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Validação de potenciais biomarcadores proteómicos na Síndrome da Apneia Obstrutiva do Sono
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Advisor(s)
Abstract(s)
[PT] A Síndrome da Apneia Obstrutiva do Sono (SAOS) é um distúrbio respiratório muito
prevalente na população em geral, com graves repercussões na saúde e na qualidade de
vida dos doentes. A Proteómica tem vindo a ser aplicada no estudo da SAOS na pesquisa
de novos biomarcadores de diagnóstico, prognóstico, monitorização e/ou alvo
terapêutico para esta doença. Estudos preliminares realizados no Laboratório de
Proteómica do INSARJ revelaram a existência de proteínas diferencialmente moduladas
em glóbulos vermelhos (GVs) de doentes com SAOS comparados com roncadores
simples. Algumas destas proteínas, como a catalase (CAT) e a peroxiredoxina 2 (Prx2),
estão envolvidas em processos antioxidantes, e este mecanismo parece estar
significativamente comprometido nos doentes com SAOS.
O principal objectivo deste trabalho foi a validação da CAT e da Prx2 por técnicas
ortogonais à Proteómica, como o western blotting (WB) e/ou por estudos de cinética
enzimática em amostras de GVs de doentes com SAOS, antes e após seis meses de
tratamento CPAP (Continuous Positive Airway Pressure), e em indivíduos roncadores
simples como controlos. Dois tipos de amostras de GVs foram avaliadas por doente,
amostra recolhida no final da tarde antes do exame de polissonografia – PSG
(denominadas de “Noite”) e amostra recolhida na manhã seguinte ao exame
(denominadas de “Manhã”). Após tratamento CPAP, apenas as amostras de GVs
“Manhã” foram preparadas e, portanto, consideradas no estudo.
O estudo da CAT, por cinética enzimática, revelou que a actividade da CAT é
significativamente menor nos GVs “Manhã” em comparação com as amostras “Noite”,
sejam de doentes SAOS ou de controlos (Anova p<0,05). Contudo, esta diminuição da
actividade da CAT durante a manhã em relação à noite estava significativamente (T
Student, p<0,05) mais acentuada nos doentes com SAOS do que nos controlos. Após seis
meses de tratamento com CPAP, observou-se um aumento muito significativo da
actividade da CAT nos GVs “Manhã”. Surpreendentemente, constatou-se por WB, não
haver diferenças quantitativas e significativas dos níveis de expressão da CAT entre os
grupos de doentes/condições analisados, e tão pouco havia, nos doentes SAOS, antes e
após o tratamento CPAP. Estes resultados sugerem que a modulação da atividade da
CAT nos GVs está essencialmente associada a modificações pós-traducionais da
XI
proteína, o que corrobora com os resultados obtidos pela Proteómica, que identificou
oito proteoformas para a CAT nestas células.
A avaliação do estado redox/oligomérico da Prx2 nos GVs por WB em SDS-PAGE não
reduzido, utilizando anticorpos específicos para a Prx2 e para formas hiperoxidadas da
Prx (PrxSO2/3), revelou que as formas monoméricas e diméricas da Prx2 estavam
significativamente hiperoxidadas nos GVs “Manhã” ou “Noite” de doentes com SAOS. O
tratamento CPAP diminui esta hiperoxidação nos monómeros e dímeros da Prx2.
Contudo, as formas multiméricas da Prx2 hiperoxidadas, descritas com actividade
chaperone/transdução de sinal associadas à protecção celular, surgiram quase que
exclusivamente nos doentes SAOS após CPAP.
A forma dissulfídica oxidada/hiperoxidada da Prx2 está associada a inativação da sua
actividade peroxidática, e por conseguinte, a estado de stresse oxidativo. A conversão
entre as diferentes funções da Prx2, ou seja, entre a actividade peroxidática
antioxidante e a atividade chaperone/transdução de sinal, é regulada pelo seu estado
redox/oligomérico na célula.
Em resumo, os resultados deste trabalho validaram os resultados anteriormente
obtidos por Proteómica, indicando nos GVs de doentes com SAOS, existência de
desregulação no seu sistema redox-homeostase envolvendo a CAT e a Prx2. Este
trabalho propõe ainda, a CAT e a Prx2, como fortes candidatos a biomarcadores de
gravidade e/ou de monitorização do tratamento CPAP. Contudo, serão necessários mais
estudos para consolidar este resultados e contribuir para uma melhor compreensão
acerca do diagnóstico, monitorização e tratamento para a SAOS.
[EN] The Obstructive Sleep Apnea (OSA) is a breathing disorder very prevalent in the general population, with serious effects on health and quality of life of the patients. The Proteomics has been applied to the study of the OSA, in order to identify new biomarkers of diagnosis, prognosis, monitoring and/or as therapeutic target for this disease. Preliminary studies at Proteomics Laboratory of INSARJ revealed the existence of proteins differentially modulated in red blood cells (RBCs) of OSA patients compared with simple snorers (controls). Some of these proteins such as catalase (CAT) and peroxiredoxin 2 (Prx2), are involved in antioxidants processes, which seem to be significantly compromised in OSA patients. The main objective of this work was the validation of these proteins by orthogonal technologies to Proteomics, such as western blotting (WB) and/or kinetic enzyme assay on RBCs samples from OSA patients, before and after six months of treatment CPAP (Continuous Positive Airway Pressure), and from simple snorers as controls. Two types of RBCs samples were evaluated per patient, sample taken late in the afternoon and before polysomnography (PSG) (denominated here "Night") and sample collected in the morning after the examination ("Morning"). After CPAP treatment, only "Morning" RBC samples were prepared and therefore considered in this study. The kinetic enzyme assay showed that CAT activity is significantly higher in RBC "Morning" compared to the "Night" samples, whatever were OSA or control (Anova p <0.05). However, this decrease in “Morning” CAT activity compared to “Night” was even greater in OSA RBC samples than in controls ones(Student's T p <0.05). After six months of CPAP treatment, there was a very significant increase in CAT activity in this "Morning" RBC OSA samples. Surprisingly, no significant differences were observed in the CAT expression level among the patient’s group/condition analysed. These results suggest that the modulation of CAT activity in RBCs is mainly associated with post-translational modifications, which supports the previous proteomics data identifying eight proteoforms for CAT in these cells. The evaluation of redox/oligomeric study of the Prx2 in RBCs by WB in SDS-PAGE, non-reduced, using antibodies specific for Prx2 and hyperoxidized forms of Prx (PrxSO2/3) demonstrated that the monomeric and dimeric form of Prx2 were significantly hyperoxidized in “Morning” or “Night” OSA RBCs. The CPAP treatment reduced this XIII hyperoxidation observed on monomers and dimers of Prx2. However, hyperoxidized multimeric forms of Prx2, described with chaperone activity, appeared almost exclusively in OSA patients after CPAP. The disulfide oxidized/hiperoxidized forms of Prx2 are associated with the inactivation of its peroxidatic activity and therefore with the state of stress-oxidative. The conversion between the different functions of Prx2, i.e. between antioxidant peroxidatic activity and chaperone activity or transduction signaling, is regulated by its redox/oligomeric state in the cell. In summary, the results of this study validated the results previously obtained by proteomics, indicating that in OSA RBCs the redox- homeostasis system involving the CAT and Prx2 is dysregulated. This work also proposes CAT and Prx2 as a promising candidates biomarkers of severity and/or to monitor CPAP treatment. However, more studies will be needed to consolidate this results and contribute to a better understanding of OSA diagnosis and therapy monitoring.
[EN] The Obstructive Sleep Apnea (OSA) is a breathing disorder very prevalent in the general population, with serious effects on health and quality of life of the patients. The Proteomics has been applied to the study of the OSA, in order to identify new biomarkers of diagnosis, prognosis, monitoring and/or as therapeutic target for this disease. Preliminary studies at Proteomics Laboratory of INSARJ revealed the existence of proteins differentially modulated in red blood cells (RBCs) of OSA patients compared with simple snorers (controls). Some of these proteins such as catalase (CAT) and peroxiredoxin 2 (Prx2), are involved in antioxidants processes, which seem to be significantly compromised in OSA patients. The main objective of this work was the validation of these proteins by orthogonal technologies to Proteomics, such as western blotting (WB) and/or kinetic enzyme assay on RBCs samples from OSA patients, before and after six months of treatment CPAP (Continuous Positive Airway Pressure), and from simple snorers as controls. Two types of RBCs samples were evaluated per patient, sample taken late in the afternoon and before polysomnography (PSG) (denominated here "Night") and sample collected in the morning after the examination ("Morning"). After CPAP treatment, only "Morning" RBC samples were prepared and therefore considered in this study. The kinetic enzyme assay showed that CAT activity is significantly higher in RBC "Morning" compared to the "Night" samples, whatever were OSA or control (Anova p <0.05). However, this decrease in “Morning” CAT activity compared to “Night” was even greater in OSA RBC samples than in controls ones(Student's T p <0.05). After six months of CPAP treatment, there was a very significant increase in CAT activity in this "Morning" RBC OSA samples. Surprisingly, no significant differences were observed in the CAT expression level among the patient’s group/condition analysed. These results suggest that the modulation of CAT activity in RBCs is mainly associated with post-translational modifications, which supports the previous proteomics data identifying eight proteoforms for CAT in these cells. The evaluation of redox/oligomeric study of the Prx2 in RBCs by WB in SDS-PAGE, non-reduced, using antibodies specific for Prx2 and hyperoxidized forms of Prx (PrxSO2/3) demonstrated that the monomeric and dimeric form of Prx2 were significantly hyperoxidized in “Morning” or “Night” OSA RBCs. The CPAP treatment reduced this XIII hyperoxidation observed on monomers and dimers of Prx2. However, hyperoxidized multimeric forms of Prx2, described with chaperone activity, appeared almost exclusively in OSA patients after CPAP. The disulfide oxidized/hiperoxidized forms of Prx2 are associated with the inactivation of its peroxidatic activity and therefore with the state of stress-oxidative. The conversion between the different functions of Prx2, i.e. between antioxidant peroxidatic activity and chaperone activity or transduction signaling, is regulated by its redox/oligomeric state in the cell. In summary, the results of this study validated the results previously obtained by proteomics, indicating that in OSA RBCs the redox- homeostasis system involving the CAT and Prx2 is dysregulated. This work also proposes CAT and Prx2 as a promising candidates biomarkers of severity and/or to monitor CPAP treatment. However, more studies will be needed to consolidate this results and contribute to a better understanding of OSA diagnosis and therapy monitoring.
Description
Dissertação de mestrado em Biologia Humana e Ambiente, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2015.
Trabalho de investigação desenvolvido no Laboratório de Proteómica do Instituto Ricardo Jorge.
Deborah Penque (orientadora), Fátima Vaz (co-orientadora) - Laboratório de Proteómica do Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge I.P.; Maria Teresa Rebelo (orientadora interna) - Faculdade de Ciências da Universidade de Lisboa.
Trabalho de investigação desenvolvido no Laboratório de Proteómica do Instituto Ricardo Jorge.
Deborah Penque (orientadora), Fátima Vaz (co-orientadora) - Laboratório de Proteómica do Departamento de Genética Humana do Instituto Nacional de Saúde Doutor Ricardo Jorge I.P.; Maria Teresa Rebelo (orientadora interna) - Faculdade de Ciências da Universidade de Lisboa.
Keywords
Apneia Sono Proteómica Doenças Genéticas Síndrome da Apneia Obstrutiva do Sono Pressão Positiva Contínua das Vias Aéreas Biomarcadores Catalase Genomica Funcional Genómica Funcional e Estrutural Peroxiredoxina 2 Continuous Positive Airway Pressure Obstructive Apnea Syndrome Proteomics Biomarkers Peroxiredoxin 2