Browsing by Author "Valentim-Coelho, Cristina"
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- Diabetes mellitus tipo 2 (DMT2) associada a Sindrome de Apneia Obstrutiva do Sono (SAOS): um estudo proteómicoPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Penque, Deborah; Barbara, CristinaIntrodução: A prevalência da SAOS é elevada em doentes com DMT2. O não tratamento da SAOS pode levar ao agravamento ou desenvolvimento da DMT2. Temos vindo a demonstrar que a SAOS altera o proteoma do glóbulo vermelho (GV). A SAOS aumenta a overoxidação da peroxirredoxina 2 (PRDX2) (enzima antioxidante), o que pode levar à desregulação da homeostasia do GV e ao desenvolvimento de doenças metabólicas. Após tratamento com ventilação não invasiva (PAP), esta overoxidação diminuiu seguida de um aumento de PRDX2 decamérica overoxidada com funções chaperone na proteção celular (Feliciano et al. 2017). No presente estudo, fomos investigar o estado redox/oligomérico da PRDX2 em doentes DMT2 com SAOS, antes/após PAP, para melhor compreender a interligação entre estas patologias. Material e métodos: Amostras de GVs de controles (n=22 sendo 3 DMT2) e doentes SAOS antes/após 6 meses de tratamento com PAP (n=29 sendo 8 DMT2) foram analisadas por western-blot não reduzido, com anticorpo para a PRDX2 e PRDXSO2/3 (overoxidada). Os grupos foram comparados estatisticamente e correlacionados com dados clínicos e bioquímicos. Resultados: Nos GVs de doentes DMT2/SAOS, o nível de monómeros da PRDX2 mostrou-se aumentado e diminuía após PAP. Contudo, o nível destes monómeros PRDXSO2/3 estava diminuído e não se alterou com o tratamento. Após PAP, o nível de decâmeros PRDX2SO2/3 foi também menor nestes doentes. Os níveis de monómeros PRDX2 e PRDX2SO2/3 correlacionaram-se negativamente com os níveis de insulina/triglicéridos e HbA1C, respetivamente. Após PAP, os níveis de decâmeros PRDX2SO2/3 correlacionou-se positivamente com os níveis de adrenalina. Conclusões: O estado redox/oligomérico da PRDX2 do GV é diferencialmente modulado nos doentes DTM2/SAOS em comparação com doentes SAOS. Decâmeros PRDXSO2/3 induzidos pelo tratamento e associadas à função protetora “chaperone” estão diminuídos em doentes DMT2/SAOS. O impacto clínico destas descobertas, necessita de mais investigação e validação.
- Evening and morning alterations in Obstructive Sleep Apnea red blood cell proteomePublication . Feliciano, Amélia; Vaz, Fátima; Valentim-Coelho, Cristina; Torres, Vukosava M.; Silva, Rita; Prosinecki, Vesna; Alexandre, Bruno M.; Almeida, Andreia; Almeida-Marques, Catarina; Carvalho, Ana S.; Matthiesen, Rune; Malhotra, Atul; Pinto, Paula; Bárbara, Cristina; Penque, DeborahThis article presents proteomics data referenced in [1] Using proteomics-based evaluation of red blood cells (RBCs), we have identified differentially abundant proteins associated with Obstructive Sleep Apnea Syndrome (OSA). RBCs were collected from peripheral blood of patients with moderate/severe OSA or snoring at pre- (evening) and post-night (morning) polysomnography, so that proteome variations between these time points could be assessed. RBC cytoplasmic fraction depleted of hemoglobin, using Hemovoid(™) system, were analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), the 2D image software-based analyzed and relevant differentially abundant proteins identified by mass spectrometry (MS). MS identified 31 protein spots differentially abundant corresponding to 21 unique proteins possibly due to the existence of post-translational modification regulations. Functional analysis by bioinformatics tools indicated that most proteins are associated with catalytic, oxidoreductase, peroxidase, hydrolase, ATPase and anti-oxidant activity. At morning a larger numbers of differential proteins including response to chemical stimulus, oxidation reduction, regulation of catalytic activity and response to stress were observed in OSA. The data might support further research in OSA biomarker discovery and validation.
- Evening and morning peroxiredoxin-2 redox/oligomeric state changes in obstructive sleep apnea red blood cells: Correlation with polysomnographic and metabolic parametersPublication . Feliciano, Amélia; Vaz, Fátima; Torres, Vukosava M.; Valentim-Coelho, Cristina; Silva, Rita; Prosinecki, Vesna; Alexandre, Bruno M.; Carvalho, Ana S.; Matthiesen, Rune; Malhotra, Atul; Pinto, Paula; Bárbara, Cristina; Penque, DeborahWe have examined the effects of Obstructive Sleep Apnea (OSA) on red blood cell (RBC) proteome variation at evening/morning day time to uncover new insights into OSA-induced RBC dysfunction that may lead to OSA manifestations. Dysregulated proteins mainly fall in the group of catalytic enzymes, stress response and redox regulators such as peroxiredoxin 2 (PRDX2). Validation assays confirmed that at morning the monomeric/dimeric forms of PRDX2 were more overoxidized in OSA RBC compared to evening samples. Six month of positive airway pressure (PAP) treatment decreased this overoxidation and generated multimeric overoxidized forms associated with chaperone/transduction signaling activity of PRDX2. Morning levels of overoxidized PRDX2 correlated with polysomnographic (PSG)-arousal index and metabolic parameters whereas the evening level of disulfide-linked dimer (associated with peroxidase activity of PRDX2) correlated with PSG parameters. After treatment, morning overoxidized multimer of PRDX2 negatively correlated with fasting glucose and dopamine levels. Overall, these data point toward severe oxidative stress and altered antioxidant homeostasis in OSA RBC occurring mainly at morning time but with consequences till evening. The beneficial effect of PAP involves modulation of the redox/oligomeric state of PRDX2, whose mechanism and associated chaperone/transduction signaling functions deserves further investigation. RBC PRDX2 is a promising candidate biomarker for OSA severity and treatment monitoring, warranting further investigation and validation.
- Impacto do fumo do cigarro passivo no proteoma humano: em busca de biomarcadores precoces de risco para a saúdePublication . Neves, Sofia; Pacheco, Solange A.; Vaz, Fátima; Valentim-Coelho, Cristina; Saraiva, Joana; James, Peter; Simões, Tânia; Penque, DeborahOs não-fumadores expostos ao fumo do cigarro passivo ou, simplesmente fumo passivo (FP), apresentam um risco acrescido de desenvolver diversas doenças graves. No entanto, os mecanismos moleculares que explicam estes efeitos continuam pouco esclarecidos, o que reforça a necessidade de identificar biomarcadores capazes de avaliar o risco associado a esta exposição. Neste estudo, analisámos o proteoma do epitélio nasal e do plasma de indivíduos não-fumadores saudáveis expostos ao FP no local de trabalho, num contexto ainda enquadrado pela Lei n.º 37/2007, utilizando uma abordagem proteómica ‘shotgun’ por espectrometria de massa. No epitélio nasal, observámos um aumento de proteínas envolvidas em vias centrais do metabolismo energético, como a Gliceraldeído-3-fosfato desidrogenase (GAPDH) e a Triosefosfato isomerase (TPI1), sugerindo uma possível reprogramação metabólica induzida pela exposição. Identificámos também uma diminuição da tubulina beta-4B (TUBB4B), relacionada com a organização do citoesqueleto, e um aumento da proteína anti-apoptótica SERPINB3, apontando para alterações em processos de morte e sobrevivência celular. No plasma, destacaram-se o aumento da Butirilcolinesterase (BChE) e a diminuição da Proteína de ligação à vitamina D (GC), ambas associadas à resposta a xenobióticos e a processos de lesão tecidular. Foram ainda detetadas alterações em proteínas reguladoras da inflamação sistémica, como C1R, C1QC, HRG e PROS1. A expressão diferencial de APOA4 e SERPINF2 sugere, adicionalmente, a ativação de mecanismos relacionados com risco aterotrombótico. Em conjunto, estes resultados contribuem para aprofundar a compreensão das vias biológicas que ligam a exposição ao fumo passivo ao risco acrescido de cancro e de doenças cardiovasculares, e apresentam um conjunto promissor de potenciais biomarcadores para avaliação do risco associado à exposição ao FP.
- Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic studyPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Feliciano, Amelia; Antunes, Marília; Pinto, Paula; Barbara, Cristina; Penque, DeborahBackground: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.
- Obstructive Sleep Apnea: a proteomics study of the effects of positive airway pressure therapyPublication . Valentim-Coelho, Cristina; Vaz, Fátima; Martins, Inês L.; Feliciano, Amélia; Pinto, Paula; Cristina, Bárbara; Penque, DeborahObstructive Sleep Apnea (OSA) syndrome is a common public health concern characterized by recurrent episodes of apneas and hypopneas during sleep. These obstructive events result in recurrent intermittent hypoxia and sleep fragmentation that can lead to metabolic and cardiovascular diseases. We recently demonstrated that OSA can cause alterations in the red blood cells (RBC) proteome that may be associated with OSA outcomes1,2. Here we intend to investigate whether the first-line therapy for OSA, the positive airway pressure (PAP) can revert or modulate these proteome alterations. RBCs from Snorers and patients with severe OSA before/after 6 months of PAP treatment (n=10/condition) were depleted of hemoglobin, analyzed by 2D-DIGE using Progenesis SameSpotsv4.5. The differentially abundant proteins were identified by MALDI-MS/MS and protein annotations acquired by DAVIDv6.8. Western blotting (WB) validation was performed for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and (overoxidized) GAPDHSO3 on a new Cohort (n=59). SPSS software was used to correlation studies with peroxiredoxin-2 (PRDX2) redox-oligomeric forms and several clinical parameters. Ten protein spots showed significant differences (Anova p<0.05) among groups and were associated with cell death, protein oligomerization and response to stress. Three proteoforms of GAPDH were identified decreased in OSA RBC (Anova p<0.05) and 6 months of PAP treatment increased these GAPDH proteoforms to the control levels. By WB, we confirmed these data by showing that the decreased GAPDH monomeric/tetrameric forms in OSA were increased by PAP treatment. PAP also increased GAPDHSO3 tetramers. In OSA, GAPDH monomers and GAPDHSO3 tetramers correlated positively with the respiratory disturbance index or triglycerides and adrenalin, respectively. After PAP, GAPDHSO3 tetramers correlated positively with PAP-induced PRDX2SO2/3 decameric forms, described having chaperone activity in cell protection. OSA induces alterations in the redox/oligomeric state of GAPDH and PRDX2 that can be reverted/modulated by PAP therapy. The clinical significant of these findings needs further validation and investigation.
- Proteomics of Obstructive Sleep Apnea with type 2 Diabetes MellitusPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Penque, DeborahBackground: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induces alterations in red blood cell (RBC) proteome (Feliciano et al 2017). Herein, we aimed to investigate redox state of PRDX2 in OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment to better understand the basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Non-reducing SDS-PAGE coomassie gel band corresponding to PRDX2 monomers from the different groups where trypsinized and investigation by LC/MS/MS for additional post-translational modifications (PTMs) are under process. Results: We confirmed previously data showing higher overoxidation on monomeric forms of PRDX2 in OSA RBC without comorbidity compared to Snorer controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although the most abundant its overoxidation level was much lower than OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of PRDX2 overoxidized monomeric/dimeric forms in RBC correlated negatively with levels of insulin / triglycerides and HbA1C, respectively.After PAP, PRDX2SO2 / 3 decamer levels correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 regulated by overoxidation of the active cysteines were differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 associated with chaperone protective function showed decreased in OSA patients with diabetes. Preliminary data pointed to polyglutamylation on PRDX2 glutamic acid91 that can be differentially modulated in OSA with and without diabetes and/or in response to PAP treatment. The clinical impact of these findings needs further investigation and validation. My perspective future encompasses the complete identification of these PTM by MS based strategies and validation by SRM approaches.
- Redox–Oligomeric State of Peroxiredoxin-2 and Glyceraldehyde-3-Phosphate Dehydrogenase in Obstructive Sleep Apnea Red Blood Cells under Positive Airway Pressure TherapyPublication . Valentim-Coelho, Cristina; Vaz, Fátima; Antunes, Marília; Neves, Sofia; Martins, Inês L.; Osório, Hugo; Feliciano, Amélia; Pinto, Paula; Bárbara, Cristina; Penque, DeborahIn this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox-oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox-oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone "gain" of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.