Protein oxidation and circadian rhythmicity: towards the identification of specific protein targets
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2015-05-07
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O ritmo circadiano existe em todos os organismos e gera ritmos com uma periodicidade de 24 h em um significante número de processos biológicos e metabólicos. Uma disrupção do relógio circadiano (BMAL1 e PER) em ratos, está diretamente associada com o aumento das espécies reativas de oxigénio (ERO) que induzem um fenótipo de envelhecimento prematuro (Kondratov, 2007; Ramsey, et al., 2007). Por outro lado, foi descrito que a acumulação de proteínas oxidadas (carboniladas) é um marcador do envelhecimento celular. Para além disso, as proteinas carboniladas, com modificação irreversível, envolve uma diminuição da qualidade do proteoma celular que pode afectar diretamente a função da célula normal.
Através da utilização de células HEK293 sincronizadas com serum shock e utilização das técnicas de electroforese de ima dimensão, o nosso grupo de trabalho demonstrou que o nível das proteínas carboniladas tinham uma ritmicidade circadiana. Essa quantificação do total de proteínas levou-nos a escolher dois picos do ritmo circadiano, correspondentes ao máximo e mínimo de nível de carbonilação. Neste trabalho, utilizamos também a electroforese de duas dimensões para caracterizar algumas proteínas em particular que pareciam sofrer oxidação circadiana.
Após a quantificação das proteínas carboniladas, observou-se que os mesmos spots eram comuns em todos os tempos, mesmo se o nível de carbonilação é notavelmente diferente. Concluindo, nós provamos, embora sem proceder a uma caracterização final, que proteínas específicas possuem oxidação circadiana. No futuro, pode ser interessante identificar as proteínas alvo específicas e compará-las com os marcadores biológicos do stress oxidativo de forma a perceber melhor o envolvimento destas na ritmicidade circadiana.
The circadian rhythmicity exists in all organisms and generates rhythms with a periodicity of 24 h of a significant number of biological and metabolic processes. A disruption of the circadian clock (BMAL1 and PER2) in mice, are directly associated to an increase of reactive oxygen species (ROS) which induce a premature aging phenotype (Kondratov, 2007; Ramsey, et al., 2007). On the other hand, it was described that the accumulation of oxidized (carbonylated) proteins is a hallmark of cellular aging. Moreover, protein carbonylation, an irreversible modification, involves a decrease quality of the cellular proteome which could directly affect the normal cell function. Using HEK-293 cells synchronized with serum shock and the 1D electrophoresis technique, our working group showed that the level of carbonylated proteins has a circadian rhythmicity. This quantification of the total proteins allowed to choose two peaks of the circadian rhythm corresponding to maximum and minimum levels of protein carbonylation. In this work 2D electrophoresis were also used, to characterise some particular proteins, which seem to suffer a circadian oxidation. After quantification of carbonylated proteins, it was observed that the same spots are common all the times, even if their level of carbonylation is noticeably different. Concluding, it was proved, although without final characterization, that some specific proteins have a circadian oxidation. For the future, it will be interesting to identify these specific proteins and compare them to biological markers of oxidative stress in order to better understand the involvement of the circadian rhythm.
The circadian rhythmicity exists in all organisms and generates rhythms with a periodicity of 24 h of a significant number of biological and metabolic processes. A disruption of the circadian clock (BMAL1 and PER2) in mice, are directly associated to an increase of reactive oxygen species (ROS) which induce a premature aging phenotype (Kondratov, 2007; Ramsey, et al., 2007). On the other hand, it was described that the accumulation of oxidized (carbonylated) proteins is a hallmark of cellular aging. Moreover, protein carbonylation, an irreversible modification, involves a decrease quality of the cellular proteome which could directly affect the normal cell function. Using HEK-293 cells synchronized with serum shock and the 1D electrophoresis technique, our working group showed that the level of carbonylated proteins has a circadian rhythmicity. This quantification of the total proteins allowed to choose two peaks of the circadian rhythm corresponding to maximum and minimum levels of protein carbonylation. In this work 2D electrophoresis were also used, to characterise some particular proteins, which seem to suffer a circadian oxidation. After quantification of carbonylated proteins, it was observed that the same spots are common all the times, even if their level of carbonylation is noticeably different. Concluding, it was proved, although without final characterization, that some specific proteins have a circadian oxidation. For the future, it will be interesting to identify these specific proteins and compare them to biological markers of oxidative stress in order to better understand the involvement of the circadian rhythm.
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Dissertação de Mestrado em Bioquímica
Palavras-chave
Proteínas (Carboniladas) , Envelhecimento , Ciclo circadiano , Serum shock , Dexametazona