Stress and ageing in yeast.

There has long been speculation about the role of various stresses in ageing. Some stresses have beneficial effects on ageing-dependent on duration and severity of the stress, others have negative effects and the question arises whether these negative effects are causative of ageing or the result of the ageing process. Cellular responses to many stresses are highly coordinated in a concerted way and hence there is a great deal of cross-talk between different stresses. Here the relevant aspects of the coordination of stress responses and the roles of different stresses on yeast cell ageing are discussed, together with the various functions that are involved. The cellular processes that are involved in alleviating the effects of stress on ageing are considered, together with the possible role of early stress events on subsequent ageing of cells.

CDP-diacylglycerol synthases regulate the growth of lipid droplets and adipocyte development.

The expansion of lipid droplets (LDs) and the differentiation of preadipocytes are two important aspects of mammalian lipid storage. In this study, we examined the role of CDP-diacylglycerol (DAG) synthases (CDSs), encoded by CDS1 and CDS2 genes in mammals, in lipid storage. CDS enzymes catalyze the formation of CDP-DAG from phosphatidic acid (PA). Knocking down either CDS1 or CDS2 resulted in the formation of giant or supersized LDs in cultured cells. Moreover, depleting CDS1 almost completely blocked the differentiation of 3T3-L1 preadipocytes, whereas depleting CDS2 had a moderate inhibitory effect on adipocyte differentiation. The levels of many PA species were significantly increased upon knocking down CDS1 In contrast, only a small number of PA species were increased upon depleting CDS2 Importantly, the amount of PA in the endoplasmic reticulum was dramatically increased upon knocking down CDS1 or CDS2 Our results suggest that the changes in PA level and localization may underlie the formation of giant LDs as well as the block in adipogenesis in CDS-deficient cells. We have therefore identified CDS1 and CDS2 as important novel regulators of lipid storage, and these results highlight the crucial role of phospholipids in mammalian lipid storage.

Ian Dawes-the third Pope-lucky to be a researcher.

Retrospective articles are an excuse for a rosy tinted view of one's life. This fully expurgated version is no exception. No "what the butler saw" or the vilification of enemies that one finds in political autobiographies - merely the account of one born to a generation of those whose forebears never had the chance to go to university and enjoy the subsequent fruits of that education - and of one who by chance stumbled into the world of yeast genetics and molecular biology, who had a lot of fun on the way and who never sought to leave it.

The AAA ATPase VPS4/SKD1 regulates endosomal cholesterol trafficking independently of ESCRT-III.

The exit of low-density lipoprotein derived cholesterol (LDL-C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann-Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol-binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N-terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well-established role in disassembling the ESCRT (endosomal sorting complex required for transport)-III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL-C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT-III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT-III.

Transcriptional and antioxidative responses to endogenous polyunsaturated fatty acid accumulation in yeast.

Pathophysiology of polyunsaturated fatty acids (PUFAs) is associated with aberrant lipid and oxygen metabolism. In particular, under oxidative stress, PUFAs are prone to autocatalytic degradation via peroxidation, leading to formation of reactive aldehydes with numerous potentially harmful effects. However, the pathological and compensatory mechanisms induced by lipid peroxidation are very complex and not sufficiently understood. In our study, we have used yeast capable of endogenous PUFA synthesis in order to understand the effects triggered by PUFA accumulation on cellular physiology of a eukaryotic organism. The mechanisms induced by PUFA accumulation in S. cerevisiae expressing Hevea brasiliensis Δ12-fatty acid desaturase include down-regulation of components of electron transport chain in mitochondria as well as up-regulation of pentose-phosphate pathway and fatty acid ß-oxidation at the transcriptional level. Interestingly, while no changes were observed at the transcriptional level, activities of two important enzymatic antioxidants, catalase and glutathione-S-transferase, were altered in response to PUFA accumulation. Increased intracellular glutathione levels further suggest an endogenous oxidative stress and activation of antioxidative defense mechanisms under conditions of PUFA accumulation. Finally, our data suggest that PUFA in cell membrane causes metabolic changes which in turn lead to adaptation to endogenous oxidative stress.

Cellular redox homeostasis, reactive oxygen species and replicative ageing in Saccharomyces cerevisiae.

Ageing cells undergo changes in redox homeostasis and acquire high levels of reactive oxygen species (ROS). Because accumulation of ROS involves a change in redox state of cells, functions that are involved in setting redox and maintaining redox homeostasis are very relevant to an understanding of the possible roles of redox homeostasis and ROS in ageing. This review discusses these aspects of ROS in relation to replicative ageing in the model organism Saccharomyces cerevisiae, with reference to ROS generated in cells; cellular responses to oxidative stress; and how cells maintain redox homeostasis in different cellular compartments. It also considers when ROS generation begins as cells age, which ROS species are relevant to ageing and which cellular compartments and processes may contribute ROS to the ageing process. The discussion also covers the heterogeneity of cells with respect to ROS accumulation at particular cell ages, and the possibility of testing the oxidative theory of ageing in yeast cells.

Actin - a biosensor that determines cell fate in yeasts.

The decision to proliferate, to activate stress response mechanisms or to initiate cell death lies at the heart of the maintenance of a healthy cell population. Within multicellular and colony-forming single-celled organisms, such as yeasts, the functionality of cellular compartments that connect signalling to cell fate must be maintained to maximise adaptability and survival. The actin cytoskeleton is involved in processes such as the regulation of membrane microcompartments, receptor internalisation and the control of master regulatory GTPases, which govern cell decision-making. This affords the actin cytoskeleton a central position within cell response networks. In this sense, a functional actin cytoskeleton is essential to efficiently connect information input to response at the level of the cell. Recent research from fungal, plant and mammalian cells systems has highlighted that actin can trigger apoptotic death in cells that become incompetent to respond to environmental cues. It may also be the case that this property has been appropriated by microorganisms competing for niche environments within a human host. Here, we discuss the research that has been carried out in yeast that links actin to signalling processes and cell fate that supports its role as a biosensor.

A role for phosphatidic acid in the formation of "supersized" lipid droplets.

Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing "supersized" LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of "supersized" LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism.

Cellular responses to L-serine in Saccharomyces cerevisiae: roles of general amino acid control, compartmentalization, and aspartate synthesis.

In addition to its other roles, L-serine functions in one-carbon metabolism and is interconvertable with glycine via serine hydroxymethyltransferases. However, the transcriptional response by Saccharomyces cerevisiae to L-serine addition is markedly different from that to glycine, with L-serine acting as a nutrient source rather than one-carbon units. Following addition of excess L-serine, 743 genes showed significant expression changes. Induced functions included amino acid synthesis, some stress responses, and FeS metabolism, while ribosomal RNA processing, ribosome biogenesis and hexose transport were repressed. A co-regulated network of ten transcription factors could together control more than 90% of the induced and repressed genes forming a general response to changes induced by other amino acids or stresses and including the general amino acid control system usually activated in response to starvation for amino acids. A specific response to L-serine was induction of CHA1 encoding serine (threonine) dehydratase. L-serine addition resulted in a substantial transient increase in L-aspartate, which is, rather than L-glutamate, the major metabolite for short-term storage of ammonia derived from degradation of L-serine. L-aspartate synthesis was exclusively through mitochondrial metabolism of L-serine to pyruvate and ammonia, involving Cha1p, cytoplasmic pyruvate carboxylases Pyc1p and Pyc2p, and the cytoplasmic aspartate aminotransferase Aat2p.

Oxidative stresses and ageing.

Oxidative damage to cellular constituents has frequently been associated with aging in a wide range of organisms. The power of yeast genetics and biochemistry has provided the opportunity to analyse in some detail how reactive oxygen and nitrogen species arise in cells, how cells respond to the damage that these reactive species cause, and to begin to dissect how these species may be involved in the ageing process. This chapter reviews the major sources of reactive oxygen species that occur in yeast cells, the damage they cause and how cells sense and respond to this damage.

The role of mitochondria in the aging processes of yeast.

This chapter reviews the role of mitochondria and of mitochondrial metabolism in the aging processes of yeast and the existing evidence for the "mitochondrial theory of aging mitochondrial theory of aging ". Mitochondria are the major source of ATP in the eukaryotic cell but are also a major source of reactive oxygen species reactive oxygen species (ROS) and play an important role in the process of apoptosis and aging. We are discussing the mitochondrial theory of aging mitochondrial theory of aging (TOA), its origin, similarity with other TOAs, and its ramifications which developed in recent decades. The emphasis is on mother cell-specific aging mother cell-specific aging and the RLS (replicative lifespan) with only a short treatment of CLS (chronological lifespan). Both of these aging processes may be relevant to understand also the aging of higher organisms, but they are biochemically very different, as shown by the fact the replicative aging occurs on rich media and is a defect in the replicative capacity of mother cells, while chronological aging occurs in postmitotic cells that are under starvation conditions in stationary phase leading to loss of viability, as discussed elsewhere in this book. In so doing we also give an overview of the similarities and dissimilarities of the various aging processes of the most often used model organisms for aging research with respect to the mitochondrial theory of aging mitochondrial theory of aging.

Cell cycle sensing of oxidative stress in Saccharomyces cerevisiae by oxidation of a specific cysteine residue in the transcription factor Swi6p.

Yeast cells begin to bud and enter the S phase when growth conditions are favorable during the G(1) phase. When subjected to some oxidative stresses, cells delay entry at G(1), allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell cycle regulator of Saccharomyces cerevisiae, Swi6p, as a redox sensor through its cysteine residue at position 404. When alanine was substituted at this position, the resultant mutant, C404A, was sensitive to several reactive oxygen species and oxidants including linoleic acid hydroperoxide, the superoxide anion, and diamide. This mutant lost the ability to arrest in G(1) phase upon treatment with lipid hydroperoxide. The Cys-404 residue of Swi6p in wild-type cells was oxidized to a sulfenic acid when cells were subjected to linoleic acid hydroperoxide. Mutation of Cys-404 to Ala abolished the down-regulation of expression of the G(1) cyclin genes CLN1, CLN2, PCL1, and PCL2 that occurred when cells of the wild type were exposed to the lipid hydroperoxide. In conclusion, oxidative stress signaling for cell cycle regulation occurs through oxidation of the G(1)/S-specific transcription factor Swi6p and consequently leads to suppression of the expression of G(1) cyclins and a delay in cells entering the cell cycle.

The yeast homolog of heme oxygenase-1 affords cellular antioxidant protection via the transcriptional regulation of known antioxidant genes.

Heme oxygenase-1 (HO-1) degrades heme and protects cells from oxidative challenge. This antioxidant activity is thought to result from the HO-1 enzymatic activity, manifested by a decrease in the concentration of the pro-oxidant substrate heme, and an increase in the antioxidant product bilirubin. Using a global transcriptional approach, and yeast as a model, we show that HO-1 affords cellular protection via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense, rather than via its oxygenase activity. Like mammalian cells, yeast responds to oxidative stress by expressing its HO-1 homolog and, compared with the wild type, heme oxygenase-null mutant cells have increased sensitivity toward oxidants that is rescued by overexpression of human HO-1 or its yeast homolog. Increased oxidant sensitivity of heme oxygenase-null mutant cells is explained by a decrease in the expression of the genes encoding γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, and methionine sulfoxide reductase, because overexpression of any of these genes affords partial, and overexpression of all four genes provides complete, protection to the null mutant. Genes encoding antioxidant enzymes represent only a small portion of the 480 differentially expressed transcripts in heme oxygenase-null mutants. Transcriptional regulation may be explained by the nuclear localization of heme oxygenase observed in oxidant-challenged cells. Our results challenge the notion that HO-1 functions simply as a catabolic and antioxidant enzyme. They indicate much broader functions for HO-1, the unraveling of which may help explain the multiple biological responses reported in animals as a result of altered HO-1 expression.

The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae.

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E(h)'. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E(h)'. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae.

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

PI3K(p110 alpha) protects against myocardial infarction-induced heart failure: identification of PI3K-regulated miRNA and mRNA.

OBJECTIVE: Myocardial infarction (MI) is a serious complication of atherosclerosis associated with increasing mortality attributable to heart failure. Activation of phosphoinositide 3-kinase [PI3K(p110 alpha)] is considered a new strategy for the treatment of heart failure. However, whether PI3K(p110 alpha) provides protection in a setting of MI is unknown, and PI3K(p110 alpha) is difficult to target because it has multiple actions in numerous cell types. The goal of this study was to assess whether PI3K(p110 alpha) is beneficial in a setting of MI and, if so, to identify cardiac-selective microRNA and mRNA that mediate the protective properties of PI3K(p110 alpha). METHODS AND RESULTS: Cardiomyocyte-specific transgenic mice with increased or decreased PI3K(p110 alpha) activity (caPI3K-Tg and dnPI3K-Tg, respectively) were subjected to MI for 8 weeks. The caPI3K-Tg subjected to MI had better cardiac function than nontransgenic mice, whereas dnPI3K-Tg had worse function. Using microarray analysis, we identified PI3K-regulated miRNA and mRNA that were correlated with cardiac function, including growth factor receptor-bound 14. Growth factor receptor-bound 14 is highly expressed in the heart and positively correlated with PI3K(p110 alpha) activity and cardiac function. Mice deficient in growth factor receptor-bound 14 have cardiac dysfunction. CONCLUSIONS: Activation of PI3K(p110 alpha) protects the heart against MI-induced heart failure. Cardiac-selective targets that mediate the protective effects of PI3K(p110 alpha) represent new drug targets for heart failure.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Complex cellular responses to reactive oxygen species.

Genome-wide analyses of yeast provide insight into cellular responses to reactive oxygen species (ROS). Many deletion mutants are sensitive to at least one ROS, but no one oxidant is representative of 'oxidative stress' despite the widespread use of a single compound such as H(2)O(2). This has major implications for studies of pathological situations. Cells have a range of mechanisms for maintaining resistance that involves either induction or repression of many genes and extensive remodeling of the transcriptome. Cells have constitutive defense systems that are largely unique to each oxidant, but overlapping, inducible repair systems. The pattern of the transcriptional response to a particular ROS depends on its concentration, and 'classical' antioxidant systems that are induced by high concentrations of ROS can be repressed when cells adapt to low concentrations of ROS.

Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae.

BACKGROUND: Protein-SH groups are amongst the most easily oxidized residues in proteins, but irreversible oxidation can be prevented by protein glutathionylation, in which protein-SH groups form mixed disulphides with glutathione. Glutaredoxins and thioredoxins are key oxidoreductases which have been implicated in regulating glutathionylation/deglutathionylation in diverse organisms. Glutaredoxins have been proposed to be the predominant deglutathionylase enzymes in many plant and mammalian species, whereas, thioredoxins have generally been thought to be relatively inefficient in deglutathionylation. RESULTS: We show here that the levels of glutathionylated proteins in yeast are regulated in parallel with the growth cycle, and are maximal during stationary phase growth. This increase in glutathionylation is not a response to increased reactive oxygen species generated from the shift to respiratory metabolism, but appears to be a general response to starvation conditions. Our data indicate that glutathionylation levels are constitutively high in all growth phases in thioredoxin mutants and are unaffected in glutaredoxin mutants. We have confirmed that thioredoxins, but not glutaredoxins, catalyse deglutathionylation of model glutathionylated substrates using purified thioredoxin and glutaredoxin proteins. Furthermore, we show that the deglutathionylase activity of thioredoxins is required to reduce the high levels of glutathionylation in stationary phase cells, which occurs as cells exit stationary phase and resume vegetative growth. CONCLUSIONS: There is increasing evidence that the thioredoxin and glutathione redox systems have overlapping functions and these present data indicate that the thioredoxin system plays a key role in regulating the modification of proteins by the glutathione system.

Reactive oxygen species and yeast apoptosis.

Apoptosis is associated in many cases with the generation of reactive oxygen species (ROS) in cells across a wide range of organisms including lower eukaryotes such as the yeast Saccharomyces cerevisiae. Currently there are many unresolved questions concerning the relationship between apoptosis and the generation of ROS. These include which ROS are involved in apoptosis, what mechanisms and targets are important and whether apoptosis is triggered by ROS damage or ROS are generated as a consequence or part of the cellular disruption that occurs during cell death. Here we review the nature of the ROS involved, the damage they cause to cells, summarise the responses of S. cerevisiae to ROS and discuss those aspects in which ROS affect cell integrity that may be relevant to the apoptotic process.