Molecular and in vivo Determinants of p66Shc-Mediated ROS Function in Cardiovascular Health

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Project Summary: p66Shc is a ShcA family member whose reactive oxygen species (ROS) production and cytochrome c (cyt c) interactions impact cardiovascular pathologies including ischemia/reperfusion injuries, endothelial dysfunction, and coronary artery disease (CAD). ShcA ROS affects CAD and stroke strongly enough that its levels can predict stroke severity or determine CAD presence in patients. Although inhibiting or removing other ROS producing proteins is fatal in mice, p66Shc knockouts are beneficial, without physiological detriments or increased compensatory ROS. The Hays lab is the first to produce full-length p66Shc, putting my project in a unique position to: 1) define the mechanism of ROS production 2) validate in vitro mechanistic findings with an in vivo model by illustrating how mechanistic manipulation can benefit pathology 3) understand how p66Shc structure mediates ROS generation and cyt c interactions. Preliminary data show that p66Shc contains four intramolecular disulfide bonds involved in producing ROS, independent of metals or cofactors. To define the mechanism, I will: 1) use Danio rerio (zebrafish) human transgenic p66Shc mutants that increase or decrease ROS production, or change p66Shc localization signals in vitro as well as p66Shc knockouts to analyze post-MI wound healing rate in relation to ROS production and p66Shc localization (increased p66Shc ROS or mitochondrial localization is associated with worse outcomes) 2) determine the order in which specific cysteines pass electrons between domains during thiol disulfide exchange 3) identify physiologically relevant initial electron donor(s) that enable ROS production. I will achieve this by performing cryoinjury of zebrafish with various p66Shc genotypes, cysteine mutagenesis combined with superoxide sensitive mass spectroscopy studies, and measuring ROS produced from potential electron donors in an oxygen free environment, respectively. My project will directly impact cardiovascular disease by identifying mechanistic and structural therapeutic targets that can affect clinical outcomes for ROS-mediated pathology. Previous studies with the isolated CH2 domain, thought to be responsible for p66Shc ROS function, indicate that the CH2 domain oxidizes cyt c to generate ROS (thermodynamically unfavorable). My full-length p66Shc and isolated CH2 domain studies show that they reduce cyt c, produce ROS independent of cyt c, and inhibit cyt c's cardiolipin-induced peroxidase activity. Cyt c peroxidase inhibition is a thermodynamically acceptable explanation for the increased ROS in the aforementioned studies. I will define the structural basis and conformational dynamics of these interactions by testing p66Shc and its CH2 domain for cyt c binding, ROS activity, and by performing Hydrogen-Deuterium exchange analysis. Lastly, I will optimize the conditions currently producing 5 Å diffractable crystals to solve the first full-length structure of the ShcA family, p66Shc.