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László Polgár - One of the best experts on this subject based on the ideXlab platform.
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Kosmotropic salt activation and substrate specificity of poliovirus protease 3C.
Biochemistry, 2006Co-Authors: Iuri E. Gouvea, Zoltán Szeltner, László Polgár, Wagner Alves De Souza Judice, Maria Helena S. Cezari, Maria A. Juliano, Tünde Juhász, Luiz JulianoAbstract:Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, Picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus Picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.
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The unusual catalytic triad of poliovirus protease 3C.
Biochemistry, 2003Co-Authors: Zsuzsa Sárkány, László PolgárAbstract:Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, Picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.
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Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of Picornain 3C.
Biochemistry, 2001Co-Authors: Zsuzsa Sárkány, Zoltán Szeltner, László PolgárAbstract:Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (Picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of Picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that Picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.
Abreu, Emanuel Felipe Medeiros - One of the best experts on this subject based on the ideXlab platform.
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Variabilidade genética do cowpea severe mosaic virus (cpsmv) e cowpea aphid-borne mosaic virus (cabmv) no Brasil
2012Co-Authors: Abreu, Emanuel Felipe MedeirosAbstract:O feijão-caupi (Vigna unguiculata (L.) Walp.) é uma importante leguminosa para o Nordeste brasileiro e tem sido tradicionalmente cultivada por pequenos agricultores. Doenças virais são consideradas um dos principais fatores limitantes da produtividade do caupi nesta região. A doença do mosaico severo do caupi é causada pelo Cowpea severe mosaic virus (CPSMV), subfamília Comovirinae, gênero Comovirus, juntamente com o Cowpea aphid-borne mosaic virus (CABMV), família Potyviridae, gênero Potyvirus, são consideradas as viroses mais prevalentes da cultura e responsáveis por grandes perdas na produção. O objetivo do presente trabalho foi estudar a variabilidade genética do CPSMV e CABMV obtidos em diferentes municípios do Nordeste brasileiro. Essa informação é crucial para o desenvolvimento de plantas resistentes a essas viroses tanto por métodos convencionais quanto moleculares de melhoramento. Isso é ainda mais crítico para o desenvolvimento de estratégias baseadas em RNA inteferente (RNAi). Plantas com sintomas de infecção pelo CPSMV e CABMV nos estados do Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Distrito Federal, Sergipe e Bahia foram coletadas, e os isolados foram identificados por Dot-blot e RT- PCR. Foram amplificados fragmentos de 2200 pb, (correspondendo a região da Helicase (Hel), Proteína ligada ao genoma viral (VPg), Picornain 3C- protease (Pro) e RNA polimerase) e 997 pb (correspondendo a região da proteína Inclusão Cilíndrica (CI) e da proteína 6K2) dos vírus CPSMV e CABMV por RT-PCR, respectivamente. Alguns fragmentos foram clonados e outros sequenciados diretamente do produto de PCR em ambas as orientações. As sequências obtidas a partir de diferentes isolados foram comparadas com sequências disponíveis no GenBank. Os alinhamentos das sequências foram obtidos usando o programa Clustal W, e uma árvore filogenética foi criado usando o software MEGA 5.1. A análise revelou baixa variabilidade entre isolados de CPSMV, variando entre 98 e 100% para sequências de nucleótidos e 96-100% para a sequência de aminoácidos deduzida. Entre os CABMV isolado, a variabilidade foi maior, variando 84-99% entre as sequências de nucleótidos 91 a 99% das sequências de aminoácidos. Este estudo fornece informações que serão a base para o desenvolvimento de estratégias para a produção de linhagens de caupi resistentes a estes vírus por RNA interferente. Os dados indicam a possibilidade de obtenção de resistência durável e aplicável ao caupi nas principais áreas produtoras no Brasil. _________________________________________________________________________________ ABSTRACTCowpea (Vigna unguiculata) is an important plant crop in Northeast Brazil being traditionally cultivated by small farmers. Virus diseases are considered to be the main factor limiting cowpea yield in the region. The severe mosaic disease caused by the Cowpea severe mosaic virus (CPSMV), subfamily Comovirinae, genus Comovirus, seems to be one of the most prevalent diseases leading to high yield losses in this crop. The Cowpea aphid borne mosaic virus (CABMV) belongs to the genus Potyvirus in the Potyviridae family, and infects cowpea worldwide. In the northeastern region of Brazil, both viruses can be found in cowpea planted areas. The aim of the present study was to access the degree of homology among regions amplified of different isolates of CPSMV and CABMV, respectively; obtained in different northeastern regions in Brazil, and to compare it to isolates throughout the world. Plants with CPSMV and CABMV symptoms from the states of Piauí, Ceará, Rio Grande do Norte, Paraiba, Pernambuco, Alagoas, Sergipe, Bahia and Distrito Federal were collected, and the isolates were identified by RT-PCR analysis. Total RNA was extracted from infected tissue and afterwards used for synthesis of cDNA fragments by RT-PCR. The synthetized primers were able to amplify fragments of 2200 and 997 bp of the CPSMV and CABMV virus, respectively. Amplification products were directly cloned into the pGEMT-Easy plasmid vector (Promega), according to the manufacturer’s instructions. Cloned fragments were sequenced in both orientations. Deduced amino acid sequences of the virus were compared to sequences available from GenBank. Multiple sequence alignments were obtained with Clustal W. Phylogenetic trees using the MEGA version 5.1 software package and the neighbour-joining method with Poisson correction. Tree branches were bootstrapped with 1000 permutations. CPSMV and CABMV diseases remain as limiting factors in this crop in Brazil, and breeding programs, either by conventional or engineered approaches, should be targeted at establishing resistance of cowpeas to CPSMV and CABMV
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Variabilidade genética do cowpea severe mosaic virus (cpsmv) e cowpea aphid-borne mosaic virus (cabmv) no Brasil
2012Co-Authors: Abreu, Emanuel Felipe MedeirosAbstract:Tese (Doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2012.O feijão-caupi (Vigna unguiculata (L.) Walp.) é uma importante leguminosa para o Nordeste brasileiro e tem sido tradicionalmente cultivada por pequenos agricultores. Doenças virais são consideradas um dos principais fatores limitantes da produtividade do caupi nesta região. A doença do mosaico severo do caupi é causada pelo Cowpea severe mosaic virus (CPSMV), subfamília Comovirinae, gênero Comovirus, juntamente com o Cowpea aphid-borne mosaic virus (CABMV), família Potyviridae, gênero Potyvirus, são consideradas as viroses mais prevalentes da cultura e responsáveis por grandes perdas na produção. O objetivo do presente trabalho foi estudar a variabilidade genética do CPSMV e CABMV obtidos em diferentes municípios do Nordeste brasileiro. Essa informação é crucial para o desenvolvimento de plantas resistentes a essas viroses tanto por métodos convencionais quanto moleculares de melhoramento. Isso é ainda mais crítico para o desenvolvimento de estratégias baseadas em RNA inteferente (RNAi). Plantas com sintomas de infecção pelo CPSMV e CABMV nos estados do Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Distrito Federal, Sergipe e Bahia foram coletadas, e os isolados foram identificados por Dot-blot e RT- PCR. Foram amplificados fragmentos de 2200 pb, (correspondendo a região da Helicase (Hel), Proteína ligada ao genoma viral (VPg), Picornain 3C- protease (Pro) e RNA polimerase) e 997 pb (correspondendo a região da proteína Inclusão Cilíndrica (CI) e da proteína 6K2) dos vírus CPSMV e CABMV por RT-PCR, respectivamente. Alguns fragmentos foram clonados e outros sequenciados diretamente do produto de PCR em ambas as orientações. As sequências obtidas a partir de diferentes isolados foram comparadas com sequências disponíveis no GenBank. Os alinhamentos das sequências foram obtidos usando o programa Clustal W, e uma árvore filogenética foi criado usando o software MEGA 5.1. A análise revelou baixa variabilidade entre isolados de CPSMV, variando entre 98 e 100% para sequências de nucleótidos e 96-100% para a sequência de aminoácidos deduzida. Entre os CABMV isolado, a variabilidade foi maior, variando 84-99% entre as sequências de nucleótidos 91 a 99% das sequências de aminoácidos. Este estudo fornece informações que serão a base para o desenvolvimento de estratégias para a produção de linhagens de caupi resistentes a estes vírus por RNA interferente. Os dados indicam a possibilidade de obtenção de resistência durável e aplicável ao caupi nas principais áreas produtoras no Brasil. _________________________________________________________________________________ ABSTRACTCowpea (Vigna unguiculata) is an important plant crop in Northeast Brazil being traditionally cultivated by small farmers. Virus diseases are considered to be the main factor limiting cowpea yield in the region. The severe mosaic disease caused by the Cowpea severe mosaic virus (CPSMV), subfamily Comovirinae, genus Comovirus, seems to be one of the most prevalent diseases leading to high yield losses in this crop. The Cowpea aphid borne mosaic virus (CABMV) belongs to the genus Potyvirus in the Potyviridae family, and infects cowpea worldwide. In the northeastern region of Brazil, both viruses can be found in cowpea planted areas. The aim of the present study was to access the degree of homology among regions amplified of different isolates of CPSMV and CABMV, respectively; obtained in different northeastern regions in Brazil, and to compare it to isolates throughout the world. Plants with CPSMV and CABMV symptoms from the states of Piauí, Ceará, Rio Grande do Norte, Paraiba, Pernambuco, Alagoas, Sergipe, Bahia and Distrito Federal were collected, and the isolates were identified by RT-PCR analysis. Total RNA was extracted from infected tissue and afterwards used for synthesis of cDNA fragments by RT-PCR. The synthetized primers were able to amplify fragments of 2200 and 997 bp of the CPSMV and CABMV virus, respectively. Amplification products were directly cloned into the pGEMT-Easy plasmid vector (Promega), according to the manufacturer’s instructions. Cloned fragments were sequenced in both orientations. Deduced amino acid sequences of the virus were compared to sequences available from GenBank. Multiple sequence alignments were obtained with Clustal W. Phylogenetic trees using the MEGA version 5.1 software package and the neighbour-joining method with Poisson correction. Tree branches were bootstrapped with 1000 permutations. CPSMV and CABMV diseases remain as limiting factors in this crop in Brazil, and breeding programs, either by conventional or engineered approaches, should be targeted at establishing resistance of cowpeas to CPSMV and CABMV
Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.
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Kosmotropic salt activation and substrate specificity of poliovirus protease 3C.
Biochemistry, 2006Co-Authors: Iuri E. Gouvea, Zoltán Szeltner, László Polgár, Wagner Alves De Souza Judice, Maria Helena S. Cezari, Maria A. Juliano, Tünde Juhász, Luiz JulianoAbstract:Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, Picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus Picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.
Zoltán Szeltner - One of the best experts on this subject based on the ideXlab platform.
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Kosmotropic salt activation and substrate specificity of poliovirus protease 3C.
Biochemistry, 2006Co-Authors: Iuri E. Gouvea, Zoltán Szeltner, László Polgár, Wagner Alves De Souza Judice, Maria Helena S. Cezari, Maria A. Juliano, Tünde Juhász, Luiz JulianoAbstract:Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, Picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus Picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.
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Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of Picornain 3C.
Biochemistry, 2001Co-Authors: Zsuzsa Sárkány, Zoltán Szeltner, László PolgárAbstract:Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (Picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of Picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that Picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.
Zsuzsa Sárkány - One of the best experts on this subject based on the ideXlab platform.
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The unusual catalytic triad of poliovirus protease 3C.
Biochemistry, 2003Co-Authors: Zsuzsa Sárkány, László PolgárAbstract:Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, Picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.
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Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of Picornain 3C.
Biochemistry, 2001Co-Authors: Zsuzsa Sárkány, Zoltán Szeltner, László PolgárAbstract:Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (Picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of Picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that Picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.