MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae

Calorie restriction (CR) robustly extends the lifespan of numerous species. In the yeast Saccharomyces cerevisiae, CR has been proposed to extend lifespan by boosting the activity of sirtuin deacetylases, thereby suppressing the formation of toxic repetitive ribosomal DNA (rDNA) circles. An alternative theory is that CR works by suppressing the TOR (target of rapamycin) signaling pathway, which extends lifespan via mechanisms that are unknown but thought to be independent of sirtuins. Here we show that TOR inhibition extends lifespan by the same mechanism as CR: by increasing Sir2p activity and stabilizing the rDNA locus. Further, we show that rDNA stabilization and lifespan extension by both CR and TOR signaling is due to the relocalization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1. These findings suggest that TOR and sirtuins may be part of the same longevity pathway in higher organisms, and that they may promote genomic stability during aging

Analysis of Cellular Responses to Heavy Metal-induced Stress in Saccharomyces cerevisiae

Chronic exposure of heavy metals is highly correlated with the epidemic of degenerative disease, nephrotoxicity, cancers and aging, while the acute response of cells to the same heavy metals provides some nuanced insights into how cells are able to handle environmental insults, and perhaps characterize specific triggers of the process, itself. Some heavy metals, such as copper, trigger an immediate accumulation of reactive oxygen species (ROS), which impair vital cellular functions by oxidative stresses; which can lead to the onset of programmed cell death, or apoptosis, which becomes an inevitable fate once the damage is too disseminative to be recovered. Other heavy metals, such as cadmium, appear to trigger similar apoptotic responses in the cell –even before ROS increases to unmanageable levels. In either instance, however, before undergoing apoptosis, there are two cellular defensive mechanisms that are able to eliminate the metal-induced oxidative stresses: 1) the neutralization of anti-oxidants, and 2) the removal of the harmful substances through a series of self-cleaning mechanisms. We have used Saccharomyces cerevisiae, or baker’s yeast, as a model organism to demonstrate the response of cells to the presence of heavy metals. In so doing, we highlight pertinent aspects of the metabolic transcriptome response of these unicellular organisms to the presence of these metals, such as changes in expression of genes involved in the pentose phosphate pathway (PPP), which facilitates the reduction of oxidative glutathione, or induction of the genes most commonly associated with autophagy. These findings serve to indicate the protective mechanisms that are triggered upon metal exposures in yeast. Curiously, we also discovered that the autophagic response may be duplicitous, in that while the autophagic process can be cyto-protective it can also enhance the self-destructive mechanisms of the apoptotic response, indeed it is appears to be a requisite part of that response. Whether or not the cells respond to the cellular stress by autophagy or apoptosis appears to be “decided” by whether or not a full-blown autophagic response to cellular stressors (which can be independently induced by the drug, rapamycin) is initiated before the same autophagic process is able to trigger activation of a caspase-induced apoptosis. In addition, in order to monitor the autophagic process more carefully, we have developed a cytometric methodology to assess the autophagy flow, that is less labor-intensive and more dynamic than the traditional Western blot-based method. In so doing we have been able to decipher the factors of cell fate decision with heavy metal-induced oxidative stress in S. cerevisiae

Yeast V-ATPase Regulation by Phosphofructokinase-1

V-ATPase is a vacuolar (lysosome-like) ATPase-dependent proton pump necessary for maintaining pH homeostasis in the organelles of the endomembrane system. It also contributes to regulation of the cytosol pH and the extracellular pH. In specialized cells (renal intercalated cells, epididymis clear cells, and osteoclasts), V-ATPase proton transport supports urinary acidification, sperm maturation, and bone resorption. Genetic mutations of V-ATPase expressed in those tissue-specific cells cause distal renal tubular acidosis, infertility, and osteopetrosis. V-ATPases are composed of a peripheral domain (V1), which hydrolyzes ATP, and a membrane-bound domain (Vo), which transports proton. V-ATPase activity is tightly regulated in vivo by numbers of mechanisms, including reversible disassembly of the V1 and Vo domains. Glucose, the nutrient oxidized in glycolysis, modulates reversible dissociation of V-ATPase. This dissertation was aimed at understanding how subunits of phosphofructokinase-1 (α subunit and β subunit) regulate V-ATPase function. Our results showed that both subunits are important for V-ATPase activity, but β subunit displayed more significant phenotypes. Deletion of β subunit reduced glucose-dependent V1Vo reassembly and altered V-ATPase binding to its assembly factor, RAVE. We additionally investigated the mechanisms by which phosphofructokinase-1 controls V-ATPase function. We concluded that glucose-dependent V1Vo reassembly and V-ATPase function at steady state were controlled by the glycolytic flux, independently of phosphofructokinase-1. Notably, V-ATPase activation in vivo correlated with the presence of phosphoglycerate kinase at vacuolar membranes. These studies further advanced our understanding how glucose controls V-ATPase pumps in vivo

Dynamic metabolic engineering: New strategies for developing responsive cell factories

Metabolic engineering strategies have enabled improvements in yield and titer for a variety of valuable small molecules produced naturally in microorganisms, as well as those produced via heterologous pathways. Typically, the approaches have been focused on up- and downregulation of genes to redistribute steady-state pathway fluxes, but more recently a number of groups have developed strategies for dynamic regulation, which allows rebalancing of fluxes according to changing conditions in the cell or the fermentation medium. This review highlights some of the recently published work related to dynamic metabolic engineering strategies and explores how advances in high-throughput screening and synthetic biology can support development of new dynamic systems. Dynamic gene expression profiles allow trade-offs between growth and production to be better managed and can help avoid build-up of undesired intermediates. The implementation is more complex relative to static control, but advances in screening techniques and DNA synthesis will continue to drive innovation in this field.National Science Foundation (U.S.) (CBET-0954986)United States. National Institutes of Health (T32GM008334

An appreciation of the prescience of Don Gilbert (1930‐2011): master of the theory and experimental unraveling of biochemical and cellular oscillatory dynamics

We review Don Gilbert's pioneering seminal contributions that both detailed the mathematical principles and the experimental demonstration of several of the key dynamic characteristics of life. Long before it became evident to the wider biochemical community, Gilbert proposed that cellular growth and replication necessitate autodynamic occurrence of cycles of oscillations that initiate, coordinate, and terminate the processes of growth, during which all components are duplicated and become spatially re‐organized in the progeny. Initiation and suppression of replication exhibit switch‐like characteristics: i.e., bifurcations in the values of parameters that separate static and autodynamic behavior. His limit cycle solutions present models developed in a series of papers reported between 1974 and 1984, and these showed that most or even all of the major facets of the cell division cycle could be accommodated. That the cell division cycle may be timed by a multiple of shorter period (ultradian) rhythms, gave further credence to the central importance of oscillatory phenomena and homeodynamics as evident on multiple time scales (seconds to hours). Further application of the concepts inherent in limit cycle operation as hypothesized by Gilbert more than 50 years ago are now validated as being applicable to oscillatory transcript, metabolite and enzyme levels, cellular differentiation, senescence, cancerous states, and cell death. Now, we reiterate especially for students and young colleagues, that these early achievements were even more exceptional, as his own lifetime's work on modeling was continued with experimental work in parallel with his predictions of the major current enterprises of biological research

Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae

To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters. Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity. Disruption of ADY2 in S. cerevisiae abolished the active transport of acetate. Microarray analyses of ady2 strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport. Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate

Regulation of the yeast metabolic cycle by transcription factors with periodic activities

<p>Abstract</p> <p>Background</p> <p>When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns.</p> <p>Results</p> <p>Transcription factor activities were estimated by linear regression using time series and genome-wide transcription factor binding data. Time-translation matrices were estimated using least squares and were used to model the interactions between the most significant transcription factors. The top transcription factors have functions involving respiration, cell cycle events, amino acid metabolism and glycolysis. Key regulators of transitions between phases of the yeast metabolic cycle appear to be Hap1, Hap4, Gcn4, Msn4, Swi6 and Adr1.</p> <p>Conclusions</p> <p>Analysis of the phases at which transcription factor activities peak supports previous findings suggesting that the various cellular functions occur during specific phases of the yeast metabolic cycle.</p

On metabolic and phenotypic diversity in yeast

This thesis explores metabolic and phenotypic diversity in the two model yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Colony screens are a classical and powerful technique for investigating these topics, but there is a lack of modern, scalable bioinformatics tools. To address this need, I have developed pyphe which greatly facilitates colony screen data acquisition and statistical analysis. I explore optimal experimental designs, especially regarding the usefulness of timecourse imaging and colony viability analysis. Pyphe is used in a functional genomics screen, aiming to find functions for a set of largely uncharacterised lincRNAs. We identify hundreds of new lincRNA-associated phenotypes across numerous conditions and compare lincRNA phenotype profiles to those of codinggene mutants. Next, I have used pyphe to investigate the respiration/fermentation balance of wild S. pombe isolates. Contrary to the expectation that glucose completely represses respiration in this Crabtree-positive species, I find that strains generally strike a balance and that individual strains differ significantly in their residual respiration activity. This is associated with an unusual miss-sense variant in S. pombe’s sole pyruvate kinase gene. Its impact is dissected in detail, revealing a change in flux through pyruvate kinase and associated changes in gene expression, metabolism, growth and stress resistance. Finally, I explore how extracellular amino acids interact with cellular metabolism, with the aim of answering the important question whether or not clonal yeast cultures segregate into heterogeneous producer/consumer populations that exchange amino acids. I develop a novel proteomics-based method that characterises amino acid labelling patterns in peptides. I find that the supplementation of some, but not all amino acids completely suppresses selfsynthesis. However, I find no evidence for heterogeneous responses of our laboratory S. cerevisiae strain, but the functionality of the method is demonstrated clearly. Overall, this work represents several advancements to our understanding of yeast metabolism and physiology, as well as new experimental and computational methods