BLR&D Research Career Scientist Award Application

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Our laboratory is focused on understanding how mitochondrial function contributes to health and disease. As the major energy generating organelle of the cell dysfunction of mitochondria has been implicated in debilitating diseases prevalent in the VA patient population. These include, neurodegenerative diseases (Parkinson's, Alzheimer's), diabetes, cancer, and heart disease. Altered mitochondrial metabolism can result in changed levels of tricarboxylic (TCA) cycle metabolites, (such as succinate or fumarate), that act as signaling molecules to promote a pro-inflammatory state. This can lead to changes in gene transcription, through the induction of reactive oxygen species (ROS), stabilization of hypoxia- inducible factor-1α (HIF-1α), or the nuclear factor erythroid-2-related factor-2 (Nrf2) transcription pathway that responds to pro-inflammatory stress. Our laboratory investigates the structure and function of two essential members of the mitochondrial respiratory chain both of which reduce ubiquinone (CoQ10) used by the oxidative phosphorylation system to generate energy. We study the function of Complex I (NADH:ubiquinone oxidoreductase) which is the largest membrane-bound component of the mitochondrion. NADH generated by the TCA cycle is used by Complex I to reduce CoQ10 and this activity controls the NADH/NAD+ ratio. The enzyme is regulated by a structural change near the membrane domain termed the Active/De-Active (A/D) transition, which we first showed occurred in vivo. We also study succinate dehydrogenase (SDH/Complex II) which is a membrane-bound heterotetramer of dual function. SDH oxidizes succinate to fumarate in the TCA cycle while reducing CoQ10 for energy generation. Malfunction of SDH results in accumulation of succinate in the cell which promotes inflammation. It has been shown that inhibitors of SDH can have a positive effect in treating damage form ischemia/reperfusion in both stroke and cardiac models. Our studies of SDH have shown how the reversible inhibitor malonate binds to the enzyme and causes inhibition. We are now focused on understanding how we can regulate the activity and structure of both Complexes I & II so that this information can be used to treat disease. One model we will use is to investigate how TCA cycle metabolites can be used to treat traumatic brain injury (TBI) or stroke. Dimethyl fumarate (DMF) is an approved drug for treating relapsing multiple sclerosis and psoriasis and Dimethyl malonate (DMM) is a cell-permeable non-toxic compound which in vivo can be used to inhibit SDH. We hypothesize that in the brain injury model that DMM will block succinate accumulation following injury and prevent the signaling that produces ROS during ischemia/reperfusion; thus, reducing inflammation, the severity of the injury, and enhance healing. We use mouse models for these studies. We will also determine if the epigenetic modifier DMF can reduce the inflammation caused by TBI thus lessening the severity of the injury and enhance neuro-regeneration. We were the first to determine the x-ray structure of SDH and have provided major insight into its catalytic mechanism and function. How the enzyme complex is assembled, however, remains and area of intense investigation. We are now studying the assembly of human Complex II using known human assembly factors, needed for incorporation of redox cofactors necessary for function of the enzyme. It has been shown that when assembly is compromised this can lead to tumor formation in humans. We have had success expressing and analyzing the three-dimensional structure of the human structural subunits of SDH expressed in bacterial models. Thus, for the first time the structure of these assembly intermediates will be known. This information is needed to develop small molecule inhibitors/activators that can be used for treatment of diseases associated with mitochondrial dysfunction and control metabolite levels in cells.