Refine
Year of publication
- 2016 (2) (remove)
Document Type
- Doctoral Thesis (2)
Language
- English (2)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Keywords
- Proteomics (2) (remove)
Heart Failure is currently the most common cardiac disorder and a major public health concern worldwide. The adult mammalian heart harbors a subpopulation of cardiac progenitor cells (CPC) that are capable of improving cardiac function. The scope of this study was to delineate the molecular phenotype of a subpopulation of CPCs characterized by the expression of the stem cells antigen-1 surface marker (Sca-1+) and to further identify molecular alterations occurring under heart failure conditions. In order to understand the underlying cellular mechanisms an integrated approach of proteomics and transcriptomics-based techniques were employed. The first step towards achieving this goal was to unravel the native Sca-1+ cell characteristics of freshly isolated progenitor cells derived from healthy adult murine hearts. The proteome map of Sca-1 cells was established using a gel-based mass-spectrometry (gel LC-MS/MS) approach. For better interpretation, a comparison with the protein profiles of cardiomyocytes and Sca-1- cells obtained under similar experimental conditions was performed. All three cell-types were morphologically different in size and structure, which was also evident from their protein expression profiles. We observed that Sca-1+ cells lack endothelial-like and cardiac contractile phenotypes, unlike Sca-1- cells and cardiomyocytes, respectively. Functional assessment of both protein and gene expression profiles revealed a possible role of Sca-1+ cells in cell adhesion, migration, and proliferation. CPC remain in a dormant state under physiological condition unless challenged by myocardial injury. Previous studies revealed that resident Sca-1+ cells home to the injured myocardium but not to the healthy heart and further differentiate into functional cardiomyocytes. We investigated the molecular background of this behavior of adult Sca-1+ cells under heart failure condition which might provide a better insight into their cardiogenic potential in a pathological milieu. The double transgenic α-myosin heavy chain (MHC)-cyclin T1/Gαq overexpressing mouse was chosen as a model for heart failure. Using the comparative gene expression profiling we could detect the differential regulation of 197 genes with at least a 2-fold difference. Among these BDNF mRNA levels were 5-fold higher in the Sca-1+ cells derived from transgenic mice (Cyc+) in comparison to that of wild-type controls (Wt+). This difference was also observed at protein level. The substantially higher expression of BDNF during heart failure prompted us to investigate its regulatory effect on Sca1+ cells. In this current study we were able to show that small amounts of exogenous BDNF stimulated the migratory potential of Cyc+ cells. This effect was not seen in treated Wt+ cells. Furthermore, pulsed SILAC was employed to monitor BDNF mediated changes following treatment. After BDNF treatment, 58 proteins were differentially regulated of which proteins related to cell proliferation were reduced in level in Cyc+ cells while they displayed increased levels in Wt+ cells. Findings from bromodeoxyuridine (BrdU) assays and immunoblotting indicated that BDNF might initiate a differentiation program by repressing cell proliferation in Cyc+ cells. Taken together, it could be shown that the BDNF effect on protein synthesis of Cyc+ and Wt+ cells varied considerably, suggesting an improvement of the cardiogenic potential of Sca-1+ cells under pathological conditions. Aldosterone levels are known to be elevated during heart failure. In this part of study it was hypothesized that endocrine factors associated with heart failure might influence the migration of CPC, thereby possibly restoring the cardiac function of diseased hearts. It could be shown that high concentrations of aldosterone, similar to those found in the plasma of heart failure patients, induced the migration of Sca-1+ cells by up to 60% when compared to control, while physiological levels had no significant influence. In addition, it could be demonstrated that the aldosterone stimulus led to the activation of the mineralocorticoid receptor (MR) expressed on Sca1+ cells, which in turn facilitated migration. This was supported by application of MR antagonist eplerenone, which significantly reduced the aldosterone-induced increase in cell migration while a glucocorticoid antagonist exhibited no inhibitory effect. Hence, the results support the potential role of aldosterone in the mobilization of CPC. It is currently believed that the beneficial effects of cell-based therapies on cardiac repair are imparted to a large degree via paracrine mechanisms. We therefore focused on understanding the influence of pathophysiological levels of aldosterone on the extracellular environment of Sca-1+ cells. MS-based secretome profiling of cells treated for 24h with aldosterone treatment revealed higher levels of proteins associated with extracellular matrix remodeling and IGF signaling. Additionally, galectin-1 and gelsolin were significantly increased in level under pathological conditions indicating a possible paracrine tissue repair of Sca-1+ cells. To conclude, the global proteome and transcriptome profiles generated here revealed the molecular phenotype of Sca-1+ cells which may be used for future reference. The comparative microarray study provided deeper insight into the endogenous changes in mRNA expression during heart failure and delineated the cardiogenic characteristics of Sca-1+ cells. Moreover, the data presented here shed new light on the potential role of BDNF in regulating the mobilization and proliferation of CPCs. Our study on the influence of aldosterone on the migration and the extracellular proteome of CPCs provided new insights on the beneficial effects of this mineralocorticoid on cardiac cells.
Members of the species Bacillus pumilus get more and more in focus of the biotechnological industry as potential new production strains. Based on secretome analysis, Bacillus pumilus strain Jo2, possessing high secretion capability, was chosen for an omics based investigation. The physiology of Bacillus pumilus cells growing either in minimal or complex medium was analyzed by a combination of proteomic and metabolomic methods. Master gels of the cytosolic and the secreted proteome covering major parts of the main metabolic pathways were created by means of 2D gel electrophoresis. Quantification of 2D gels allowed displaying the most abundant proteins in these sub-proteomes. Application of the GeLC-MS/MS technique tripled the number of identified proteins and enabled detection of many intrinsic membrane proteins. In total, 1542 proteins were identified in growing B. pumilus cells, among them 1182 cytosolic proteins, 297 membrane and lipoproteins and 63 secreted proteins. This accounts for about 43 % of the 3616 proteins encoded in the B. pumilus Jo2 genome sequence. By using GC-MS, IP-LC/MS and H-NMR methods numerous metabolites were analyzed and assigned to the reconstructed metabolic pathways. Our data indicate that applying a combination of proteomic and metabolomic techniques a comprehensive view of the physiology of growing B. pumilus cells can be gained. In addition, selected production-relevant genome features such as the restriction modification system, NRPS clusters and the secretory system of B. pumilus Jo2 are discussed. In their natural habitat, the soil, B. pumilus cells are often exposed to growth limiting conditions due to the lack of sufficient amounts of nutrients. Such limitations can also occur during fermentation conditions and will negatively influence the efficiency of the process. Glucose is the main carbon and energy source of B. pumilus. Thus, a deficiency of glucose has an enormous impact on cell growth. A 1D LC-MS/MS approach was performed to quantify the proteins using an N14/N15 labeling and to analyze the changes in the protein equipment when B. pumilus cells stop their exponential growth and become stationary due to limitation of glucose. 1033 proteins in the cytosolic fraction of B. pumilus cells were quantified and 272 of them appeared to be upregulated when the cells experience glucose starvation. 2D-PAGE was used to analyze the exoproteome of those cells. Glucose starving B. pumilus cells seemed to focus on usage of proteins and peptides as alternative carbon and energy sources instead of other carbohydrates. Especially the exoproteome of glucose starving cells is dominated by proteases and peptidases. Furthermore, cells used fatty acids as carbon source indicated by upregulation of enzymes involved in β-oxidation and the methylcitrate pathway. Bacillus pumilus is characterized by a higher oxidative stress resistance than other comparable industrially relevant Bacilli such as B. subtilis or B. licheniformis. In this study the response of B. pumilus to oxidative stress was investigated during a treatment with high concentrations of hydrogen peroxide at the proteome, transcriptome and metabolome level. Genes/proteins belonging to regulons, which are known to have important functions in the oxidative stress response of other organisms, were found to be upregulated, such as the Fur, Spx, SOS or CtsR regulon. Strikingly, parts of the fundamental PerR regulon responding to peroxide stress in B. subtilis are not encoded in the B. pumilus genome. Thus, B. pumilus misses the catalase KatA, the DNA-protection protein MrgA or the alkyl hydroperoxide reductase AhpCF. Data of this study suggests that the catalase KatX2 takes over the function of the missing KatA in the oxidative stress response of B. pumilus. The genome-wide expression analysis revealed an induction of bacillithiol (Cys-GlcN-malate, BSH) relevant genes. An analysis of the intracellular metabolites detected high intracellular levels of this protective metabolite, which indicates the importance of bacillithiol in the peroxide stress resistance of B. pumilus. Using the physiological knowledge gained during our studies, we analyzed samples taken during an industrial fermentation process. Five samples were taken during the processes using a protease overexpressing B. pumilus strain and a non-overexpressing B. pumilus reference strain. 2D-PAGE was employed to analyze the samples. 448 proteins could be identified in the samples from the protease overexpressing stain as well as 453 proteins in the reference strain. The proteins were quantified relatively comparing the different growth phases of each strain as well as comparing the strains to each other. The physiological knowledge gained from the shake flask studies enabled us to interpret the findings. Both strains showed an induction of proteins involved in acquisition of alternative carbon sources and of proteins involved in degradation and usage of fatty acids, e.g. the methylcitrate pathway, when they stop exponential growth. This is comparable to the results gained from the analysis of B. pumilus cells under glucose limitation, indicating similar conditions during the processes. Especially in the late phases of the fermentation processes the cells were obviously exposed to severe stress conditions. Our results demonstrated that overexpressing cells showed a significantly stronger oxidative stress response at the end of the fermentation process compared to non-overexpressing cells, which indicated that not only the high cell densities but also the overproduction of the target protein might be responsible for these conditions.