Metabolic switches regulating NADPH availability and stress resistance
Cells protect themselves against oxidative stress by dynamic rerouting of metabolic fluxes. Metabolic reconfigurations occur both as a rapid response to acute oxidative stress or as an anticipatory mechanism that renders cells more robust while the metabolic environment is favorable. In this project, we will address how both mechanisms serve to either regenerate or save redox coenzymes, NADPH in particular, to reverse the oxidation of bio-molecules, and to protect cells from oxidative stress. Combining the expertise of both labs, we will screen for novel metabolic switches in yeast, and elaborate the mechanisms and functions of metabolic switches in mammalian cells. In the first part, we will investigate how cells use metabolic adaptations as a preventive protective measure in anticipation of future stress situations. We recently observed that microbial cells harvest large amounts of lysine when available in the environment and do so to re-configure metabolism for enhanced stress protection (Olin-Sandoval et al, Nature, 2019). Hence, metabolite uptake not only serves cell growth and proliferation, but also to enhance robustness. Mechanistically, preventive stress protection is mediated by a re-configuration of redox metabolism that saves reducing equivalents. NADPH is generated at standard flux (i.e. in the pentose phosphate pathway) but required at lower flux by anabolism. Instead of re- ducing flux into the non-required metabolite, which would be the conventional cellular reaction to a metabolite overproduction, cells however create an NADPH overflow. The NADPH overflow is then channeled into pro- tective pathways, in particular glutathione biosynthesis, to render cells stress tolerant. We will address several new hypotheses about how metabolism is influencing stress tolerance by increasing robustness. Which me- tabolites are taken up to confer stress tolerance rather than growth? How does the natural genetic diversity impact metabolite uptake and stress tolerance? What are the metabolic switches that reconfigure the cellular metabolome to enable stress protection, and how is the process regulated both at the genetic as well as the biochemical level? We will address these questions by screening a collection of 1011 yeast wild strains for differences in stress tolerance, and by using metabolomic, proteomic, and biochemical methods to identify the underlying metabolic differences. Emphasis is placed on the reconfiguration of NADPH metabolism. The second part of the proposal aims to bring the knowledge obtained from the analysis of microorganisms to mam- malian cells. As the yeast screening results will not be available from the start of the project, we will first focus on a previously defined molecular switch involved in the reorganization of metabolism in response to oxidative stress. This binary switch, originally discovered by the Ralser lab (J Biol, 2007) in yeast, and explained mech- anistically by the Dick lab (Nat Chem Biol, 2015) is the redox state of the active site cysteine of GAPDH. GAPDH is oxidized (and hence inactivated) upon elevation of endogenous H2O2 above homeostatic levels. Inhibition of GAPDH supports the rerouting of glucose into the oxidative pentose phosphate pathway, hence boosting NADPH regeneration from NADP+. Highly cnserved side chains catalyze the reaction between H2O2 and the active site thiol. Their mutagenesis selectively abolishes oxidation sensitivity without affecting canonical enzyme activity. This insight now allows for the engineering of mammalian cell lines and transgenic mice which differ from their isogenic wild type counterparts only by the inability of GAPDH to respond to H2O2. We are now able to ask how the presence or absence of GAPDH oxidation sensitivity affects metabolic flux under various metabolic and stress conditions. Furthermore, we are asking if GAPDH oxidation sensitivity is important for tumor cells to grow and form metastases. The Dick and Ralser labs have highly complementary expertise. The Dick lab has a long-standing expertise in redox biochemistry, e.g. they developed redox bio- sensors and characterized thiol-based redox switches, including the one that facilitates oxidative GAPDH in- activation. The Ralser lab has long-standing expertise in the analysis of dynamic metabolic adaptations, e.g. they described for the first time the role of metabolic switches in stress protection, and with lysine harvesting, also described the first preventative metabolic adaptation. The Dick lab will profit from the expertise of the Ralser lab in metabolomics and biological mass spectrometry, to be used in this project to discover and quantify stress-responsive metabolites at the system level. The Ralser lab will profit from the expertise of the Dick lab in the detailed analysis of redox changes and oxidative stress phenomena, e.g. by using biosensor tools developed by the Dick lab, to screen for new metabolic anti-stress mechanisms in yeast.