Expanding the set of genetically encoded tools for compartment-specific manipulation of redox metabolism in living cells

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Abstract The metabolic environment that cells face has profound effects on cellular behavior. This is especially true for the reduction-oxidation (redox) environment, but many aspects of how redox metabolism is regulated and how it directs cellular decisions are poorly understood. In order to systematically address these pressing questions, it is necessary to have tools with which key contributors to the cellular redox environment can be safely and directly modulated with spatial and, most importantly, temporal resolution. We previously used a H2O-forming NADH oxidase from Lactobacillus brevis (LbNOX) to decrease the NADH/NAD+ ratio when ectopically expressed in cytoplasm or mitochondria of mammalian cells. Furthermore, we engineered a variant of this enzyme with strict specificity towards NADPH (TPNOX). We subsequently employed both LbNOX and TPNOX as genetically encoded tools to show that NAD+ regeneration but not ATP production is a critical requirement of proliferation of mammalian cells. In our original MIRA ESI application, we plan to continue development of evolution-inspired, genetically encoded tools for spatiotemporal modulation of key cellular redox parameters. In Project 1, we plan to expand our toolkit by developing a genetically encoded tool for the direct modulation of NADH reductive stress (i.e. increased NADH/NAD+ ratio). In Project 2, we will elucidate the metabolic and cellular consequences of the NADH reductive stress in various backgrounds. We will use Drosophila flies to directly test whether redox modulation in either the oxidative or reductive direction is correlated with stress resistance, healthspan and lifespan. In Project 3 we will combine protein engineering and imaging techniques to develop versions of our tools where the corresponding enzymatic activity is controlled by small molecule or light stimulation to achieve temporal control of the corresponding redox pairs. Using our tools, we will also illuminate the role of various redox active small molecules, including systemic mitochondrial complex I inhibition and associated redox imbalance, in the progression of neuronal loss in Parkinson’s disease (PD). This Administrative Supplement requests the acquisition of a BioTek Cytation C10 confocal imaging reader, which would allow us to use automated microscopy to quantify multiple cell parameters simultaneously, including cellular size and shape, morphological and functional changes in subcellular structures, inter-organelle communication and to image fluorescence-based biosensors. In summary, access to a BioTek Cytation C10 instrument will significantly accelerate experiments described in Projects 1-3.