Influência do uso de hidroxicarbamida sobre o papel do sistema ubiquitina-proteassomo na homeostasia redox de células falcêmicas.
Data
2024-03-06
Autores
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A anemia falciforme (AF) é uma doença hemolítica caracterizada por uma série de complicações decorrentes do estresse oxidativo constante. Esse fenômeno desencadeia a produção exacerbada de pró-oxidantes, os quais promovem danos severos às macromoléculas celulares, culminando na formação de eritrócitos densos, com uma densidade superior a 1,11 g/mL. Sabe-se que o sistema ubiquitina-proteassomo (UPS) é um dos principais mecanismos de degradação de proteínas com anomalias estruturais ou com danos oxidativos, sendo fundamental para a homeostasia celular. Diante desse cenário, este estudo se propôs a investigar a relação entre o estresse oxidativo característico das células falciformes e a atividade do sistema UPS, com um enfoque especial na influência do tratamento com hidroxicarbamida (HC).Para tal, foram avaliados 9 indivíduos controles (CG), 10 com AF tratados (HC+) e 10 sem tratamento (HC-). Dos indivíduos com AF obteve-se, por centrifugação, os subconjuntos celulares de baixa densidade (LDSC – low dense sickle cells) e de alta densidade (HDSC - high dense sickle cells), associadas ao envelhecimento celular. A atividade proteassômica do tipo quimiotripsina (ChT-L) e tripsina (T-L) foram analisadas nos extratos proteicos sem purificação e purificados por coluna DEAE- Sepharose, enquanto a expressão do core do proteassomo 20S e imunoproteassomo foi avaliada por imunomarcação. Nossos resultados demonstraram, no conjunto completo de hemácias, um aumento da atividade T-L para o grupo HC- em relação ao CG (p<0,01). No entanto, este grupo apresentou a menor expressão do proteassomo 20S em relação aos demais grupos de estudo (p<0,01). Após a eliminação da hemoglobina e outras proteínas do eritrócito pela purificação, verificou-se uma mudança do perfil de atividade ChT-L, uma vez CG apresentou maior atividade em relação a HC+ e atividades semelhantes entre HC+ e HC- (p=0,04). O efeito do envelhecimento eritrocitário foi verificado, indicando um aumento na atividade Cht-L (p<0,01) e T-L (p<0,01) nos conjuntos LDSC-HC- em relação aos de HDSC-HC-. No extrato proteico purificado, notou-se a mudança do perfil da atividade, com o grupo CG apresentando os maiores valores de atividade, porém sem qualquer efeito estatisticamente significativo entre os subconjuntos celulares independente do uso de HC (p=0,22). Por fim, encontramos, alta expressão de subunidades do imunoproteassomo nas células do grupo HC-. Deste modo, embora a HC tenha demonstrado melhorar o controle de qualidade das proteínas nas células falciformes, esse efeito antioxidante não se traduziu em mudanças significativas na atividade proteassômica, independentemente do tipo de extrato proteico ou do envelhecimento celular. Além disso, fornecemos a evidência preliminar da presença do imunoproteassomo nas hemácias e a influência do tratamento com HC em sua expressão, sugerindo que a relação entre o tratamento com HC e o UPS na manutenção da homeostase redox nas células falcêmicas é muito mais complexa do que foi explorado até o momento.
Sickle cell anemia (SCA) is a hemolytic disease characterized by complications from constant oxidative stress. This phenomenon triggers the exacerbated production of pro-oxidants, which promote severe damage to cellular macromolecules, culminating in dense erythrocytes with a density of more than 1.11 g/mL. The ubiquitin-proteasome system (UPS) is known to be one of the primary mechanisms for the degradation of proteins with structural anomalies or oxidative damage and is essential for cellular homeostasis. Given this scenario, this study set out to investigate the relationship between the oxidative stress characteristic of sickle cells and the activity of the UPS system, with a special focus on the influence of treatment with hydroxycarbamide (HC). To this end, nine control individuals (CG), 10 treated AF individuals (HC+), and ten untreated individuals (HC-) were evaluated. The low-dense sickle cells (LDSC) and high-dense sickle cells (HDSC), associated with cellular aging, were obtained by centrifugation. Chymotrypsin-like (ChT-L) and trypsin-like (T-L) proteasome activity was analyzed in the protein extracts without purification and purified by DEAE-Sepharose column, while the expression of the 20S proteasome core and immunoproteasome was evaluated by immunolabeling. Our results showed an increase in T-L activity for the HC- group compared to the CG group (p<0.01). However, this group showed the lowest expression of the 20S proteasome compared to the other study groups (p<0.01). After the elimination of hemoglobin and other erythrocyte proteins by purification, there was a change in the ChT-L activity profile since CG showed higher activity compared to HC+ and similar activities between HC+ and HC- (p=0.04). The effect of erythrocyte aging was verified, indicating an increase in Cht-L (p<0.01) and T-L (p<0.01) activity in LDSC-HC- sets compared to HDSC-HC- sets. In the purified protein extract, there was a change in the activity profile, with the CG group showing the highest activity values but without any statistically significant effect between the cell subsets regardless of the use of HC (p=0.22). Finally, we found high expression of immunoproteasome subunits in the cells of the HC- group. Thus, although HC has been shown to improve protein quality control in sickle cells, this antioxidant effect has yet to translate into significant changes in proteasome activity, regardless of the type of protein extract or cell aging. In addition, we provide preliminary evidence of the presence of the immunoproteasome in RBCs and the influence of HC treatment on its expression, suggesting that the relationship between HC treatment and the UPS in maintaining redox homeostasis in sickle cells is much more complex than has been explored to date.
Sickle cell anemia (SCA) is a hemolytic disease characterized by complications from constant oxidative stress. This phenomenon triggers the exacerbated production of pro-oxidants, which promote severe damage to cellular macromolecules, culminating in dense erythrocytes with a density of more than 1.11 g/mL. The ubiquitin-proteasome system (UPS) is known to be one of the primary mechanisms for the degradation of proteins with structural anomalies or oxidative damage and is essential for cellular homeostasis. Given this scenario, this study set out to investigate the relationship between the oxidative stress characteristic of sickle cells and the activity of the UPS system, with a special focus on the influence of treatment with hydroxycarbamide (HC). To this end, nine control individuals (CG), 10 treated AF individuals (HC+), and ten untreated individuals (HC-) were evaluated. The low-dense sickle cells (LDSC) and high-dense sickle cells (HDSC), associated with cellular aging, were obtained by centrifugation. Chymotrypsin-like (ChT-L) and trypsin-like (T-L) proteasome activity was analyzed in the protein extracts without purification and purified by DEAE-Sepharose column, while the expression of the 20S proteasome core and immunoproteasome was evaluated by immunolabeling. Our results showed an increase in T-L activity for the HC- group compared to the CG group (p<0.01). However, this group showed the lowest expression of the 20S proteasome compared to the other study groups (p<0.01). After the elimination of hemoglobin and other erythrocyte proteins by purification, there was a change in the ChT-L activity profile since CG showed higher activity compared to HC+ and similar activities between HC+ and HC- (p=0.04). The effect of erythrocyte aging was verified, indicating an increase in Cht-L (p<0.01) and T-L (p<0.01) activity in LDSC-HC- sets compared to HDSC-HC- sets. In the purified protein extract, there was a change in the activity profile, with the CG group showing the highest activity values but without any statistically significant effect between the cell subsets regardless of the use of HC (p=0.22). Finally, we found high expression of immunoproteasome subunits in the cells of the HC- group. Thus, although HC has been shown to improve protein quality control in sickle cells, this antioxidant effect has yet to translate into significant changes in proteasome activity, regardless of the type of protein extract or cell aging. In addition, we provide preliminary evidence of the presence of the immunoproteasome in RBCs and the influence of HC treatment on its expression, suggesting that the relationship between HC treatment and the UPS in maintaining redox homeostasis in sickle cells is much more complex than has been explored to date.
Descrição
Palavras-chave
Anemia Falciforme, Hidroxiureia, Proteassomo, Sickle cell anemia, Hydroxyurea, Proteasome
Como citar
ZUCÃO, Ana Clara Albertin. Influência do uso de hidroxicarbamida sobre o papel do sistema ubiquitina-proteassomo na homeostasia redox de células falcêmicas.. 2024. 58 f. Dissertação (Mestrado em Biociências ) – Universidade Estadual Paulista (Unesp), Instituto de Biociências Letras e Ciências Exatas (Ibilce), São José do Rio Preto, 2024.