Avaliação funcional dos inibidores químicos da NAD(P)+ transidrogenase mitocondrial [recurso eletrônico] = Functional evaluation of chemical inhibitors of the mitochondrial NAD(P)+ transhydrogenase
Rafaela Bicego
DISSERTAÇÃO
Multilíngua
T/UNICAMP B471a
[Functional evaluation of chemical inhibitors of the mitochondrial NAD(P)+ transhydrogenase ]
Campinas, SP : [s.n.], 2021.
1 recurso online ( 60 p.) : il., digital, arquivo PDF.
Orientadores: Roger Frigério Castilho, Annelise Francisco
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Resumo: A NAD(P)+ transidrogenase mitocondrial, também conhecida como transidrogenase de nucleotídeos de nicotinamida (NNT), é uma proteína integral da membrana mitocondrial interna e catalisa uma reação reversível que consiste na transferência de um hidreto entre NAD(H) e NADP(H) acoplada ao...
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Resumo: A NAD(P)+ transidrogenase mitocondrial, também conhecida como transidrogenase de nucleotídeos de nicotinamida (NNT), é uma proteína integral da membrana mitocondrial interna e catalisa uma reação reversível que consiste na transferência de um hidreto entre NAD(H) e NADP(H) acoplada ao transporte de um próton entre o espaço intermembranar e a matriz mitocondrial. Em condições fisiológicas, a reação da NNT ocorre predominantemente no sentido da redução de NADP+ (i.e., reação direta), fazendo com que essa enzima seja uma importante fonte mitocondrial de NADPH, o qual é utilizado por sistemas antioxidantes da organela e em vias de biossíntese. A supressão da atividade da NNT tem relevância não só como abordagem experimental para avaliação da contribuição desta enzima em diferentes contextos fisiológicos e patológicos, como também consiste em uma possível abordagem terapêutica em condições patológicas específicas. A inibição da atividade NNT pode ser obtida por modificações moleculares como silenciamento de sua expressão, mutações ou com o uso de inibidores químicos. O presente trabalho objetivou avaliar a eficácia de alguns dos principais compostos propostos como inibidores químicos da NNT, diante de possíveis efeitos inespecíficos destes sobre a respiração mitocondrial. Foram avaliados os compostos: 4-cloro-7-nitrobenzofuran (NBD-Cl), N,N?-diciclo-hexilcarbodiimida (DCC), palmitoil-CoA, palmitoil-L-carnitina e rhein. Diferentes concentrações desses compostos foram testadas em mitocôndrias isoladas de fígado de camundongo quanto a (1) capacidade de inibir as reações direta e reversa da NNT em meio contendo detergente, e (2) efeitos sobre a função respiratória de mitocôndrias intactas, estimada pelo consumo de oxigênio estimulado por ADP e pelo estado respiratório não-fosforilante após adição de oligomicina. De forma geral, concentrações destes compostos capazes de gerar uma inibição parcial das reações direta e reversa da NNT em mitocôndrias, ocasionaram um prejuízo significativo na função respiratória mitocondrial. Dentre os compostos testados, o NBD-Cl mostrou uma melhor relação entre a inibição das reações da NNT e menor impacto sobre a função respiratória mitocondrial. Tendo em vista esse resultado, foram realizados testes adicionais com NBD-Cl em cultura primária de astrócitos sem NNT funcional, onde foi feita a contagem de células viáveis, medida da metabolização de MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazolium) e dosagem de ATP após 48 h de exposição dessa cultura a diferentes concentrações de NDB-Cl. Nesses experimentos, as concentrações de NBD-Cl que causaram somente uma inibição parcial das reações direta e reversa da NNT (i.e., 10 e 20 µM) diminuíram significativamente a proliferação e viabilidade de astrócitos, demonstrando haver um efeito tóxico do NBD-Cl. Em outro conjunto de experimentos, observou-se que o NBD-Cl inibiu parcialmente a capacidade de mitocôndrias isoladas de fígado de camundongos com NNT intacta metabolizarem o peroxido orgânico tert-butil hidroperóxido, o que pode ser devido a inibição da NNT, mas também devido a efeitos tóxicos e inibitórios sobre outras proteínas mitocondriais. Os dados aqui apresentados permitem concluir que, embora os compostos testados realmente tenham apresentado efeitos inibitórios sobre as reações direta e reversa da NNT, concentrações efetivas desses compostos causam efeitos indesejáveis significativos sobre a função respiratória mitocondrial e viabilidade celular.
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Abstract: The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the...
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Abstract: The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the intermembrane space and mitochondrial matrix. In most physiological conditions, the NNT reaction occurs towards NADP+ reduction (i.e., forward reaction); because of that this enzyme is an important mitochondrial source of NADPH, which is used by antioxidant systems in the organelle and in biosynthetic pathways. The suppression of NNT activity is relevant not only as an experimental approach to assess the contribution of this enzyme in different physiological and pathological contexts, but also consists of a possible therapeutic approach in specific pathological conditions. The inhibition of NNT activity can be achieved by molecular modifications such as silencing its expression, mutations or with the use of chemical inhibitors. The present study aimed at evaluating the efficacy of the main chemical compounds proposed as NNT inhibitors in view of their possible effects on other mitochondrial components. The compounds 4-chloro-7-nitrobenzofuran (NBD-Cl), N,N?-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-L-carnitine and rhein were evaluated. Different concentrations of these compounds were tested in isolated mitochondria from mouse liver regarding (1) the ability to inhibit the direct and reverse reactions of NNT in detergent-solubilized mitochondria, and (2) effects on respiratory function of intact mitochondria, estimated by ADP-stimulated oxygen consumption and non-phosphorylating respiratory state due to oligomycin addition. In general, concentrations of these compounds capable of generating a partial inhibition of the direct and reverse activities of NNT in mitochondria, significantly impaired mitochondrial respiratory function. Among the compounds assayed, NBD-Cl showed a better relationship between NNT reaction inhibition and lower impact on mitochondrial oxygen consumption. In view of this result, additional experiments were carried out with NBD-Cl in a primary culture of astrocytes devoid of functional NNT, where it was evaluated cell number, metabolism of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP content after 48 h of exposure to different NDB-Cl concentrations. In these experiments, NBD-Cl concentrations able to partially inhibit the direct and reverse NNT reactions (i.e., 10 and 20 µM) significantly decreased the proliferation and viability of astrocytes, demonstrating that there is a toxic effect of NBD-Cl. In another set of experiments, NBD-Cl partially decreased the ability of isolated mouse liver mitochondria with intact NNT to metabolize the organic peroxide tert-butyl hydroperoxide, which might be explained by inhibition of NNT, but also can be due to toxicity and inhibitory effects on other mitochondrial proteins. The data presented here allow us to conclude that, although the tested compounds indeed presented inhibitory effects on the direct and reverse activities of NNT, at effective concentrations they caused significant undesirable effects on mitochondrial respiratory function and cell viability.
The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the intermembrane space and mitochondrial matrix. In most physiological conditions, the NNT reaction occurs towards NADP+ reduction (i.e., forward reaction); because of that this enzyme is an important mitochondrial source of NADPH, which is used by antioxidant systems in the organelle and in biosynthetic pathways. The suppression of NNT activity is relevant not only as an experimental approach to assess the contribution of this enzyme in different physiological and pathological contexts, but also consists of a possible therapeutic approach in specific pathological conditions. The inhibition of NNT activity can be achieved by molecular modifications such as silencing its expression, mutations or with the use of chemical inhibitors. The present study aimed at evaluating the efficacy of the main chemical compounds proposed as NNT inhibitors in view of their possible effects on other mitochondrial components. The compounds 4-chloro-7-nitrobenzofuran (NBD-Cl), N,N?-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-L-carnitine and rhein were evaluated. Different concentrations of these compounds were tested in isolated mitochondria from mouse liver regarding (1) the ability to inhibit the direct and reverse reactions of NNT in detergent-solubilized mitochondria, and (2) effects on respiratory function of intact mitochondria, estimated by ADP-stimulated oxygen consumption and non-phosphorylating respiratory state due to oligomycin addition. In general, concentrations of these compounds capable of generating a partial inhibition of the direct and reverse activities of NNT in mitochondria, significantly impaired mitochondrial respiratory function. Among the compounds assayed, NBD-Cl showed a better relationship between NNT reaction inhibition and lower impact on mitochondrial oxygen consumption. In view of this result, additional experiments were carried out with NBD-Cl in a primary culture of astrocytes devoid of functional NNT, where it was evaluated cell number, metabolism of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP content after 48 h of exposure to different NDB-Cl concentrations. In these experiments, NBD-Cl concentrations able to partially inhibit the direct and reverse NNT reactions (i.e., 10 and 20 µM) significantly decreased the proliferation and viability of astrocytes, demonstrating that there is a toxic effect of NBD-Cl. In another set of experiments, NBD-Cl partially decreased the ability of isolated mouse liver mitochondria with intact NNT to metabolize the organic peroxide tert-butyl hydroperoxide, which might be explained by inhibition of NNT, but also can be due to toxicity and inhibitory effects on other mitochondrial proteins. The data presented here allow us to conclude that, although the tested compounds indeed presented inhibitory effects on the direct and reverse activities of NNT, at effective concentrations they caused significant undesirable effects on mitochondrial respiratory function and cell viability. Ver menos
The mitochondrial NAD(P)+ transhydrogenase, also known as nicotinamide nucleotide transhydrogenase (NNT), is an integral protein of the inner mitochondrial membrane that catalyzes a reversible transfer of a hydride between NAD(H) and NADP(H) coupled to the transport of a proton between the intermembrane space and mitochondrial matrix. In most physiological conditions, the NNT reaction occurs towards NADP+ reduction (i.e., forward reaction); because of that this enzyme is an important mitochondrial source of NADPH, which is used by antioxidant systems in the organelle and in biosynthetic pathways. The suppression of NNT activity is relevant not only as an experimental approach to assess the contribution of this enzyme in different physiological and pathological contexts, but also consists of a possible therapeutic approach in specific pathological conditions. The inhibition of NNT activity can be achieved by molecular modifications such as silencing its expression, mutations or with the use of chemical inhibitors. The present study aimed at evaluating the efficacy of the main chemical compounds proposed as NNT inhibitors in view of their possible effects on other mitochondrial components. The compounds 4-chloro-7-nitrobenzofuran (NBD-Cl), N,N?-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-L-carnitine and rhein were evaluated. Different concentrations of these compounds were tested in isolated mitochondria from mouse liver regarding (1) the ability to inhibit the direct and reverse reactions of NNT in detergent-solubilized mitochondria, and (2) effects on respiratory function of intact mitochondria, estimated by ADP-stimulated oxygen consumption and non-phosphorylating respiratory state due to oligomycin addition. In general, concentrations of these compounds capable of generating a partial inhibition of the direct and reverse activities of NNT in mitochondria, significantly impaired mitochondrial respiratory function. Among the compounds assayed, NBD-Cl showed a better relationship between NNT reaction inhibition and lower impact on mitochondrial oxygen consumption. In view of this result, additional experiments were carried out with NBD-Cl in a primary culture of astrocytes devoid of functional NNT, where it was evaluated cell number, metabolism of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ATP content after 48 h of exposure to different NDB-Cl concentrations. In these experiments, NBD-Cl concentrations able to partially inhibit the direct and reverse NNT reactions (i.e., 10 and 20 µM) significantly decreased the proliferation and viability of astrocytes, demonstrating that there is a toxic effect of NBD-Cl. In another set of experiments, NBD-Cl partially decreased the ability of isolated mouse liver mitochondria with intact NNT to metabolize the organic peroxide tert-butyl hydroperoxide, which might be explained by inhibition of NNT, but also can be due to toxicity and inhibitory effects on other mitochondrial proteins. The data presented here allow us to conclude that, although the tested compounds indeed presented inhibitory effects on the direct and reverse activities of NNT, at effective concentrations they caused significant undesirable effects on mitochondrial respiratory function and cell viability. Ver menos
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Avaliação funcional dos inibidores químicos da NAD(P)+ transidrogenase mitocondrial [recurso eletrônico] = Functional evaluation of chemical inhibitors of the mitochondrial NAD(P)+ transhydrogenase
Rafaela Bicego
Avaliação funcional dos inibidores químicos da NAD(P)+ transidrogenase mitocondrial [recurso eletrônico] = Functional evaluation of chemical inhibitors of the mitochondrial NAD(P)+ transhydrogenase
Rafaela Bicego