Mechanisms of Longevity Extension by Caloric Restriction and Lithocholic Acid in the Yeast Saccharomyces Cerevisiae

The objective of studies described in this thesis was to elucidate molecular and cellular mechanisms by which caloric restriction (CR) and lithocholic acid (LCA) extend longevity of the budding yeast Saccharomyces cerevisiae. Recent studies of how CR influences a pattern of metabolism and organelle dynamics in the chronologically aging yeast S. cerevisiae have revealed that this low-calorie diet alters age-related dynamics of ethanol metabolism, lipid synthesis and degradation, trehalose metabolism, ROS homeostasis maintenance, mitochondrial morphology control, mitochondrial functionality preservation, stress response control, cell cycle regulation, quiescence maintenance, and apoptotic and liponecrotic death subroutines. Our hypothesis was that CR may delay yeast chronological aging by altering the age-related dynamics of some or all these cellular processes. Findings presented here support this hypothesis. Indeed, we found that CR slows yeast chronological aging by mechanisms that coordinate the spatiotemporal dynamics of various cellular processes before entry into a non-proliferative state and after such entry. CR causes a stepwise establishment of an aging-delaying cellular pattern by tuning a network that assimilates the following: 1) pathways of carbohydrate and lipid metabolism; 2) communications between the endoplasmic reticulum, lipid droplets, peroxisomes, mitochondria and the cytosol; and 3) a balance between the processes of mitochondrial fusion and fission. Through different phases of the aging process, the CR-dependent remodeling of this intricate network 1) postpones \n \nthe age-related onsets of apoptotic and liponecrotic modes of regulated cell death; and 2) actively increases the chance of cell survival by supporting the maintenance of cellular proteostasis. Because CR decreases the risk of cell death and actively increases the chance of cell survival throughout chronological lifespan, this dietary intervention extends longevity of chronologically aging yeast. \nWe also used a mass spectrometry-based quantitative analysis of the water-soluble cellular metabolome for the investigation of how CR and the longevity-extending tor1Δ mutation (which eliminates the Tor1 protein kinase known to orchestrate the nutrient- and energy-sensing TOR [target of rapamycin] pro-aging signaling pathway) influence the concentrations of various water- soluble metabolites at consecutive stages of the chronological aging process in S. cerevisiae. Our investigation provided the first evidence that both the longevity-extending diet CR and the- longevity extending mutation tor1Δ establish a similar pattern of relative concentrations of methionine metabolism intermediates through the entire process of chronological aging in S. cerevisiae. We proposed a hypothesis that the observed redirection of metabolite flow from the biosynthesis of methionine and spermidine to the biosynthesis of cysteine and glutathione may represent an anti-aging pattern characteristic of the ʺmetabolic signatureʺ of longevity extension in chronologically aging yeast cells placed on the CR diet or having the TOR pro-aging signaling pathway being inactivated. \nBased on recent findings from the Titorenko laboratory, we hypothesized that the LCA- driven changes in mitochondrial lipidome may have a causal role in the age-related remodeling of proteome, thus eliciting changes in mitochondrial functionality and delaying yeast chronological aging. To test this hypothesis, we used a mass spectrometry-based quantitative analysis to investigate how certain mutations that eliminate enzymes involved in mitochondrial phospholipid metabolism influence the mitochondrial proteome and how they affect the geroprotective efficiency of LCA in chronologically aging yeast. Our investigation provided the first evidence that LCA-driven specific changes in the composition of mitochondrial membrane lipids cause a distinct remodeling of mitochondrial proteome by decreasing and increasing concentration of many mitochondrial proteins. These proteins have been implicated in such vital mitochondrial functions as the ETC and respiration, the TCA cycle, ribosome assembly, amino acid metabolism, carbohydrate metabolism, protein import, proteostasis, metabolite synthesis, protein synthesis, ATP synthesis, metabolite transport, lipid metabolism, contact sites and cristae maintenance, redox homeostasis, mtDNA maintenance, stress response, mRNA synthesis and processing, the maintenance of contact sites between mitochondria and vacuoles, and mitochondrial fusion. We provided evidence that the LCA-dependent remodeling of mitochondrial lipidome and the resulting changes in mitochondrial proteome are essential for the ability of LCA to delay aging. \nOur recent studies have indicated that under CR conditions LCA influences not only the composition and functionality of mitochondria but also some cellular processes confined to other cellular compartments. We therefore hypothesized that LCA may delay chronological aging of yeast limited in calorie supply also because it affects these other cellular processes taking place in various cellular locations. To test this hypothesis, we investigated mechanisms through which LCA controls the spatiotemporal dynamics of these other cellular processes in different cellular locations under CR conditions. Our investigation provided important new insights into the mechanisms by which LCA delays yeast chronological aging under CR conditions by altering the spatiotemporal dynamics of a cellular network that integrates certain pathways of lipid and carbohydrate metabolism, some intercompartmental communications, specific aspects of mitochondrial morphology and functionality, and liponecrotic and apoptotic modes of regulated cell death. \nBecause LCA triggers major changes in the age-related chronology of several vital processes taking place in mitochondria, we hypothesized that LCA may cause these changes by eliciting a reversible phosphorylation of some mitochondrial proteins. To test this hypothesis, we investigated if an exposure of chronologically aging yeast to exogenous LCA can trigger such phosphorylation. We found that LCA elicits the establishment of a distinct phosphoprotein profile of mitochondria, which significantly differs from the phosphoprotein profile of mitochondria in yeast cells cultured without LCA.

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