Exploring the metabolism beyond cell aging in yeast

All living organisms undergo a progressive physiological decline with age that results in an increased risk of the development of many diseases. Up to now, many factors have been shown to be involved in aging, like oxidative damage, telomere erosion, mitochondrial dysfunction, genomic instability or epigenetic changes, but, although many efforts made, none of them is commonly recognized as the primary molecular cause of this phenomenon. The budding yeast Saccharomyces cerevisiae has proven to be an experimental useful model for studying the aging process leading to the identification of pathways whose counterparts can be found in higher eukaryotes and in particular in humans. In yeast, the two paradigms of aging are described: the replicative aging and the chronological aging. The latter refers to the aging of cells in a quiescent-like state, with chronological lifespan (CLS) defined as the length of time that non-dividing yeast cells can maintain replicative potential. CLS is influenced by several factors, both intrinsic and extrinsic. The former group includes the signal transduction pathways involved in stress and nutrient responses, such as the TOR/Sch9 and the Ras/PKA pathways. Among the latter, in yeast two byproducts of the cell metabolism seem to play a determinant role: acetate and ethanol. In particular, their presence in the exhausted growth medium has a pro-aging effect, even though the exact role they play is still a matter of debate. Moreover, a key role in lifespan regulation is widely recognized for Sir2, the founder of Sirtuins, a family of highly conserved NAD-dependent histone deacetylases. Unlike the other families of deacetylases, Sirtuins couple protein deacetylation with the cleavage of NAD+, a feature that makes them key elements for the interconnection between cell homeostasis and aging. In addition, Sirtuins also influence the activity of many metabolic enzymes in humans by modulating their acetylation state, strengthening the linkage between the metabolic status of the cell and Sirtuin function. In this context, this work aims to deepen the knowledge on some extrinsic and intrinsic regulators of chronological aging. Among intrinsic factors, we analyzed Sir2, which plays a pro-aging role in CLS. We found that the lack of this deacetylase influences some aspects of the cell metabolism, with a particular regard to ethanol and acetate. Among the extrinsic factors, and particularly dealing with acetate, we focused on Ach1, a mitochondrial enzyme, whose function has not been well characterized yet but that may play a key role in the metabolism of acetic acid at mitochondrial level. To analyze the interconnections among Sir2 activity, lifespan and the cell metabolism, we performed experiments in batch with fermentative and non-fermentative carbon sources, during chronological aging and in chemostat (glucose-limited cultures pulsed with ethanol and acetate) where growth parameters together with metabolite contents were analyzed. During chronological aging, CLS was also determined. Since in cells lacking Sir2 we determined an increase in acetate and ethanol catabolism, we focused on gluconeogenesis (in particular on Pck1, the rate limiting enzyme) and on the glyoxylate cycle (in particular on one of the two unique enzymes of this cycle, Icl1). In fact, both processes are fundamental when cells are growing on acetate and ethanol. By measuring the activity of Pck1 and Icl1, we found that in sir2D cells both these enzymatic activities were enhanced. In particular, the increased activity of Pck1 correlated with a higher acetylation level of this protein, giving also experimental evidence of a model where it has been proposed that Pck1, acetylated by Esa1, could be the target of Sir2- mediated deacetylation. Moreover, to check whether the activity of these enzymes was related with the CLS phenotype of sir2D cells, we inactivated ICL1 and PCK1 in sir2D cells to analyze the longevity and the metabolite level of the double mutant strains during chronological aging: both icl1D and pck1D mutants accumulated high level of extracellular acetate and ethanol and were short-lived mutants. In addition, ICL1 and PCK1 inactivation had epistatic effects on sir2D cells. Moreover, consistent with an increased gluconeogenetic flux, trehalose levels in sir2D stationary phase cells were higher compared with wild type cells. On the whole, we provided evidence that SIR2 inactivation positively affects acetate metabolism by enhancing the glyoxylate-requiring gluconeogenesis. In the aging context, this implies lower levels of negative extracellular factors and a major accumulation of protective trehalose which create a beneficial environment for long-term survival of non-dividing cells. Then, we focused on acetate, and in particular on Ach1, a mitochondrial enzyme whose exact function has not been well characterized. In the ’90, Ach1 was identified as mitochondrial hydrolase, even though the physiological role of acetyl-CoA hydrolysis was not clear. Recently, in Aspergillus nidulans an enzyme with high sequence identity with Ach1 was identified which is involved in the process of propionate detoxification. On the basis of this, the hypothesis arose that in S. cerevisiae as well this enzyme could catalyze a transferase reaction by activating, rather than hydrolyzing, acetyl-CoA from acetate. In this work of thesis, we characterized the phenotype of ach1D cells, with the aim of better understanding the role of this protein in acetate metabolism and any potential implications on mitochondrial functionality and on CLS modulation. We found that chronologically aging ach1D cells accumulated a high amount of extracellular acetate which correlated with a short-lived phenotype. This phenotype was strictly dependent on extracellular acetate since, when the acid stress was abrogated either by a calorie restricted regimen (no acetic acid production) or by transferring chronologically aging ach1D cells to water, cell survival was restored. Moreover, the short-lived phenotype of chronological aging ach1D cells is accompanied by ROS accumulation, a compromised mitochondrial activity and a precocious activation of the Yca1-dependent apoptotic pathway. In agreement with this compromised mitochondrial activity, we also saw that ach1D cells had severe problems to grow in media containing acetate as carbon source, underlining a primary role of Ach1 enzymatic activity in acetic acid detoxification which is important for mitochondrial functionality. Mitochondrial functionality which also plays an essential role for chronological cell survival. Since we found an inverse correlation between extracellular ethanol and acetic acid level and CLS, further experiments were performed to clarify the role played by these two pro-aging factors. Data obtained support the hypothesis that at physiological levels is not the mere presence of ethanol and acetic acid to influence the CLS but it is their metabolism. Thus, both these C2 compounds act as carbon sources that prevent entry of cells into a calorie restriction-like state, the only one in which cells are able to maintain a long term survival.