Publicação
Chronic obstructive pulmonary disease : a proteomics approach
| Resumo: | Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation that is not fully reversible even under bronchodilators effect, caused by a mixture of small airway disease – obstructive bronchiolitis – and parenchymal destruction – emphysema. At the present time, COPD is the fourth leading cause of death and its prevalence and mortality are expected to continue increasing in next decade. Spirometry is the most reproducible way to measure lung function and is nowadays the best tool to diagnose airflow limitation and, consequently, diagnose COPD itself. Biomarkers for diagnosis and/or prognosis as well as novel targets for the development of more effective therapies for COPD are still needed. Proteomics has the capacity to provide large-scale information and consequently it has the potential to expand previous knowledge on COPD. Surprisingly, given the need for new biomarkers in COPD and the power of proteomics, proteomics have been quite neglected so far. Up to date only 50 reports (14 are reviews) match the search at Pubmed (http://www.ncbi.nlm.nih.gov/pubmed, accessed June 23, 2011) for COPD proteomics. Hence, there is a clear need to engage clinically valuable proteomics studies in order to match the need for new biomarkers in COPD. In last decade, the shotgun proteomics approach has become the method of choice for identifying and quantifying proteins in most large-scale studies. Compared with 2DE, shotgun proteomics allows higher data throughput and better protein detection sensitivity. This strategy is based on trypsin digestion of proteins into peptides. This produces a complex peptide mixture that is then separated by one- or multiple dimensional liquid chromatography (LC) and subjected to peptide sequencing using tandem mass spectrometry (MS/MS) before automated database searching. In the present work we have employed different methodologies within shotgun proteomics to generate solid and comprehensive data in COPD. There are quite a lot biological materials that can be used to investigate biomarkers for this disease. Although COPD is now known to possess a systemic inflammation xii component which is responsible for affecting other organs, it is in the lung that the events that lead to breathless take place. Investigating to the lung directly is therefore an optimal strategy to be able to identify proteins that may not be detectable elsewhere either because they are not present or diluted into undetectable concentrations. But this means that lung tissue has to be collected by biopsy which is an extremely invasive technique. But besides tissue biospecimens, other sources of biological materials used to study COPD is biofluids which includes sputum, bronchoalveolar and nasal lavage fluid, exhaled breath condensate, and blood. It had been observed before by means of microscopy that red blood cells (RBCs) from COPD patients showed deformations in their shape. RBCs are crucial to the uptake of oxygen from the lungs to the cells and this transport is dependent on their ability to change shapes rapidly while navigating through blood vessels. In addition, RBCs play a crucial role in antioxidant defense when fighting against oxidative stress, which has long been recognized as feature of COPD. In this work we made use of a RBC membrane fractionation procedure, stable isotope labeling and bidimensional liquid chromatography (strong cation exchange / reverse phase) before sample acquisition using a high-resolution fourier transform - ion cyclotron resonance (FT-ICR) mass spectrometer (Chapter III). A total of 4697 peptides were quantified as present in both COPD and control spectra corresponding to 1083 proteins. Three-hundred and fourteen proteins possessing at least two peptides were identified, 46% of which were annotated as membrane proteins. Golgin-245/p230 (GOLGA4), was identified as overexpressed in COPD, a protein which is reported to be essential for intracellular trafficking and cell surface delivery of tumor necrosis factor-α (TNF), the main proinflammatory cytokine made and secreted by inflammatory macrophages enhancing activation and recruitment of T-cells and ensuring robust innate and acquired immune responses. Chorein or Vacuolar protein sorting-associated protein 13A (VPS13A) is reported to play a role in the cytoskeleton organization has been associated with thorny deformations of circulating erythrocytes, possibly due to red cell membranes deformation. This protein was found to be underexpressed in COPD patients when compared to controls by MS and this underexpression was confirmed by WB. Consequently, underexpression of chorein may play an important role in the deformation of COPD RBCs. Many other interesting proteins were identified in the xiii context of COPD and, additionally, there were a considerable number of proteins described in RBC for the first time (Chapter III). To overcome the difficulty of acquiring fresh biopsies of well characterized patients, we have established in our laboratory a procedure to collect human fresh nasal epithelial cells. We have shown previously that these cells presented similar proteome of epithelial cells presented in the lower airway. Here, two different types of studies were presented using these cells: a study performed on the effects of cigarette smoke, which is the main risk factor for developing COPD (Chapter IV) and a comparative proteomic study between COPD patients and healthy individuals (Chapter V). Both were pioneer studies by investigating the proteome of fresh nasal epithelial cells from cigarette smoker subjects (Chapter IV) or COPD patients (Chapter V). In both studies a high-resolution mass analyzer, the orbitrap, was employed increasing the number of confident peptide/protein identifications. In Chapter IV, ninety-six proteins were found to be differentially expressed between the proteomes of healthy smokers and nonsmokers. These proteins were related to processes of antigen presentation, cell-to-cell signaling and interaction, cell morphology, drug metabolism, DNA repair, energy production or mitochondrial dysfunction. Although requiring further orthogonal validation, our data was consistent with previous evidences showing CD44, MUC5AC or SOD2 differential modulation in smokers due to inflammatory response pathways. In Chapter V, 89968 peptides and 1475 proteins were identified in total, of which 1173 proteins were identified by at least two peptides. We were able to confirm previous evidences that UPR is activated in COPD patients since we were able to observe overexpression in a considerable number of proteins involved in different protein complexes involved in UPR. This includes overexpression of VCP, both components of the Hsp10/Hsp60 chaperone complex (HSPD1 and HSPE1), CALR and two members of a large ER-localized multiprotein complex of at least 11 proteins, PPIB and ERP29. We also observed an increase in expression of proteins related to Nrf2-mediated oxidative stress response such as GSTP1, TXNRD1 and GSR. Additionally, we also report an increase in drug metabolism, as all significantly differentially expressed proteins related to this biofunction were overexpressed in COPD: GSTP1, GSR, AKR1C3 and ANXA2. Further validation by orthogonal methods is needed so that the activation of xiv UPR and Nrf2-mediated oxidative stress response and the increase in drug metabolism on the nasal epithelial cells of COPD patients is fully confirmed. In Chapter VI, serum collected from COPD patients was divided into 4 different groups in all different combinations of presence/absence of the two main features of COPD, chronic bronchitis and emphysema, to study their impact in the serum proteome. Due to its complex protein mixture, serum was first immunodepleted from its most abundant proteins, comprising about 94% of total protein content, before being analyzed by 1D-PAGE – LC-MS/MS (GeLC-MS/MS) in a linear ion trap mass spectrometer. This powerful strategy was able to identify as many as 2856 proteins, of which 929 were identified by two or more peptides. Plasminogen was found to be underexpressed in COPD patients that suffer simultaneously from emphysema and chronic bronchitis, while it maintained about the same expression level over the three other groups of COPD patients and this differential expression was successfully validated by ELISA. It was possible to identify other interesting proteins as TRAF3IP2, which is associated with innate immunity in response to pathogens, inflammatory signals and stress and has also been implicated in airway hyperresponsiveness or Isoform 1 of phosphatidylinositol-glycan-specific phospholipase D (GPLD1), which is GPI degrading enzyme that was described to be responsible for secretion of prostasin, which was the first of several membrane serine peptidases found to activate the epithelium-sodium channel (ENaC). Prostasin was also reported to have a critical role in regulating epithelial sodium transport in normal and pathological conditions in the lung. The work herein presented confirmed a few findings that had already been reported and more important revealed new insights into COPD disease mechanisms as well as provided new candidate biomarkers for these diseases. Further validation and integration of all data obtained into a systems biology approach will certainly contribute to increase knowledge of COPD and ultimately bringing the well being of patients. |
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| Autores principais: | Alexandre, Bruno Miguel Coelho, 1980- |
| Assunto: | Doença pulmonar obstrutiva crónica Proteómica Biologia molecular Teses de doutoramento - 2012 |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation that is not fully reversible even under bronchodilators effect, caused by a mixture of small airway disease – obstructive bronchiolitis – and parenchymal destruction – emphysema. At the present time, COPD is the fourth leading cause of death and its prevalence and mortality are expected to continue increasing in next decade. Spirometry is the most reproducible way to measure lung function and is nowadays the best tool to diagnose airflow limitation and, consequently, diagnose COPD itself. Biomarkers for diagnosis and/or prognosis as well as novel targets for the development of more effective therapies for COPD are still needed. Proteomics has the capacity to provide large-scale information and consequently it has the potential to expand previous knowledge on COPD. Surprisingly, given the need for new biomarkers in COPD and the power of proteomics, proteomics have been quite neglected so far. Up to date only 50 reports (14 are reviews) match the search at Pubmed (http://www.ncbi.nlm.nih.gov/pubmed, accessed June 23, 2011) for COPD proteomics. Hence, there is a clear need to engage clinically valuable proteomics studies in order to match the need for new biomarkers in COPD. In last decade, the shotgun proteomics approach has become the method of choice for identifying and quantifying proteins in most large-scale studies. Compared with 2DE, shotgun proteomics allows higher data throughput and better protein detection sensitivity. This strategy is based on trypsin digestion of proteins into peptides. This produces a complex peptide mixture that is then separated by one- or multiple dimensional liquid chromatography (LC) and subjected to peptide sequencing using tandem mass spectrometry (MS/MS) before automated database searching. In the present work we have employed different methodologies within shotgun proteomics to generate solid and comprehensive data in COPD. There are quite a lot biological materials that can be used to investigate biomarkers for this disease. Although COPD is now known to possess a systemic inflammation xii component which is responsible for affecting other organs, it is in the lung that the events that lead to breathless take place. Investigating to the lung directly is therefore an optimal strategy to be able to identify proteins that may not be detectable elsewhere either because they are not present or diluted into undetectable concentrations. But this means that lung tissue has to be collected by biopsy which is an extremely invasive technique. But besides tissue biospecimens, other sources of biological materials used to study COPD is biofluids which includes sputum, bronchoalveolar and nasal lavage fluid, exhaled breath condensate, and blood. It had been observed before by means of microscopy that red blood cells (RBCs) from COPD patients showed deformations in their shape. RBCs are crucial to the uptake of oxygen from the lungs to the cells and this transport is dependent on their ability to change shapes rapidly while navigating through blood vessels. In addition, RBCs play a crucial role in antioxidant defense when fighting against oxidative stress, which has long been recognized as feature of COPD. In this work we made use of a RBC membrane fractionation procedure, stable isotope labeling and bidimensional liquid chromatography (strong cation exchange / reverse phase) before sample acquisition using a high-resolution fourier transform - ion cyclotron resonance (FT-ICR) mass spectrometer (Chapter III). A total of 4697 peptides were quantified as present in both COPD and control spectra corresponding to 1083 proteins. Three-hundred and fourteen proteins possessing at least two peptides were identified, 46% of which were annotated as membrane proteins. Golgin-245/p230 (GOLGA4), was identified as overexpressed in COPD, a protein which is reported to be essential for intracellular trafficking and cell surface delivery of tumor necrosis factor-α (TNF), the main proinflammatory cytokine made and secreted by inflammatory macrophages enhancing activation and recruitment of T-cells and ensuring robust innate and acquired immune responses. Chorein or Vacuolar protein sorting-associated protein 13A (VPS13A) is reported to play a role in the cytoskeleton organization has been associated with thorny deformations of circulating erythrocytes, possibly due to red cell membranes deformation. This protein was found to be underexpressed in COPD patients when compared to controls by MS and this underexpression was confirmed by WB. Consequently, underexpression of chorein may play an important role in the deformation of COPD RBCs. Many other interesting proteins were identified in the xiii context of COPD and, additionally, there were a considerable number of proteins described in RBC for the first time (Chapter III). To overcome the difficulty of acquiring fresh biopsies of well characterized patients, we have established in our laboratory a procedure to collect human fresh nasal epithelial cells. We have shown previously that these cells presented similar proteome of epithelial cells presented in the lower airway. Here, two different types of studies were presented using these cells: a study performed on the effects of cigarette smoke, which is the main risk factor for developing COPD (Chapter IV) and a comparative proteomic study between COPD patients and healthy individuals (Chapter V). Both were pioneer studies by investigating the proteome of fresh nasal epithelial cells from cigarette smoker subjects (Chapter IV) or COPD patients (Chapter V). In both studies a high-resolution mass analyzer, the orbitrap, was employed increasing the number of confident peptide/protein identifications. In Chapter IV, ninety-six proteins were found to be differentially expressed between the proteomes of healthy smokers and nonsmokers. These proteins were related to processes of antigen presentation, cell-to-cell signaling and interaction, cell morphology, drug metabolism, DNA repair, energy production or mitochondrial dysfunction. Although requiring further orthogonal validation, our data was consistent with previous evidences showing CD44, MUC5AC or SOD2 differential modulation in smokers due to inflammatory response pathways. In Chapter V, 89968 peptides and 1475 proteins were identified in total, of which 1173 proteins were identified by at least two peptides. We were able to confirm previous evidences that UPR is activated in COPD patients since we were able to observe overexpression in a considerable number of proteins involved in different protein complexes involved in UPR. This includes overexpression of VCP, both components of the Hsp10/Hsp60 chaperone complex (HSPD1 and HSPE1), CALR and two members of a large ER-localized multiprotein complex of at least 11 proteins, PPIB and ERP29. We also observed an increase in expression of proteins related to Nrf2-mediated oxidative stress response such as GSTP1, TXNRD1 and GSR. Additionally, we also report an increase in drug metabolism, as all significantly differentially expressed proteins related to this biofunction were overexpressed in COPD: GSTP1, GSR, AKR1C3 and ANXA2. Further validation by orthogonal methods is needed so that the activation of xiv UPR and Nrf2-mediated oxidative stress response and the increase in drug metabolism on the nasal epithelial cells of COPD patients is fully confirmed. In Chapter VI, serum collected from COPD patients was divided into 4 different groups in all different combinations of presence/absence of the two main features of COPD, chronic bronchitis and emphysema, to study their impact in the serum proteome. Due to its complex protein mixture, serum was first immunodepleted from its most abundant proteins, comprising about 94% of total protein content, before being analyzed by 1D-PAGE – LC-MS/MS (GeLC-MS/MS) in a linear ion trap mass spectrometer. This powerful strategy was able to identify as many as 2856 proteins, of which 929 were identified by two or more peptides. Plasminogen was found to be underexpressed in COPD patients that suffer simultaneously from emphysema and chronic bronchitis, while it maintained about the same expression level over the three other groups of COPD patients and this differential expression was successfully validated by ELISA. It was possible to identify other interesting proteins as TRAF3IP2, which is associated with innate immunity in response to pathogens, inflammatory signals and stress and has also been implicated in airway hyperresponsiveness or Isoform 1 of phosphatidylinositol-glycan-specific phospholipase D (GPLD1), which is GPI degrading enzyme that was described to be responsible for secretion of prostasin, which was the first of several membrane serine peptidases found to activate the epithelium-sodium channel (ENaC). Prostasin was also reported to have a critical role in regulating epithelial sodium transport in normal and pathological conditions in the lung. The work herein presented confirmed a few findings that had already been reported and more important revealed new insights into COPD disease mechanisms as well as provided new candidate biomarkers for these diseases. Further validation and integration of all data obtained into a systems biology approach will certainly contribute to increase knowledge of COPD and ultimately bringing the well being of patients. |
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