Elucidating the Effects on Proteome Regulation under Caloric Restriction
Cellular energy homeostasis contributes to normal cell growth, functioning as a biological checkpoint of life. There have been numerous genes and metabolic pathways known to regulate the energy status of the cell, and how they contribute to cellular maintenance is of great interest; Caloric Restriction (CR) takes advantage of these metabolic pathways, retarding the aging process by slowing down the metabolic rate. However, despite the many studies on cellular energy homeostasis, much work still needs to be done.
Many of the applications of CR in various organisms have established life-extending benefits by regulating age-related diseases through convergent mechanisms. In fact, a number of signaling pathways, as well as master regulator proteins, act through these mechanisms to interact and regulate protein expressions. CR, as well as other lifespan extending interventions such as rapamycin treatment and inhibition of insulin growth factors, are subject to translational regulation. Increased caloric intake and obesity related comorbidities, in turn, can also work through these pathways to induce translation and cause disease, indicating the role of protein synthesis in health and disease is of great importance. Stable isotope-labeling proteomics are a powerful strategy that enable the assessment of proteome-wide dynamic fluxes in energy regulating biological interventions such as CR and mimetics. Using mass spectrometric (MS) strategies has afforded scientists a never before seen understanding of how proteome dynamics stand at the center of phenotype, physiologic adaptation, and disease pathogenesis. Through this approach, researches have been able to measure protein synthesis and turnover rates, both for targeted proteins an unbiased screening.
In this dissertation, I present a thorough review of our current understanding of metabolic pathways in the context of lifespan extension interventions like CR. I also discuss the underlying principles for measuring protein dynamics, focusing on metabolic labeling with 2H2O (heavy water) combined with tandem MS analysis of mass isotopomer abundances. Next, I demonstrate its application in four separate studies. First, I use 2H2O labeling in CR mice in a time-course to identify whether the protein turnover rate slowdown occurs immediately, gradually, or within a discreate time period. Next, I explore separately whether two potential CR mimetics, exercise and metformin administration, are able to slow down the fractional synthesis rate of proteins in a manner comparable to CR. Finally, I discuss a potential CR modulator in Nitric Oxide, a short-lived bioactive molecule known to be induced under CR conditions and reduced during aging and disease. Overall, the work reported in this dissertation demonstrates how global proteome dynamics require NO for proper regulation, CR mimetics influence the proteome in similar mode, aerobic exercise has very different effects and CR effects on proteome fluxes are activated within a narrow and discrete time period.