Repositório Institucional - Unifesp :: Navegando por Palavras-chave "protease"

Navegando por Palavras-chave "protease"

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Acesso aberto (Open Access)
Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides
(Frontiers Media Sa, 2017) Hamin Neto, Youssef A. A.; de oliveira, Lilian C. G.[INIFESP]; de oliveira, Juliana R.[INIFESP]; Juliano, Maria A.[INIFESP]; Juliano, Luiz[INIFESP]; Arantes, Eliane C.; Cabral, Hamilton
Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60 degrees C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The primed side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S'(1) subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P'(1). These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.
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Angiotensin I-converting enzyme (ACE) activity and expression in rat central nervous system after sleep deprivation
(Walter de Gruyter & Co, 2011-04-01) Visniauskas, Bruna [UNIFESP]; Oliveira, Vitor [UNIFESP]; Carmona, Adriana K. [UNIFESP]; D'Almeida, Vania [UNIFESP]; Melo, Robson L. de; Tufik, Sergio [UNIFESP]; Chagas, Jair R. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Butantan Inst
Proteases are essential either for the release of neuropeptides from active or inactive proteins or for their inactivation. Neuropeptides have a fundamental role in sleep-wake cycle regulation and their actions are also likely to be regulated by proteolytic processing. Using fluorescence resonance energy transfer substrates, specific protease inhibitors and real-time PCR we demonstrate changes in angiotensin I-converting enzyme (ACE) expression and proteolytic activity in the central nervous system in an animal model of paradoxical sleep deprivation during 96 h (PSD). Male rats were distributed into five groups (PSD, 24 h, 48 h and 96 h of sleep recovery after PSD and control). ACE activity and mRNA levels were measured in hypothalamus, hippocampus, brainstem, cerebral cortex and striatum tissue extracts. in the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96 h of sleep recovery. in the brainstem and hippocampus, although significant, changes in mRNA do not parallel changes in ACE specific activity. Changes in ACE activity could affect angiotensin II generation, angiotensin 1-7, bradykinin and opioid peptides metabolism. ACE expression and activity modifications are likely related to some of the physiological changes (cardiovascular, stress, cognition, metabolism function, water and energy balance) observed during and after sleep deprivation.
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Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity
(Blackwell Publishing, 2005-06-01) Oliveira, Vitor; Garrido, Paula AG; Rodrigues, Claudia C.; Colquhoun, Alison; Castro, Leandro M.; Almeida, Paulo C.; Shida, Claudio S.; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Camargo, Antonio CM; Hyslop, Stephen; Roberts, James L.; Grum-Tokars, Valerie; Glucksman, Marc J.; Ferro, Emer S.; Univ Cidade São Paulo; Universidade de São Paulo (USP); Univ Mogi das Cruzes; Universidade Federal de São Paulo (UNIFESP); Inst Butantan; Universidade Estadual de Campinas (UNICAMP); Univ Texas; Rosalind Franklin Univ Med & Sci
The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). the constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. in the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium.. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.
Acesso aberto (Open Access)
Desenvolvimento de antivirais: Triagem de inibidores da protease do vírus Zika
(Universidade Federal de São Paulo, 2022-12-08) Coelho, Camila de Morais [UNIFESP]; Würtele, Martin [UNIFESP]; http://lattes.cnpq.br/0625673643875019; http://lattes.cnpq.br/2584731188791527
O crescente surgimento e ressurgimento mundial de doenças virais tornou o desenvolvimento de novos antivirais extremamente importante. No caso do vírus Zika, vários surtos importantes foram relatados desde sua primeira identificação em 1947, incluindo o surto de Zika entre 2015-2016. Como a protease do vírus Zika é um alvo essencial e bem estabelecido para o desenvolvimento de agentes antivirais, este trabalho teve como objetivo realizar uma varredura bioquímica de pequenos compostos utilizando uma forma recombinante desta enzima. Como resultado, foi possível identificar 12 inibidores de protease do vírus Zika. Esses compostos são todos produtos naturais e apresentaram forte inibição nos ensaios bioquímicos. Os valores das constantes inibitórias (Ki) para os compostos variaram de 5 nM a 8 μM. Entre os inibidores mais potentes estão alguns flavonóides como o irigenol hexa-acetato (1, Ki = 0,28 μM), katacina (2, Ki = 0,26 μM) e teaflavina galato (5, Ki = 0,40 μM). Os inibidores de outros grupos de produtos naturais incluem senosídeo A (9, Ki = 0,19 μM) e gossipol (10, Ki = 0,70 μM). Vários dos compostos obtidos são conhecidos pelos seus efeitos benéficos para a saúde humana. Portanto, eles podem constituir compostos líderes relevantes para o desenvolvimento de futuros medicamentos antivirais contra o Zika.
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Differences in substrate and inhibitor sequence specificity of human, mouse and rat tissue kallikreins
(Portland Press, 2004-06-15) Fogaça, Sandro E. [UNIFESP]; Melo, Robson L. [UNIFESP]; Pimenta, Daniel C. [UNIFESP]; Hosoi, Kazuo; Juliano, Luiz [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Tokushima
The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikrems were assayed with the bradykinin-containing synthetic peptides NH2-MTEMARRPPGFSPFRSVTVQ-NH2 (where Abz stands for o-aminobenzoyl) and Abz-MTS-VIRRPPGFSPFRAPRV-NH2, which correspond to fragments Met(374)-Gln(393) and Met(375)-Val(393) of mouse and rat LMWKs (low-molecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1- and rK1-mediated hydrolysis of Arg-Arg and Arg-Ser (or Arg-Ala) peptide bonds. However, owing to preferential hydrolysis of Phe-Arg compared with the Arg-Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg(9)]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH2). the formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N- or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe-Arg bond, confirming the potential des-[Arg(9)]bradykinin-releasing activity of hK1 on rat kininogen. the proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg-Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S-1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg(9)] bradykinin. for further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.
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Effects of magnesium ions on recombinant human furin: selective activation of hydrolytic activity upon substrates derived from virus envelope glycoprotein
(Walter de Gruyter & Co, 2010-09-01) Izidoro, Mario A. [UNIFESP]; Assis, Diego M. [UNIFESP]; Oliveira, Vitor [UNIFESP]; Santos, Jorge A. N. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Lindberg, Iris; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Maryland
Here we report a detailed analysis of magnesium (Mg(2+)) ion effects on furin hydrolysis of fluorescent resonance energy transfer decapeptide substrates derived from canonical R-X-K/R-R furin cleavage motifs within certain viral envelope glycoproteins and eukaryotic proproteins. Using virus-derived sequences a selective activation of furin by Mg(2+) ions was observed as a result of cooperativity between furin subsites. Furin hydrolysis of the peptides Abz-SRRHKR down arrow FAGV-Q-EDDnp (from measles virus fusion protein F(o)) and Abz-RERRRKKR down arrow GLFG-Q-EDDnp (from Asian avian influenza A, H5N1) was activated between 60- and 80-fold by MgCl(2). It appears that virus envelope glycoprotein mutations have been selected to increase their susceptibility to furin within cells, a location where Mg(2+) is present in adequate concentrations for activation. Both the pH profile of furin and its intrinsic fluorescence were modified by Mg(2+) ions, which bind to furin with a K(d) value of 1.1 mM.
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Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid
(Wiley-Blackwell, 1998-01-01) Ito, A. S.; Turchiello, RDF; Hirata, I. Y.; Cezari, MHS; Meldal, M.; Juliano, L.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Dept Chem
ortho-Aminobenzoic acid (Abz) has been used as a convenient fluorescent donor group in internally quenched fluorescent peptides, which are employed as substrates for several proteolytic enzymes. As Abz is usually bound to the N-amino terminal of these peptides, it is of interest to investigate the Abz group fluorescent properties bound to different amino acids. We report in this article the optical absorption and fluorescent properties, in aqueous media, of Abz bound to the alpha-amino group of Ala, Gly, Leu, ne, Val, Pro, Phe, Arg, Glu, Met, Asn, Tyr, and Trp, with monomethyl-amidated alpha-carboxyl group, in order to explore the origin of the drastic reduction of Abz attached to N-alpha amino group of prolyl-peptides, we also examined the fluorescence properties of Abz-NHCH3, Abz-N(CH3)(2), and Abz-pyrrolidine. Molecular dynamics simulation and NMR data indicated a lack of periplanarity of the Abz-dimethylamide, which could be the origin of low fluorescence quantum yield of Abz-prolyl-peptides. (C) 1998 John Wiley & Sons, Inc.
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Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition
(Walter de Gruyter & Co, 2010-12-01) Okamoto, Debora N. [UNIFESP]; Oliveira, Lilian C. G. [UNIFESP]; Kondo, Marcia Y. [UNIFESP]; Cezari, Maria H. S. [UNIFESP]; Szeltner, Zoltan; Juhasz, Nde; Juliano, Maria A. [UNIFESP]; Polgar, Laszlo; Juliano, Luiz [UNIFESP]; Gouvea, Iuri E. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Hungarian Acad Sci
The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. in contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. in this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. the activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na(2)SO(4), whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. the activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. the viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.
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Irreversible inhibition of human cathepsins B, L, S and K by hypervalent tellurium compounds
(Walter de Gruyter & Co, 2009-11-01) Cunha, Rodrigo L. O. R. [UNIFESP]; Gouvea, Iuri E. [UNIFESP]; Feitosa, Geovana P. V. [UNIFESP]; Alves, Marcio F. M. [UNIFESP]; Broemme, Dieter; Comasseto, Joao V.; Tersariol, Ivarne L. S.; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade Federal do ABC (UFABC); Univ British Columbia; Universidade de São Paulo (USP); Univ Mogi das Cruzes
The inhibition of human cysteine cathepsins B, L, S and K was evaluated by a set of hypervalent tellurium compounds (telluranes) comprising both organic and inorganic derivatives. All telluranes studied showed a time-and concentration-dependent irreversible inhibition of the cathepsins, and their second-order inactivation rate constants were determined. the organic derivatives were potent inhibitors of the cathepsins and clear specificities were detected, which were parallel to their known substrate specificities. in all cases, the activity of the tellurane-inhibited cathepsins was recovered by treatment of the inactivated enzymes with reducing agents. the maximum stoichiometry of the reaction between cysteine residues and telluranes were also determined. the presented data indicate that it is possible to design organic compounds with a tellurium(IV) moiety as a novel warhead that covalently modifies the catalytic cysteine, and which also form strong interactions with subsites of cathepsins B, L, S and K, resulting in more specific inhibition.
Acesso aberto (Open Access)
Isolamento, identificação e caracterização de bactérias cultiváveis presentes na compostagem de resíduos orgânicos do zoológico de São Paulo e produtoras de amilases e proteases
(Universidade Federal de São Paulo, 2014-03-18) Magron, Claudio Fernando [UNIFESP]; Oliveira, Julio Cezar Franco de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
A Fundação Parque Zoológico de São Paulo (FPZSP) possui uma Unidade de Produção de Composto Orgânico (UPCO) que recicla material orgânico, como: excremento de aproximadamente 3.500 animais (cerca de 400 espécies) oriundos de várias partes do mundo, cama de animais, resíduos de poda dos jardins do Parque Zoológico, restos vegetais da Mata Atlântica e resíduos alimentares. O composto final, cerca de 600 toneladas/ano, é utilizado como fertilizante orgânico na fazenda do Zoológico, para produção de cerca de 70% dos alimentos consumidos pelos animais do Parque, fechando assim um ciclo virtuoso de sustentabilidade. Além disso, a compostagem evita que a matéria orgânica seja depositada em aterros sanitários, o que causaria problemas ambientais. A riqueza microbiológica desse material é incalculável, visto que ali poderão ser encontradas espécies que ainda não foram descritas e/ou cultivadas, bem como espécies produtoras de enzimas de interesse industrial. Dentro de um acordo de parceria estabelecida entre a UNIFESP e a FPZSP, e dentro de colaboração com grupos de pesquisa do IQ-USP, este projeto de mestrado envolveu o isolamento de bactérias em meios de cultura ricos não seletivos provenientes de diferentes etapas de um processo de compostagem efetuado numa célula de compostagem da UPCO do Zoológico de São Paulo. Uma vez purificados e estocados no banco de microrganismos do LIMic (Laboratório de Interações Microbianas, Unifesp ? Diadema), os isolados microbianos foram triados para aqueles secretores de amilase ou protease, seguido de caracterização taxonômica por espectrometria de massa (MALDI/TOF-MS). Nove isolados bacterianos secretores de amilases foram selecionados para estudo quanto à atividade da atividade das amilases secretadas em meio de cultura líquido, e tiveram respectivos fragmentos do 16SrDNA amplificados por PCR, sequenciados e submetidos a análise de sequências para caracterização taxonômica.
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Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa
(Bentham Science Publ Ltd, 2006-01-01) Cabral, H.; Leopoldino, A. M.; Tajara, E. H.; Greene, L. J.; Faca, V. M.; Mateus, R. P.; Ceron, C. R.; Judice, WAD; Juliano, Luiz [UNIFESP]; Bonilla-Rodriguez, G. O.; UNESP; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)
The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. the present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. the product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).
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The presence of resistance mutations to protease and polymerase inhibitors in Hepatitis C virus sequences from the Los Alamos databank
(Wiley-Blackwell, 2013-06-01) Alves, R. [UNIFESP]; Queiroz, A. T. L.; Pessoa, M. G.; Silva, E. F. da; Mazo, D. F. C.; Carrilho, F. J.; Carvalho-Filho, R. J. [UNIFESP]; Carvalho, I. M. V. G. de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); CPqGM FIOCRUZ; Universidade de São Paulo (USP); Inst Butantan
Several new direct-acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase have been the major targets. HCV variants displaying drug-resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment-naive HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a=680, 1b=498 and 3a=205) and 806 NS5B polymerase sequences (genotypes 1a=471, 1b=329, 3a=6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low-level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre-existence of HCV variants resistant to first-generation protease inhibitors and to non-nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre-existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.
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Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART
(Elsevier B.V., 2002-07-01) Tanuri, A.; Caridea, E.; Dantas, M. C.; Morgado, M. G.; Mello, DLC; Borges, S.; Tavares, M.; Ferreira, S. B.; Santoro-Lopes, G.; Martins, CRF; Esteves, ALC; Diaz, R. S.; Andreo, SMS; Ferreira, LAP; Rodrigues, R.; Reuter, T.; Cavalcanti, AMS; Oliveira, S. M. de; Barbosa, H. B. de; Teixeira, P. R.; Chequer, P. N.; Universidade Federal do Rio de Janeiro (UFRJ); Brazilian Minist Hlth; Fiocruz MS; Univ Fed Espirito Santos; Universidade de Brasília (UnB); Lab Cent Saude Publ; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP); Universidade Federal de Santa Catarina (UFSC)
Background: Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. Objective: in an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. the mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. Study Design: Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pal RT (palm finger region) genes. the mutations related to drug resistance in RT gene was analyzed using a line probe assay (LIPA(R)) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. Results: There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. the main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). in the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. Conclusions: This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance. (C) 2002 Elsevier Science B.V. All rights reserved.
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S(1)' and S(2)' subsite specificities of human plasma kallikrein and tissue kallikrein 1 for the hydrolysis of peptides derived from the bradykinin domain of human kininogen
(Walter de Gruyter & Co, 2008-12-01) Lima, Aurelio Resende [UNIFESP]; Alves, Fabiana M. [UNIFESP]; Angelo, Pedro Francisco [UNIFESP]; Andrade, Douglas [UNIFESP]; Blaber, Sachiko I.; Blaber, Michael; Juliano, Luiz [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Florida State Univ
The S(1)' and S(2)' subsite specificities of human tissue kallikrein 1 (KLK1) and human plasma kallikrein (HPK) were examined with the peptide series Abz-GFSPFRXSRIQ-EDDnp and Abz-GFSPFRSXRIQ-EDDnp [X=natural amino acids or S( PO 3 H 2) x. KLK1 efficiently hydrolyzed most of the peptides except those containing negatively charged amino acids at P 19 and P 29 positions. Abz-GFSPFRSSRIQ-EDDnp, as in human kininogen, is the best substrate for KLK1 and exclusively cleaved the R-S bond. All other peptides were cleaved also at the F-R bond. the synthetic human kininogen segment Abz-MISLMKRPPGFSPFRS(390)S(391)RI-NH(2) was hydrolyzed by KLK1 first at R-S and then at M-K bonds, releasing Lys-bradykinin. in the S(390) and S(391) phosphorylated analogs, this order of hydrolysis was inverted due to the higher resistance of the R-S bond. Abz-MISLMKRPPGFSPFRSS(PO(3)H(2))(391)RI-NH(2) was hydrolyzed by KLK1 at M-K and mainly at the F-R bond, releasing des-(Arg(9))-Lys-Bk which is a B1 receptor agonist. HPK cleaved all the peptides at R and showed restricted specificity for S in the S(1)' subsite, with lower specificity for the S(2)' subsite. Abz-MISLMKRPPGFSPFRSSRI-NH(2) was efficiently hydrolyzed by HPK under bradykinin release, while the analogs containing S(PO(3)H(2)) were poorly hydrolyzed. in conclusion, S(1)' and S(2)' subsite specificities of KLK1 and HPK showed peculiarities that were observed with substrates containing the amino acid sequence of human kininogen.
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Sexual transmission of HIV-1 isolate showing G -> A hypermutation
(Elsevier B.V., 2002-01-01) Caride, E.; Brindeiro, R. M.; Kallas, Esper Georges [UNIFESP]; Sa, CAM de; Eyer-Silva, W. A.; Machado, E.; Tanuri, A.; Universidade Federal do Rio de Janeiro (UFRJ); Universidade Federal de São Paulo (UNIFESP); Hosp Univ Gaffree & Guinle
Retroviral genomes with a high frequency of G --> A mutations are thought to originate during reverse transcription (RT). Here we present a case report of an AIDS patient infected with a subtype F variant where extensive G --> A hypermutation (G --> A Hypm) sequences were found in the protease gene. This patient was failing HAART at the time the hypermutation was found. These sequences were basically encountered in the proviral compartment on two occasions and were persistently absent in the plasma viral population. the patient's viral genotype showed several mutations related to antiretroviral drug resistance in RT (T69N, N184V, T215F, K219Q) and protease (M36I, G48V, I54V, T63L, V82A) genes. the drug regimen was changed and the viral load dropped 0.9 Logs and CD4 count increased by 200 cells/ml. the hypermutation was not found any more in a 1-year follow up. the patient's wife was infected with a similar virus strain and G A Hypm sequences were also detected in the RT gene. This is the first report of sexual transmitted G --> A Hypermutation in HIV-1 and suggest that this phenomenon can be genetically coded by the viral RT molecule. (C) 2002 Elsevier Science B.V. All rights reserved.
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Specificity of human tissue kallikrein towards substrates containing Phe-Phe pair of amino acids
(Portland Press, 1999-04-15) Pimenta, Daniel C.; Chao, Julie; Chao, Lee; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Med Univ S Carolina
We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK(1)) of substrates containing the Phe-Phe amino acid pair, after which hK(1) cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1-14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P-9 to P'(13), and the kinetic parameters of their hydrolysis by hK(1) were determined. All these peptides were cleaved after the Phe-Phe pair. for comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe-Arg and Leu-Lys bonds, respectively. Hybrid peptides containing kallistatin-PCI sequences showed that the efficiency of hK(1) activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P-1 amino acid as well as on residues at the P- and PI-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe-Phe, and on Lys and lie at the P-3 and P-4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp, All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe-Arg pair; however, hydrolysis after Phe-Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme-substrate interaction and/or particular substrate conformations.
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Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: Influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates
(Elsevier B.V., 2007-01-15) Gouvea, I. E.; Izidoro, M. A.; Judice, W. A. S.; Cezari, M. H. S.; Caliendo, G.; Santagada, V.; Santos, C. N. D. dos; Queiroz, M. H.; Juliano, M. A.; Young, P. R.; Fairlie, D. P.; Juliano, L.; Universidade Federal de São Paulo (UNIFESP); Univ Naples Federico II; Inst Biol Mol Parana; Univ Queensland
A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native Substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. the hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S-4 and S'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on K-m and k(cat) suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors. (c) 2006 Elsevier Inc. All rights reserved.
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Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase
(Blackwell Publishing Ltd, 2002-09-01) Oliveira, Vitor [UNIFESP]; Gatti, R; Rioli, V; Ferro, Emer Suavinho [UNIFESP]; Spisni, A.; Camargo, Antonio Carlos Martins de [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); CBME; Universidade de São Paulo (USP); Univ Parma; Inst Butantan
We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. the kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. the relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.054.2 M for NaCl, KCl, NH4Cl and NaI, and 0.025-0.1 M for Na2SO4. Concentration higher than 0.2 N NH4Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na2SO4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na2SO4, while NH4Cl and NaI have very modest effect. High positive values; of enthalpy (DeltaH*) and entropy (DeltaS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. the effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.
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Triapsin, an unusual activatable serine protease from the saliva of the hematophagous vector of Chagas' disease Triatoma infestans (Hemiptera : Reduviidae)
(Elsevier B.V., 2001-03-15) Amino, R.; Tanaka, A. S.; Schenkman, S.; Universidade Federal de São Paulo (UNIFESP)
Salivary anticoagulant activities are widely distributed among hematophagous arthropods. Most of them are inhibitors of the serine proteases of the coagulation cascade. Here we show that the saliva of the exclusively hematophagous insect Triatoma infestans, an important Vector in the transmission of Chagas' disease, contains an uncommon trypsin-like activity, triapsin. This novel enzyme was purified and characterized. It is a serine protease that is stored as a zymogen in the luminal content of the salivary glands D2. Triapsin is activated by trypsin treatment, or when the saliva is ejected during the insect bite. the enzyme was purified 300-fold from the released saliva by anion exchange chromatography in a HiTrap Q column, followed by chromatography in Phenyl-Superose, and Superdex HR75. the purified triapsin shows an apparent molecular mass of around 40 kDa in non-reduced SDS gels and in sieving chromatography, and 33 kDa in reduced SDS-gels, Its activity is lost after incubation with dithiothreitol indicating that cysteine bridges are essential for activity. Triapsin cleaves gelatin and synthetic substrates showing preference for arginine at P1 residues. the best p-nitroanilide substrate is isoleucyl-prolyl-arginine. It does not cleave bradykinin, angiotensin and other lysine containing substrates. the triapsin amidolytic activity against chromogenic substrates is similar to plasminogen activators, such as urokinase and tissue plasminogen activator. However, it does not activate plasminogen. the fact that triapsin is released at the bite in its active form suggests that it has a role in blood feeding. (C) 2001 Elsevier B.V. All rights reserved.

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