Littérature scientifique sur le sujet « Yeast, Metabolism, Aging »

This review of sphingolipid metabolism in the budding yeast Saccharomyces cerevisiae contains information on the enzymes and the genes that encode them, as well as connections to other metabolic pathways. Particular attention is given to yeast homologs, domains, and motifs in the sequence, cellular localization of enzymes, and possible protein–protein interactions. Also included are genetic interactions of special interest that provide clues to the cellular biological roles of particular sphingolipid metabolic pathways and specific sphingolipids.Key words : yeast, sphingolipid metabolism, subcellular localization, protein–protein interactions, stress response, aging.

SUMMARY When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.

The aromatic metabolites derived from yeast metabolism determine the characteristics of aroma and taste in wines, so they are considered of great industrial interest. Volatile esters represent the most important group and therefore, their presence is extremely important for the flavor profile of the wine. In this work, we use and compare two Saccharomyces cerevisiae yeast strains: P29, typical of sparkling wines resulting of second fermentation in a closed bottle; G1, a flor yeast responsible for the biological aging of Sherry wines. We aimed to analyze and compare the effect of endogenous CO2 overpressure on esters metabolism with the proteins related in these yeast strains, to understand the yeast fermentation process in sparkling wines. For this purpose, protein identification was carried out using the OFFGEL fractionator and the LTQ Orbitrap, following the detection and quantification of esters with gas chromatograph coupled to flame ionization detector (GC-FID) and stir-bar sorptive extraction, followed by thermal desorption and gas chromatography-mass spectrometry (SBSE-TD-GC-MS). Six acetate esters, fourteen ethyl esters, and five proteins involved in esters metabolism were identified. Moreover, significant correlations were established between esters and proteins. Both strains showed similar behavior. According to these results, the use of this flor yeast may be proposed for the sparkling wine production and enhance the diversity and the typicity of sparkling wine yeasts.

Abstract In this work, we set out to develop a high throughput screening method, SEBYL (SEquencing Based Yeast replicative Lifespan screen), in order to identify new aging regulators in budding yeast. By utilizing SEBYL on yeast knockout collection, we were able to identify 285 long-lived gene deletions, of which a significant portion was proven to have extended lifespan by previous classical experiments. To demonstrate the ability of our method to discover new genes and pathways involved in aging process, we focused on characterizing one newly identified long-lived candidate emerged from the screening, histone deacetylase complex HDA, and found it regulates aging through mediating stress response pathways, especially DNA damage stress response. Presence of HDA complex inhibits expression of trehalose metabolism genes, which act as stress protectant. When HDA complex is mutated, trehalose genes are de-repressed, enhancing stress response and eventually promotes longevity. In summary, we conclude SEBYL to be time and energy saving, robust, and suitable for discovery of aging regulating genes using various preexisting yeast mutant collection resource.

Yeast multicellular colonies possess several traits that are absent from individual yeasts. These include the ability to synchronize colony population development and adapt its metabolism to different environmental changes, such as nutrient depletion. This, together with cell diversification to cell variants with distinct metabolic and other properties, contributes to the main goal of the colony population: to achieve longevity. In this respect, a benefit to individual cells is subordinated to the benefit to the whole population, exhibiting a kind of altruistic behaviour. For example, some colony cells located at particular positions undergo regulated cell dying and provide components to other cells located in more propitious areas. The enhancement of techniques that enable the in vivo investigation of three-dimensional spatiotemporal colony development may lead to new discoveries on metabolic differentiation and regulation in the near future.

Nicotinamide, nicotinic acid and nicotinamide riboside are vitamin B3 precursors of NAD+ in the human diet. NAD+ has a fundamental importance for cellular biology, that derives from its essential role as a cofactor of various metabolic redox reactions, as well as an obligate co-substrate for NAD+-consuming enzymes which are involved in many fundamental cellular processes including aging/longevity. During aging, a systemic decrease in NAD+ levels takes place, exposing the organism to the risk of a progressive inefficiency of those processes in which NAD+ is required and, consequently, contributing to the age-associated physiological/functional decline. In this context, dietary supplementation with NAD+ precursors is considered a promising strategy to prevent NAD+ decrease and attenuate in such a way several metabolic defects common to the aging process. The metabolism of NAD+ precursors and its impact on cell longevity have benefited greatly from studies performed in the yeast Saccharomyces cerevisiae, which is one of the most established model systems used to study the aging processes of both proliferating (replicative aging) and non-proliferating cells (chronological aging). In this review we summarize important aspects of the role played by nicotinamide, nicotinic acid and nicotinamide riboside in NAD+ metabolism and how each of these NAD+ precursors contribute to the different aspects that influence both replicative and chronological aging. Taken as a whole, the findings provided by the studies carried out in S. cerevisiae are informative for the understanding of the complex dynamic flexibility of NAD+ metabolism, which is essential for the maintenance of cellular fitness and for the development of dietary supplements based on NAD+ precursors.

Flor velum yeast growth activators during biological aging are currently unknown. In this sense, this research focuses on the use of bee pollen as a flor velum activator. Bee pollen influence on viable yeast development, surface hydrophobicity, and yeast assimilable nitrogen has already been studied. Additionally, bee pollen effects on the main compounds related to flor yeast metabolism and wine sensory characteristics have been evaluated. “Fino” (Sherry) wine was supplemented with bee pollen using six different doses ranging from 0.1 to 20 g/L. Its addition in a dose equal or greater than 0.25 g/L can be an effective flor velum activator, increasing yeast populations and its buoyancy due to its content of yeast assimilable nitrogen and fatty acids. Except for the 20 g/L dose, pollen did not induce any significant effect on flor velum metabolism, physicochemical parameters, organic acids, major volatile compounds, or glycerol. Sensory analysis showed that low bee pollen doses increase wine’s biological aging attributes, obtaining the highest score from the tasting panel. Multiflora bee pollen could be a natural oenological tool to enhance flor velum development and wine sensory qualities. This study confirms association between the bee pollen dose applied and the flor velum growth rate. The addition of bee pollen could help winemakers to accelerate or reimplant flor velum in biologically aged wines.

Proline is a pivotal and multifunctional amino acid that is used not only as a nitrogen source but also as a stress protectant and energy source. Therefore, proline metabolism is known to be important in maintaining cellular homeostasis. Here, we discovered that proline oxidation, catalyzed by the proline oxidase Put1, a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate, controls the chronological lifespan of the yeast Saccharomyces cerevisiae. Intriguingly, the yeast strain with PUT1 deletion showed a reduced chronological lifespan compared with the wild-type strain. The addition of proline to the culture medium significantly increased the longevity of wild-type cells but not that of PUT1-deleted cells. We next found that induction of the transcriptional factor Put3-dependent PUT1 and degradation of proline occur during the aging of yeast cells. Additionally, the lifespan of the PUT3-deleted strain, which is deficient in PUT1 induction, was shorter than that of the wild-type strain. More importantly, the oxidation of proline by Put1 helped maintain the mitochondrial membrane potential and ATP production through the aging period. These results indicate that mitochondrial energy metabolism is maintained through oxidative degradation of proline and that this process is important in regulating the longevity of yeast cells.

Volatile sulfur compounds (VSCs) are associated with unpleasant reductive aromas and are responsible for an important reduction in wine quality, causing major economic losses. Understanding the origin of these compounds in wine remains a challenge, as their formation and further evolution during winemaking can involve both chemical and biological reactions. Comparing the VSCs profile (i) of fermenting synthetic grape juices supplemented with a selected VSC (eight compounds tested) and incubated in presence or absence of yeast, and (ii) during storage of wines under an accelerated aging procedure, allowed us to elucidate the chemical and metabolic connections between VSCs during fermentation and aging. Yeast metabolism, through the Ehrlich pathway and acetylation reactions, makes an important contribution to the formation of compounds such as methionol, 3-methylthiopropionate, 3-methylthiopropylacetate, 3-mercaptopropanol, 2-mercaptoethanol and thioesters. By contrast, chemical reactions are responsible for interconversions between thiols and disulfides, the formation of thiols from thioesters or, more surprisingly, the formation of ethylthiopropanol from methionol during fermentation. During aging, variations in heavy VSC concentrations, such as an increase in 3-methylthiopropylacetate and a decrease in ethyl-3-methylthiopropionate formation, were evidenced. Overall, this study highlights that it is essential to consider both yeast metabolism and the high chemical reactivity of VSCs to understand their formation and evolution during winemaking.

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.

I composti intermedi generati dal metabolismo del carbonio sono precursori fondamentali per la biosintesi di lipidi e carboidrati, la cui importanza nella regolazione della longevità è sempre più rilevante. Per comprendere il ruolo di tali composti nel contesto dell’invecchiamento diventa necessario indagare le relazioni tra i pathway che generano i precursori metabolici e come i flussi dei singoli metaboliti impattano sulla sopravvivenza cellulare. Per affrontare questa tematica, il mio progetto di dottorato sfrutta il lievito gemmante Saccharomyces cerevisiae, organismo modello ampiamente affermato per lo studio di meccanismi molecolari evolutivamente conservati. In particolare, si è studiato l’invecchiamento cronologico, ossia quello a cui vanno incontro cellule di lievito quiescenti in fase stazionaria e che rappresenta un efficace modello per lo studio dell’invecchiamento di cellule di mammifero post-mitotiche quali neuroni e miociti. In questo contesto, un metabolita di fondamentale interesse è l’acetil-CoA, ossia la forma attivata dell’acetato tramite un legame tioestere con il coenzima A. L’acetil-CoA rappresenta un importante snodo per il metabolismo, anche in relazione alla sua localizzazione. Ad esempio, l’acetil-CoA mitocondriale, generato per decarbossilazione ossidativa del piruvato nel mitocondrio, entra nel TCA per foraggiare il metabolismo biosintetico e quello energetico. L’altro grande pool di acetil-CoA è quello nucleocitosolico, rilevante nel metabolismo lipidico: l’acetil-CoA può essere infatti utilizzato per convogliare unità di carbonio alla biosintesi di macromolecole lipidiche, inclusi acidi grassi i quali in seguito, tramite β-ossidazione nei perossisomi, possono fornire nuovamente acetil-CoA. Inoltre, lo stesso pool nucleocitosolico è anche usato come donatore di gruppi acetili, per regolare tramite l’acetilazione attività enzimatiche e stato della cromatina, quindi lo stato funzionale della cellula. Procedendo, tre punti diventano rilevanti: 1) la regolazione del flusso dei precursori dell'acetil-CoA tra compartimenti cellulari; 2) il controllo del metabolismo attraverso il meccanismo di (de)acetilazione; 3) il ruolo degli acidi grassi, intimamente connessi con l’acetil-CoA. Questi punti sono stati indagati generando mutanti ad hoc tramite la delezione di geni, esaminando diversi aspetti sia metabolici che funzionali e adoperando approcci nutrizionali mirati. Complessivamente, i risultati hanno permesso chiarire come il ruolo dell’acetil-CoA non sia rilegato ad una specifica fase di crescita o ad un particolare metabolismo ma abbia un valore più ampio. La sua corretta produzione e distribuzione tra i compartimenti cellulari, favorita anche dalla somministrazione di molecole di interesse nutrizionale, garantisce una modulazione efficace del metabolismo anche attraverso modificazioni post-traduzionali, incrementando la longevità cellulare. Infine, i mitocondri sono emersi come organelli estremamente sensibili alla gestione di acetil-CoA, quindi come sensori per monitorare lo stato cellulare nel tempo. In conclusione, i dati ottenuti sottolineano la complessità del metabolismo dell’acetil-CoA durante l’invecchiamento, e come la gestione di questo metabolita chiave sia strettamente legata alla sopravvivenza cellulare.
Carbon metabolism intermediates are known to be fundamental precursors in the biosynthesis of carbohydrates and lipids, whose metabolism is being increasingly acknowledged as a relevant lifespan regulator. Therefore, to understand the importance of these compounds in the context of aging, it is necessary to deepen the relations among the pathways involved in the production of metabolic precursors and how fluxes of single key metabolites impact on cellular survival. In order to reach this goal, we employ the budding yeast Saccharomyces cerevisiae, a model organism successfully used to study evolutionarily conserved molecular mechanisms. In particular, the interest is in chronological aging, defined as the aging process of quiescent yeast cells during the stationary phase, that is, after all carbon sources are consumed. Currently, this model is extensively used as a paradigm for the aging process of mammalian post-mitotic cells such as neurons and myocytes. In this context, a particularly interesting metabolite is acetyl-CoA, the activated form of acetate due to a thioester bond with coenzyme A. Acetyl-CoA is indeed important for many cellular events, depending on its cellular localization. For instance, mitochondrial acetyl-CoA, generated from the uptake of pyruvate in the mitochondria, enters the TCA cycle and fosters both biosynthesis and energy metabolism. The other great distinct pool, represented by the nucleocytoplasmic acetyl-CoA, provides instead the fundamental bricks for the biosynthesis of lipid macromolecules, including fatty acids, which in turn, if processed by β-oxidation, can provide again acetyl-CoA. In addition, the available nucleocytosolic pool is also used as a source of acetyl groups to modulate, through post-translational modifications, enzyme activities and chromatin state, therefore the functional state of a cell. Proceeding through steps, three key points become relevant in the context of acetyl-CoA and aging: 1) the regulation of the flux of acetyl-CoA precursors among compartments; 2) the control of metabolism through (de)acetylation; 3) the role of fatty acid metabolism, intimately linked to acetyl-CoA. These key points were investigated generating ad hoc mutants through gene deletion, analysing different metabolic and functional aspects and using peculiar nutritional approaches. Overall, results gave insights on the role of acetyl-CoA, highlighting that the importance of this metabolite is not restricted to a particular growth phase or metabolism but has a broader value. A correct production and distribution of acetyl-CoA among compartments, supported by the administration of nutritionally relevant molecules, guarantees an efficient modulation of metabolism with the contribute of post-translational modifications, increasing longevity. Also, mitochondria emerged as extremely sensitive on how acetyl-CoA is managed during aging, therefore as sensors to monitor the cellular state through time. In conclusion, the obtained data underline the complexity of acetyl-CoA metabolism during aging, and how the management of this key metabolite is strictly related to cellular survival.

L’aumento dell’aspettativa di vita non è associato con un altrettanto aumento delle condizioni di salute nella popolazione anziana. Oggigiorno, un’ampia parte di popolazione al di sopra dei 65 anni soffre di molteplici malattie, molte delle quali debilitanti, come le malattie cardiovascolari, i tumori o i disordini neurodegenerativi. Questo aspetto ha aumentato l’interesse per le tematiche legate all’invecchiamento, enfatizzando l’importanza di ridurre il gap tra longevità salute durante l’invecchiamento. A questo proposito, gli sforzi di molte linee di ricerca sono focalizzati nel tentativo di comprendere quali sono i principali fattori che influenzano l’invecchiamento, allo scopo di sviluppare approcci capaci di mitigare gli effetti dannosi dell’invecchiamento sulla salute. Molti pathway associati all’invecchiamento sono evolutivamente conservati dagli organismi unicellulari a quelli più complessi. Questo ci ha permesso di semplici organismi modello per studiare questo complesso fenomeno biologico. In questo lavoro abbiamo utilizzato l’eucariote unicellulare Saccharomyces cerevisiae, che va incontro sia all’invecchiamento replicativo che a quello cronologico, due modelli complementari di invecchiamento, che rispettivamente simulano il processo di invecchiamento delle cellule mitoticamente attive e quello delle cellule post-mitotiche. In questo contesto la replicative lifespan (RLS) è definita come il numero di cellule figlie generate da una cellula madre in presenza di nutrienti prima della morte. Al contrario, la chronological lifespan (CLS) è il periodo di sopravvivenza medio e massimo di una popolazione di cellule di lievito in fase stazionaria. Essa è determinata, partendo tre giorni dallo shift diauxico, dalla capacità di cellule quiescenti di riprendere la crescita una volta tornate su terreno fresco ricco. Considerata l’esistenza di una forte connessione tra invecchiamento cellulare, nutrienti e metabolismo, abbiamo studiato i possibili effetti di alcuni composti nutraceutici, allo scopo di identificare molecole per sviluppare interventi anti-aging, oltre che aggiungere informazioni utili per comprendere meglio il processo di invecchiamento. A questo scopo, durante il primo e il secondo anno del mio progetto di dottorato, ho studiato gli effetti del resveratrolo (RSV) sulla CLS. RSV è un composto polifenolico annoverato tra i composti attivatori delle Sirtuine (STAC) ed è riconosciuto per conferire benefici su molte patologie legate all’invecchiamento. Le Sirtuine sono una famiglia di deacetilasi NAD+-dipendenti, il cui capostipite è Sir2 di S. cerevisiae, la cui attività è coinvolta sia nell’RLS che nella CLS. Inaspettatamente, abbiamo osservato che il trattamento con RSV incrementava lo stress ossidativo, in concomitanza con una notevole riduzione del pathway anti-aging della gluconeogenesi. L’attività deacetilasica di Sir2 sul suo target gluconeogenico Pck1 era incrementata, determinandone la sua inattivazione e indicando che RSV effettivamente agisce come STAC. Come conseguenza, questo causava effetti negativi sul metabolismo, determinando un fenotipo short-lived. Successivamente, ci siamo focalizzati sulla quercitina (QUER), un composto nutraceutico con proprietà benefiche su diverse patologie, incluse le malattie cardiovascolari, il cancro e la dislipidemia. Ciononostante, i target cellulari della QUER devono essere ancora esplorati. Abbiamo visto che la QUER possiede proprietà anti-aging che favoriscono un’estensione della CLS. Tutti i dati indicano un’inibizione dell’attività deacetilasica di Sir2 a seguito del trattamento con la QUER, determinando un incremento dei livelli di acetilazione e di attività di Pck1. Questo determina un rimodellamento metabolico a favore del pathway della gluconeogenesi, incrementando le riserve di trealosio e garantendo un miglioramento del processo di invecchiamento.
Since the second half of past century in many developed Countries, life expectancy has gradually increased, reaching, and in some extreme cases exceeding, the threshold of 85 years. However, the increase of life expectancy is not associated with a corresponding increment of healthy conditions for the older population. Nowadays, a huge part of population over 65 years suffers a multitude of diseases, most of them highly disabling, like cardiovascular diseases, tumour or neurodegenerative disorders. This aspect has increased the interest on age-related issues, emphasizing the importance of reducing the gap between longevity and health during aging. For this purpose, efforts of many research lines have focused on studying which are the main factors that affect aging, in order to develop approaches that mitigate the detrimental effects of aging on health. Many aging-related pathways are evolutionarily conserved from some single-celled organisms to complex multicellular ones. Such knowledge has allowed us the use of simple model organisms to study this complex biological phenomenon. In this work we used the single-celled eukaryote Saccharomyces cerevisiae, which undergoes both replicative and chronological aging, two complementary models of aging, which respectively resemble the aging process of mitotically active and post-mitotic mammalian cells. In this context, replicative lifespan (RLS) is defined as the number of buds generated by a single mother cell in the presence of nutrients before death. On the contrary, chronological lifespan (CLS) is the mean and maximum period of time of surviving cells in stationary phase. It is determined, starting three days from the diauxic shift, by the capability of quiescent cells to resume growth once returning to rich fresh medium. Considering that there is a strong connection between cellular aging, nutrients and metabolism, we investigated the possible effects of some nutraceutical compounds, in order to identify molecules for anti-aging interventions, as well as add useful information to understand the aging process. To this end, during the first and second year of my PhD project, I studied the effects of resveratrol (RSV) on CLS. RSV is a polyphenolic compound counted among the Sirtuin Activator Compounds (STACs), which has been proposed to confer health benefits on different age-related diseases. Sirtuins are a family of NAD+-dependent deacetylases, the founding member of which is Sir2 of S. cerevisiae, whose activity is involved in both RLS and CLS. Unexpectedly, we found that RSV supplementation increased oxidative stress in concert with a strong reduction of the anti-aging gluconeogenesis pathway. The deacetylase activity of Sir2 on its gluconeogenic target Pck1 was enhanced, resulting in its inactivation and indicating that RSV really acts as a STAC. As a consequence, this brought about detrimental effects on the survival metabolism resulting in a short-lived phenotype. Next, we focused on the study of quercetin (QUER), a nutraceutical compound with health-promoting properties on different pathologies, including cardiovascular disorders, cancer and dyslipidaemia. Nevertheless, QUER cellular targets are still being explored. We found that QUER displays anti-aging properties favouring CLS extension. All data point to an inhibition of the deacetylase activity of Sir2 following QUER supplementation, resulting in increased levels of acetylation and activity of Pck1. This determines a metabolic remodelling in favour of the pro-longevity gluconeogenesis pathway, increasing trehalose storage and ensuring healthy aging improvement.

Caloric restriction (CR) extends lifespan from yeast to mammals. In budding yeast, inhibition of the conserved TOR and/or PKA pathways has been shown to mediate lifespan extension by CR partly through the activation of stress response. However, how the stress response is regulated at the systems level is poorly understood. In this study, by using fluorescent reporters whose expression is dependent on the transcription factors Msn2/4 and Gis1, two separate screenings were conducted to reveal novel regulators of the stress response induced by starvation. A 'focused' screening on the 272 'signalling' mutants revealed that, apart from the previously identified Rim15, Yak1 and Mck1 kinases, the SNF1/AMPK complex, the cell wall integrity (CWI) pathway and a number of cell cycle regulators are necessary to elicit appropriate stress response. The chronological lifespan (CLS) of these signalling mutants correlates well with the amount of accumulated storage carbohydrates but poorly with transition-phase cell cycle status. Subsequent analyses reveal that the levels of intracellular reactive oxygen species are controlled by Rim15, Yak1 and Mck1. Furthermore, CLS extension enabled by tor1 deletion is dependent on the above three kinases. These data suggest that the signalling pathways (SNF1 and CWI) and the kinases downstream of TOR/PKA (Rim15, Yak1 and Mck1) coordinate the metabolic reprogramming (to accumulate storage carbohydrates) and the activation of anti-oxidant defence systems (to control ROS levels) to extend chronological lifespan. A 'genome-wide' screening of a haploid deletion library indicates that less than 10% of the non-essential genes are implicated in the regulation of starvation-induced stress response. Gene ontology analysis suggests that they can be grouped into major clusters including mitochondrial function, r-RNA processing, DNA damage and repair, transcription from RNA polymerase and cell cycle regulation. Further phenotypic assays confirm the previous observation that CLS extension is mostly correlated with the accumulation of storage carbohydrates. Compromised expression of stress response reporters is confirmed by FACS in a variety of mitochondrial mutants, suggesting that mitochondrial respiration also plays a key role in the activation of stress response. Put together, the above findings indicate that stress response and metabolic reprogramming induced by glucose starvation are coordinated by multiple signalling pathways and the activation of mitochondrial respiration is essential to both cellular processes and to CLS extension.

A variety of genes that influence aging have been identified in a broad selection of organisms including Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worms), Drosophila (fruit flies), Macaca Mulatta (rhesus monkeys), and even Homo sapiens. Many of these genes, such the TOR’s, FOXO’s, AKT’s, and S6K’s are conserved across different organisms. All of these genes participate in nutrient sensing networks. Other conserved genetic networks may similarly affect lifespan. In this thesis, I explored genes from an iron metabolism family and a heat shock protein (HSP) gene family that have been identified, but not confirmed, to influence lifespan. Yeast is a reliable model for mitotic (replicative) aging. Using yeast, I tested whether the FET-genes, encoding a family of iron importer-related genes, are required for mitotic lifespan. I also tested whether another family of genes, the yeast SSA HSP70- encoding genes, related to mammalian HSP70s, influence mitotic aging. I primarily used the replicative lifespan (RLS) assay, in which I measured the mitotic capacity of multiple FET and SSA yeast mutants. I hypothesize that aging occurs when iron transport is misregulated, which may lead to an over-reliance on HSPs for lifespan maintenance. The results presented in this thesis support the hypothesis. First, FET3 was primarily involved in lifespan maintenance under normal conditions (2% glucose), while FET5 was primarily involved in the cellular lifespan extension characteristic of caloric restriction (0.01% glucose), a known anti-aging intervention. In addition, SSA2 appeared to facilitate lifespan maintenance in the absence of FET4, while the presence of SSA1 limited lifespan length. That the aging genes identified in this study are involved in iron metabolism or heat stress suggests that protein aggregation or reactive oxidative species production are common processes through which these genes interact.

In the budding yeast Saccharomyces cerevisiae, the evolutionarily conserved Target of Rapamycin (TOR) signaling network regulates cell growth in accordance with nutrient and stress conditions. In this work, I present evidence that the TOR complex 1 (TORC1)-interacting proteins Nnk1, Fmp48, Mks1, and Sch9 link TOR to various facets of nitrogen metabolism and mitochondrial function. The Nnk1 kinase controlled nitrogen catabolite repression-sensitive gene expression via Ure2 and Gln3, and physically interacted with the NAD+-linked glutamate dehydrogenase Gdh2 that catalyzes deamination of glutamate to alpha-ketoglutarate and ammonia. In turn, Gdh2 modulated rapamycin sensitivity, was phosphorylated in Nnk1 immune complexes in vitro, and was relocalized to a discrete cytoplasmic focus in response to NNK1 overexpression or respiratory growth. The Fmp48 kinase regulated respiratory function and mitochondrial morphology, while Mks1 linked TORC1 to the mitochondria-to-nucleus retrograde signaling pathway. The Sch9 kinase appeared to act as both an upstream regulator and downstream sensor of mitochondrial function. Loss of Sch9 conferred a respiratory growth defect, a defect in mitochondrial DNA transmission, lower mitochondrial membrane potential, and decreased levels of reactive oxygen species. Conversely, loss of mitochondrial DNA caused loss of Sch9 enrichment at the vacuolar membrane, loss of Sch9 phospho-isoforms, and small cell size suggestive of reduced Sch9 activity. Sch9 also exhibited dynamic relocalization in response to stress, including enrichment at mitochondria under conditions that have previously been shown to induce apoptosis in yeast. Taken together, this work reveals intimate connections between TORC1, nitrogen metabolism, and mitochondrial function, and has implications for the role of TOR in regulating aging, cancer, and other human diseases.