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

项目总结： P66shc是ShcA家族中的一个成员，其活性氧簇(Ros)的产生和细胞色素c(Cytc)的表达。 相互作用影响心血管病理，包括缺血/再灌注损伤，内皮功能障碍， 和冠心病(CAD)。SHCA ROS对冠心病和卒中的影响非常强烈，其水平可以预测 中风的严重程度或确定患者是否存在冠心病。虽然抑制或去除了其他ROS的产生 蛋白质对小鼠是致命的，p66Shc基因敲除是有益的，没有生理上的损害或增加 补偿性ROS。Hays实验室是第一个生产全长p66Shc的实验室，使我的项目成为一个独特的 定位于：1)确定ROS产生的机制2)用体内验证体外机制的发现 通过说明机械性操作如何使病理受益的模型3)了解p66Shc的结构 调节ROS的产生和细胞色素C的相互作用。 初步数据显示，p66Shc含有四个与产生ROS有关的分子内二硫键， 独立于金属或辅因的。为了定义这种机制，我将：1)使用Danio Rerio(斑马鱼)人类 增加或减少ROS产生或改变p66Shc定位信号的转基因p66Shc突变体 体外和p66Shc基因敲除分析心肌梗死后创面愈合率与ROS产生和 P66shc定位(p66Shc ROS增加或线粒体定位与更差的结果相关)2) 确定在硫醇二硫化物交换过程中特定半胱氨酸在结构域间传递电子的顺序 3)确定能够产生ROS的生理上相关的初始电子供体(S)。我将通过以下方式实现这一目标 对不同p66Shc基因的斑马鱼进行冷冻损伤，半胱氨酸诱变联合 超氧化物敏感质谱学研究和潜在电子供体产生的ROS的测量 分别在无氧环境中。我的项目将通过确定 可以影响ROS介导的病理的临床结果的机械性和结构性治疗靶点。 先前对分离的CH2结构域的研究表明，该结构域被认为与p66Shc ROS功能有关 CH2结构域氧化Cytc生成ROS(热力学不利)。我的全长p66Shc和 分离的CH2结构域研究表明，它们降低了Cytc，产生了不依赖Cytc的ROS，并抑制了Cytc 心磷脂诱导的过氧化物酶活性。细胞色素c过氧化物酶抑制在热力学上是可以接受的。 对上述研究中ROS增加的解释。我将定义结构基础和 通过测试p66Shc及其CH2结构域的细胞色素c结合、ROS来研究这些相互作用的构象动力学 活度，并进行氢-氚交换分析。最后，我会优化目前的条件 生产5？可衍射晶体，以解决SHCA系列的第一个全长结构P66Shc。

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.