasite acquires nutrients such as sugars, lipids and amino acids from their hosts. Amino acids are used to generate energy and for protein and lipid synthesis. However, it is still unknown how T. brucei catabolizes most of the acquired amino acids. Here we explored the role of an enzyme of the leucine catabolism, the 3-methylglutaconyl-Coenzyme A hydratase (3-MGCoA-H). It catalyzes the hydration of 3-methylglutaconyl-Coenzyme A (3-MGCoA) into 3-hydroxymethylglutaryl-Coenzyme A (3-HMGCoA). We found that 3-MGCoA-H localizes in the mitochondria) matrix and is expressed in both insect and mammalian bloodstream forms of the parasite. The depletion of 3-MGCoA-H by RNA interference affected minimally the proliferation of both forms. However, an excess of leucine in the culture medium caused growth defects in cells depleted of 3-MGCoA-H, which could be reestablished by mevalonate, a precursor of isoprenoids and steroids. Indeed, procyclics depleted of the 3-MGCoA-H presented reduced levels of synthesized steroids relative to cholesterol that is scavenged by the parasite, and these levels were also reestablished by mevalonate. These results suggest that accumulation of leucine catabolites could affect the level of mevalonate and consequently inhibit the sterol biosynthesis, required for T. brucei growth. (C) 2017 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Univ Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, Sao Paulo, SP, Brazil[Univ Fed Sao Paulo, Dept Biofis, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, SP, BrazilFAPESP: 11/51973-3FAPESP: 2015/22031-0CNPq: 445655/2014-3CNPq: 306815/2015-0Web of Scienc