Proteome Turnover in Mouse Aging: In Vivo Studies by Stable Isotope Mass Spectrometr

Introduction: Maintenance of proper protein homeostasis (proteostasis) is essential to cellular and organismal health. A wealth of research has shown that age-related diseases and conditions are associated with the inability of the cell to maintain healthy proteins or get rid of defective proteins, including: neurodegenerative disease, cardiac dysfunction, cataracts, and sarcopenia. Calorie restriction (CR) and rapamycin (RP), two interventions known to alter protein degradation and synthesis rates, effectively extend lifespan and improve health in many model organisms. Overexpression of mitochondria-targeted catalase (mCAT), an antioxidant enzyme, similarly extends lifespan and healthspan in mammals, protects against protein damage and unfolding, and preserves proteostasis machinery. While a general observation of improved proteome quality and activation of proteostasis machinery can be observed in all three aging-interventions, the downstream proteins targeted by the affected processes remain unknown. Identifying these targets, their functional relevance to health and aging, and the mechanisms by which they change is the focus of this thesis.;Methods: Utilizing a combination of stable isotope labeling and nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS), we perform large-scale estimation of changes in protein abundances and turnover rates simultaneously for each treatment, followed by clustering, pathway enrichment, correlations, and statistical analysis.;Calorie restriction and rapamycin in the aging heart and liver: In several tissues, we examine changes in protein abundances and in vivo turnover rates of young and old mice treated with either CR or RP for 10 weeks. Our data shows that global protein turnover tends to increase with age in the heart and liver, while both RP and CR reverse this aging effect. In old mice treated with CR, protein abundances closely recapitulated young levels in both heart and liver among the top significantly altered pathways. Treatment with RP recapitulated young levels in heart, but had the opposite effect in liver. The top pathways altered by treatment consisted mostly of metabolic pathways with increases in mitochondrial function, oxidative phosphorylation, and fatty acid beta-oxidation and decreases in glycolytic pathways.;Poly-ubiquitin mediated proteostasis: To determine the relative contribution of ubiquitin-mediated homeostasis to changes in turnover, we also performed an antibody enrichment of the poly-ubiquitin modified fraction (ubiquitinome) of liver tissue, followed by a novel analysis of "poly-ubiquitinome" kinetics. Our data demonstrates that a large number of poly-ub-proteins show a preferential increase in abundance compared to their unmodified counterparts, in several cases, significantly correlate with previously observed increases in half-lives (HLs). These correlations extended to pathway-level changes during aging and treatment. Furthermore, we demonstrate how label kinetics can be used to distinguish accumulating poly-ub-proteins from readily degraded ones. Overall, our results demonstrate that proteomic analysis of sub-populations of proteins may be a promising approach to identifying the targets and dynamics of major cellular processes.;Mitochondrial respiratory chain turnover: The turnover rates of subunits of the mitochondrial respiratory chain are observed from over 40 unique experimental conditions representing a wide range of proteome compositions and turnover rates. We show that across highly divergent conditions, the relative differences in half-lives of respiratory proteins are highly conserved. This conserved heterogeneity can be partly explained by several factors.;Mitochondria-targeted catalase (mCAT) and reverse antagonistic pleiotropy: A comprehensive examination of changes in abundance and turnover during normal and mCAT aging. The effects of mCAT on global turnover rates as well as a number of changes in top pathways is comparable to CR and RP. Interestingly, the YmCAT proteome is largely different in abundances and turnover rates from the YWT proteome and more closely resembles the OWT proteome -- exhibiting a pattern of "reverse antagonistic pleiotropy".