Living cells continuously degrade and resynthesize their constituent proteins. The maintenance of protein homeostasis is fundamental to cell survival and function. Recent advances in mass spectrometry, especially the development of stable isotope labeling with amino acids in cell culture (SILAC), have enabled proteome-wide analyses of cellular protein turnover and elucidation of protein homeostasis maintenance mechanisms. However, more efficient methods are still needed for higher precision analysis of proteome dynamics. This work first clarified one overlooked issue in the interpretation of dynamic SILAC experiments and indicated that in typical experiments conducted in culture, the aminoacyl-tRNA precursor pool is near completely labeled in a few hours and protein turnover is the limiting factor in establishing the labeling kinetics of most proteins. Second, a methodology that combines metabolic isotopic labeling (SILAC) with isobaric tagging (Tandem Mass Tags - TMT) was established for analysis of multiplexed samples. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. By globally quantifying the kinetics of protein clearance and synthesis, this approach provided important insights into the regulation of the proteome as fibroblasts transit from a dividing to a quiescent state. Our results indicated that, in quiescent cells, protein synthesis decreases, while protein degradation increases by up-regulation of autophagy and lysosome biogenesis. Lastly, by measuring protein degradation rates in wildtype and autophagy-deficient cells, we investigated the selectivity of macroautophagy on a global scale. Together, this work developed a more efficient methodology for measuring protein synthesis and turnover rates on a global scale and revealed an important mechanism of protein homeostasis in quiescent fibroblasts.
|Commitee:||Dumont, Mark E., Goldfarb, David, Seluanov, Andrei|
|School:||University of Rochester|
|Department:||School of Arts and Sciences|
|School Location:||United States -- New York|
|Source:||DAI-B 78/10(E), Dissertation Abstracts International|
|Keywords:||Autophagy, Long-lived proteins, Protein turnover, Quiescent cells, Selectivity of autophagy, TMT-SILAC|
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