The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions.

Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease-age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.Work in the Ralser lab is funded from the Wellcome Trust (RG 093735/Z/10/Z), the ERC (Starting grant 260809). Markus A. Keller is supported by the Austrian Science Funds by an Erwin Schrödinger postdoctoral fellowship (FWF, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This is the final version of the article. It first appeared from MDPI via http://dx.doi.org/10.3390/biom503210

The widespread role of non-enzymatic reactions in cellular metabolism.

Enzymes shape cellular metabolism, are regulated, fast, and for most cases specific. Enzymes do not however prevent the parallel occurrence of non-enzymatic reactions. Non-enzymatic reactions were important for the evolution of metabolic pathways, but are retained as part of the modern metabolic network. They divide into unspecific chemical reactivity and specific reactions that occur either exclusively non-enzymatically as part of the metabolic network, or in parallel to existing enzyme functions. Non-enzymatic reactions resemble catalytic mechanisms as found in all major enzyme classes and occur spontaneously, small molecule (e.g. metal-) catalyzed or light-induced. The frequent occurrence of non-enzymatic reactions impacts on stability and metabolic network structure, and has thus to be considered in the context of metabolic disease, network modeling, biotechnology and drug design.We acknowledge funding from the Wellcome Trust (RG 093735/Z/10/Z), the ERC (starting Grant 260809). Markus A Keller is supported by the Austrian Science Funds by an Erwin Schroeder postdoctoral fellowship (FWF, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This paper was originally published in Current Opinion in Biotechnology (Keller MA, Piedrafita G, Ralser M, Current Opinion in Biotechnology 2015, 34, 153–161, doi:10.1016/j.copbio.2014.12.020)

Reductive stress on life span extension in C. elegans

Recently, Schulz and colleagues have contributed to the ongoing controversy on the unproven role of oxidative stress in the aging process in their well-performed study 'Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress' (Cell Metab 2007, 6: 280–293). Here, we suggest an alternative hypothesis that reductive stress can prevent calorie-restriction induced life span extension. We draw attention to this condition as an explanation for some contradictory observations including the deleterious effects from antioxidants

Surviving in the cold: yeast mutants with extended hibernating lifespan are oxidant sensitive

Metabolic activity generates oxidizing molecules throughout life, but it is still debated if the resulting damage of macromolecules is a causality, or consequence, of the aging process. This problem demands for studying growth- and longevity phenotypes separately. Here, we assayed a complete collection of haploid Saccharomyces cerevisiae knock-out strains for their capacity to endure long periods at low metabolic rates. Deletion of 93 genes, predominantly factors of primary metabolism, allowed yeast to survive for more than 58 months in the cold. The majority of these deletion strains were not resistant against oxidants or reductants, but many were hypersensitive. Hence, survival at low metabolic rates has limiting genetic components, and correlates with stress resistance inversely. Indeed, maintaining the energy consuming anti-oxidative machinery seems to be disadvantageous under coldroom conditions

Cell-to-cell heterogeneity emerges as consequence of metabolic cooperation in a synthetic yeast community.

Cells that grow together respond heterogeneously to stress even when they are genetically similar. Metabolism, a key determinant of cellular stress tolerance, may be one source of this phenotypic heterogeneity, however, this relationship is largely unclear. We used self-establishing metabolically cooperating (SeMeCo) yeast communities, in which metabolic cooperation can be followed on the basis of genotype, as a model to dissect the role of metabolic cooperation in single-cell heterogeneity. Cells within SeMeCo communities showed to be highly heterogeneous in their stress tolerance, while the survival of each cell under heat or oxidative stress, was strongly determined by its metabolic specialization. This heterogeneity emerged for all metabolite exchange interactions studied (histidine, leucine, uracil, and methionine) as well as oxidant (H2 O2 , diamide) and heat stress treatments. In contrast, the SeMeCo community collectively showed to be similarly tolerant to stress as wild-type populations. Moreover, stress heterogeneity did not establish as sole consequence of metabolic genotype (auxotrophic background) of the single cell, but was observed only for cells that cooperated according to their metabolic capacity. We therefore conclude that phenotypic heterogeneity and cell to cell differences in stress tolerance are emergent properties when cells cooperate in metabolism.Wellcome Trust (Grant ID: RG 093735/Z/10/Z) and the European Research Council (Starting grant 260809)This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/biot.20150030

A haploproficient interaction of the transaldolase paralogue NQM1 with the transcription factor VHR1 affects stationary phase survival and oxidative stress resistance.

BACKGROUND: Studying the survival of yeast in stationary phase, known as chronological lifespan, led to the identification of molecular ageing factors conserved from yeast to higher organisms. To identify functional interactions among yeast chronological ageing genes, we conducted a haploproficiency screen on the basis of previously identified long-living mutants. For this, we created a library of heterozygous Saccharomyces cerevisiae double deletion strains and aged them in a competitive manner. RESULTS: Stationary phase survival was prolonged in a double heterozygous mutant of the metabolic enzyme non-quiescent mutant 1 (NQM1), a paralogue to the pentose phosphate pathway enzyme transaldolase (TAL1), and the transcription factor vitamin H response transcription factor 1 (VHR1). We find that cells deleted for the two genes possess increased clonogenicity at late stages of stationary phase survival, but find no indication that the mutations delay initial mortality upon reaching stationary phase, canonically defined as an extension of chronological lifespan. We show that both genes influence the concentration of metabolites of glycolysis and the pentose phosphate pathway, central metabolic players in the ageing process, and affect osmolality of growth media in stationary phase cultures. Moreover, NQM1 is glucose repressed and induced in a VHR1 dependent manner upon caloric restriction, on non-fermentable carbon sources, as well as under osmotic and oxidative stress. Finally, deletion of NQM1 is shown to confer resistance to oxidizing substances. CONCLUSIONS: The transaldolase paralogue NQM1 and the transcription factor VHR1 interact haploproficiently and affect yeast stationary phase survival. The glucose repressed NQM1 gene is induced under various stress conditions, affects stress resistance and this process is dependent on VHR1. While NQM1 appears not to function in the pentose phosphate pathway, the interplay of NQM1 with VHR1 influences the yeast metabolic homeostasis and stress tolerance during stationary phase, processes associated with yeast ageing.We thank the Max Planck Society, Wellcome Trust (RG 093735/Z/10/Z), the ERC (Starting grant 260809), and the Isaac Newton Trust for funding. Markus A Keller is supported by an Erwin Schroedinger postdoctoral fellowship (FWF, Austria, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This is the final version of the article. It first appeared from BMC via http://dx.doi.org/10.1186/s12863-015-0171-

MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells.

Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available.This work was supported by funding from the Wellcome Trust (RG 093735/Z/10/Z) and the ERC (Starting grant 260809). M.R. is a Wellcome Trust Research Career Development and Wellcome-Beit prize fellow.This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S1567724915300088

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis.

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK-TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress

THADA regulates the organismal balance between energy storage and heat production

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. We uncover here the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. We report here that THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution