Análise bioquímica, estudos da relação estrutura-função e de expressão da proteína desacopladora mitocondrial de plantas
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Data
2008-05-30
Autores
Fávaro, Regiane Degan [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Proteínas desacopladoras (UCPs; do inglês uncoupling proteins) são proteínas especializadas no transporte mitocondrial que desacoplam a respiração da síntese de ATP. UCPs geram um fluxo de prótons através da membrana mitocondrial interna, dependente de ácidos graxos e sensível a nucleotídeos purínicos (PN). No presente trabalho, foram realizados vários estudos empregando UCPs de plantas (pUCP). Inicialmente, foi empreendida a caracterização bioquímica de uma UCP de milho (Zea mays; ZmUCP), representativa de espécies monocotiledôneas. Essa proteína, quando expressa em Escherichia coli e reconstituída em lipossomos, foi capaz de induzir um fluxo de prótons dependente de ácido linoléico (LA) e sensível a ATP com valores de Km, Vmax e Ki similares àqueles observados para pUCPs de espécies dicotiledôneas. A ZmUCP foi utilizada para investigar a importância de um par de histidinas presente no segundo loop matricial da UCP1 de mamíferos e ausente nas pUCPs. A introdução do referido par de histidinas na ZmUCP (Lys155His e Ala157His) provocou um aumento na afinidade por LA enquanto que a sua atividade permaneceu inalterada. Em um estudo mais abrangente de estrutura-função, mutações pontuais nos resíduos Lys147, Arg155 e Tyr269, localizados nas chamadas assinaturas das UCPs, e Cys28 e His83, específicos para pUCPs, foram introduzidas na proteína AtUCP1 de Arabidopsis. Os efeitos de tais mutações nas propriedades bioquímicas da AtUCP1 foram examinados usando sistema de reconstituição em lipossomos. O resíduo Arg155 parece ser crucial para a afinidade da AtUCP1 por LA enquanto que His83 tem importante função na atividade de transporte. Os resíduos Cys28, Lys147, e também Tyr269, são importantes para a correta funcionalidade da AtUCP1, já que suas substituições...
Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins, which uncouple respiration from ATP synthesis. UCPs mediate a fatty acid (FA)-dependent, purine nucleotide (PN)-inhibitable proton flux across the inner membrane mitochondrial. In the present study, several assays on plant UCPs (pUCP) were performed. Firstly, we biochemically characterized an UCP from maize (Zea mays; ZmUCP), a representative uncoupling protein from monocot species. This protein was expressed in Escherichia coli and reconstituted in liposomes. ZmUCP was fully active and induced a linoleic acid-dependent proton flux that was sensitive to ATP. The obtained Km, Vmax and Ki values were similar to those observed for dicot pUCPs. ZmUCP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in pUCPs. ZmUCP with the introduced histidine pair (Lys155His and Ala157His) displayed increased LA-affinity while its activity remained unchanged. In a subsequent study using AtUCP1, point mutations were introduced in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and Cys28 and His83, specific for pUCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted liposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to LA whereas His83 plays an important role in transport activity. Residues Cys28, Lys147, and also Tyr269 are important for correct AtUCP1 function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to PN inhibitors. Furthermore, we analyzed the expression profiles of the six genes encoding pUCP in Arabidopsis thaliana (AtUCP1-6) in response to salt (NaCl) and osmotic (mannitol)... (Complete abstract click electronic access below)
Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins, which uncouple respiration from ATP synthesis. UCPs mediate a fatty acid (FA)-dependent, purine nucleotide (PN)-inhibitable proton flux across the inner membrane mitochondrial. In the present study, several assays on plant UCPs (pUCP) were performed. Firstly, we biochemically characterized an UCP from maize (Zea mays; ZmUCP), a representative uncoupling protein from monocot species. This protein was expressed in Escherichia coli and reconstituted in liposomes. ZmUCP was fully active and induced a linoleic acid-dependent proton flux that was sensitive to ATP. The obtained Km, Vmax and Ki values were similar to those observed for dicot pUCPs. ZmUCP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in pUCPs. ZmUCP with the introduced histidine pair (Lys155His and Ala157His) displayed increased LA-affinity while its activity remained unchanged. In a subsequent study using AtUCP1, point mutations were introduced in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and Cys28 and His83, specific for pUCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted liposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to LA whereas His83 plays an important role in transport activity. Residues Cys28, Lys147, and also Tyr269 are important for correct AtUCP1 function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to PN inhibitors. Furthermore, we analyzed the expression profiles of the six genes encoding pUCP in Arabidopsis thaliana (AtUCP1-6) in response to salt (NaCl) and osmotic (mannitol)... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Genetica vegetal, Proteínas desacopladoras, Uncoupling proteins
Como citar
FÁVARO, Regiane Degan. Análise bioquímica, estudos da relação estrutura-função e de expressão da proteína desacopladora mitocondrial de plantas. 2008. 80 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Instituto de Biociências de Botucatu, 2008.