Navegando por Palavras-chave "Serine protease"
Agora exibindo 1 - 7 de 7
Resultados por página
Opções de Ordenação
- ItemAcesso aberto (Open Access)Characterization, subsite mapping and N-terminal sequence of miliin, a serine-protease isolated from the latex of Euphorbia milii(Elsevier B.V., 2013-04-01) Moro, Luigi P.; Cabral, Hamilton; Okamoto, Débora Noma [UNIFESP]; Hirata, Izaura Yoshico [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Juliano, Luiz [UNIFESP]; Bonilla-Rodriguez, Gustavo Orlando; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Miliin is a serine protease purified from the latex of Euphorbia milii. This work reports the effect of pH and temperature on the catalytic activity of miliin, using fluorescence resonance energy transfer (FRET) substrates. Miliin displayed the highest activity at pH 9 and 35 degrees C. Subsite mapping shows that subsites S-2 to S-2' prefer uncharged residues. the S-2 subsite prefers hydrophobic aliphatic amino acids (Val, Pro and Ile) and defines the cleavage site. This work is the first one that reports subsite mapping of Euphorbiacea proteases. the N-terminal sequence showed higher similarity (40%) with the serine protease LIM9 isolated from Lilium. the presence of Tyr, Pro and Lys at positions 2, 5 and 10 respectively, were observed for most of the serine proteases used for comparison. the N-terminal sequence has striking differences with those reported previously for milin and eumiliin, other serine proteases isolated from the latex of E. milii. (C) 2013 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosEnzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors(Elsevier B.V., 2014-03-01) Yonamine, Camila M. [UNIFESP]; Kondo, Marcia Y. [UNIFESP]; Nering, Marcela B. [UNIFESP]; Gouvea, Iuri E. [UNIFESP]; Okamoto, Debora [UNIFESP]; Andrade, Douglas [UNIFESP]; Silva, Jose Alberto A. da; Prieto da Silva, Alvaro R. B.; Yamane, Tetsuo; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Lapa, Antonio J. [UNIFESP]; Hayashi, Mirian A. F. [UNIFESP]; Lima-Landman, Maria Teresa R. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); IPEN; Inst Butantan; Univ Estado AmazonasGyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. the serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. in the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. the k(cat)/K-M values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. for the sh and lg PAR-2 mimetic peptides the k(cat)/K-M values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. the k(cat)/K-M values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-I, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. the determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects. (C) 2013 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosFunctional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP)(Elsevier Science Bv, 2018) Marem, Alyne [UNIFESP]; Okamoto, Débora Noma [UNIFESP]; Oliveira, Lilian Caroline Gonçalves de [UNIFESP]; Ruiz, Diego M.; Paggi, Roberto A.; Kondo, Marcia Yuri [UNIFESP]; Gouvea, Iuri Estrada [UNIFESP]; Juliano, Maria Aparecida [UNIFESP]; Castro, Rosana E. de; Juliano, Luiz [UNIFESP]; Icimoto, Marcelo Yudi [UNIFESP]Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0 M NaCI. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (Nep Delta KTE), that was more active in 4 M NaCI than the non-truncated wild type enzyme. Comparable to the wild type, Nep Delta CTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated Nep Delta CTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior. (C) 2018 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosIsomannide derivatives as new class of inhibitors for human kallikrein 7(Elsevier B.V., 2012-10-01) Freitas, Renato F.; Teixeira, Thiago S. P.; Barros, Thalita G.; Santos, Jorge A. N.; Kondo, Marcia Y. [UNIFESP]; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Blaber, Michael; Antunes, Octavio A. C.; Abrahao, Odonirio; Pinheiro, Sergio; Muri, Estela M. F.; Puzer, Luciano; Univ Fed Triangulo Mineiro; Universidade Federal Fluminense (UFF); Universidade Federal de São Paulo (UNIFESP); Florida State Univ; Universidade Federal do Rio de Janeiro (UFRJ); Universidade Federal do ABC (UFABC)Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. of the inhibitors tested, two inhibited KLK7 with K-i values in the low micromolar range (9g = 1.8 mu M; 9j = 3.0 mu M). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB) SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. the promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors. (C) 2012 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosIsomannide-Based Peptidomimetics as Inhibitors for Human Tissue Kallikreins 5 and 7(Amer Chemical Soc, 2014-02-01) Oliveira, Jocelia P. C.; Freitas, Renato F.; Melo, Leandro Silva de; Barros, Thalita G.; Santos, Jorge A. N.; Juliano, Maria A. [UNIFESP]; Pinheiro, Sergio; Blaber, Michael; Juliano, Luiz [UNIFESP]; Muri, Estela M. F.; Puzer, Luciano; Universidade Federal do ABC (UFABC); Johns Hopkins Univ; Universidade Federal Fluminense (UFF); Inst Fed Educ Ciencia & Tecnol Sul Minas Gerais; Universidade Federal de São Paulo (UNIFESP); Florida State UnivHuman kallikrein 5 (KLK5) and 7 (KLK7) are potential targets for the treatment of skin inflammation and cancer. Previously, we identified isomannide derivatives as potent and competitive KLK7 inhibitors. the introduction of N-protected amino acids into the isomannide-based scaffold was studied. Some KLK5 inhibitors with submicromolar affinity (K-i values of 0.3-0.7 mu M) were identified, and they were 6- to 13-fold more potent than our previous hits. Enzyme kinetics studies and the determination of the mechanism of inhibition confirmed that the new isomannide-based derivatives are competitive inhibitors of both KLK5 and KLK7. Molecular docking and MD simulations of selected inhibitors into the KLK5 binding site provide insight into the molecular mechanism by which these compounds interact with the enzyme. the promising results obtained in this study open new prospects on the design and synthesis of highly specific KLK5 and KLK7 inhibitors.
- ItemSomente MetadadadosKinetic analysis of salting activation of a subtilisin-like halophilic protease(Elsevier B.V., 2009-02-01) Okamoto, Debora N. [UNIFESP]; Kondo, Marcia Y. [UNIFESP]; Santos, Jorge A. N. [UNIFESP]; Nakajima, Sawa; Hiraga, Kazumi; Oda, Kohei; Juliano, Maria A. [UNIFESP]; Juliano, Luiz [UNIFESP]; Gouvea, Iuri E. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Kyoto Inst TechnolThe secreted extracellular subtilase SR5-3 from Halobacillus sp. bacterium, isolated from the high-salt environment of Thai fish sauce. was utilized as a model halophilic serine protease. the dependence of salt activation on the size and structure of substrates was evaluated assaying the enzyme with Suc-AAPF-MCA and with the Fluorescence Resonance Energy Transfer (FRET) peptide Abz-AAPFSSKQ-EDDnp. Solvent isotope effects (SIE) and the thermodynamic parameters for activation of the hydrolysis of Suc-AAPF-MCA and Abz-AAPFSSKQ-EDDnp by SR5-3 protease in the presence of salts were also performed. All the obtained results support the notion that the salting out effect is responsible for the halophilic character of SR5-3, and the magnitude of its hydrolytic activity is mainly derived from the improvement of catalytic and/or interaction steps depending on the nature and size of the substrates, principally if they occupy the substrate prime subsites. (C) 2008 Published by Elsevier B.V.
- ItemSomente MetadadadosThe natural flavone fukugetin as a mixed-type inhibitor for human tissue kallikreins(Pergamon-Elsevier Science Ltd, 2016) Santos, Jorge A. N.; Kondo, Marcia Y. [UNIFESP]; Freitas, Renato F.; dos Santos, Marcelo H.; Ramalho, Teodorico C.; Assis, Diego M. [UNIFESP]; Juliano, Luiz [UNIFESP]; Juliano, Maria A. [UNIFESP]; Puzer, LucianoThe human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate. (C) 2016 Elsevier Ltd. All rights reserved.