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

p66Shc 介导的 ROS 功能在心血管健康中的分子和体内决定因素

• 批准号: 10471855
• 负责人: Landon H. Haslem
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-24 至 2023-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10471855
• 关键词: Affect; Amino Acids; Binding; Biochemistry; Biological Assay; Biological Markers; Cardiolipins; Cardiovascular Diseases; Cardiovascular Pathology; Cardiovascular system; Cell Survival; Clinical; Collagen; Coronary Arteriosclerosis; Crystallization; Cysteine; Data; Deuterium; Disulfides; Drug Targeting; Electron Transport; Electrons; Environment; Family; Family member; Foundations; Future; Generations; Genotype; Health; Human; Hydrogen; Image; Impaired wound healing; In Vitro; Infarction; Knock-out; Length; Location; Mammals; Mass Spectrum Analysis; Measures; Mediating; Metals; Mitochondria; Modeling; Molecular; Molecular Conformation; Molecular Structure; Mus; Mutagenesis; Mutation; Myocardial; Myocardial Infarction; Outcome; Oxides; Oxidoreductase; Oxygen; PTB Domain; Pathology; Patients; Peroxidases; Physiological; Play; Positioning Attribute; Production; Protein Dynamics; Protein Family; Proteins; Reactive Oxygen Species; Reperfusion Injury; Role; Severities; Signal Transduction; Stroke; Structure; Sulfhydryl Compounds; Superoxides; Testing; Therapeutic; Thermodynamics; Transgenic Organisms; Variant; X-Ray Crystallography; Zebrafish; biophysical analysis; cardiovascular health; cofactor; cytochrome c; cytochrome c oxidase; design; diabetic; disulfide bond; electron donor; endothelial dysfunction; experimental study; heart function; human tissue; improved; in vivo; in vivo Model; injury recovery; insight; ischemic injury; mutant; myocardial injury; novel therapeutics; p66(ShcA) protein; targeted treatment; therapeutic target; wound healing

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.

项目概要： p66 Shc是ShcA家族成员，其活性氧（ROS）产生和细胞色素c（cyt c） 相互作用影响心血管病理学，包括缺血/再灌注损伤，内皮功能障碍， 和冠状动脉疾病（CAD）。ShcA ROS对CAD和中风的影响足够强烈，其水平可以预测 中风严重程度或确定患者中CAD的存在。虽然抑制或去除其他ROS产生 p66 Shc蛋白在小鼠中是致命的，p66 Shc敲除是有益的，没有生理学上的损害或增加 代偿性ROS。海斯实验室是第一个生产全长p66 Shc的实验室， 位置：1）定义ROS产生的机制2）用体内 3）理解p66 Shc结构如何影响p66 Shc的表达， 介导ROS的产生和细胞色素C的相互作用。 初步数据显示p66 Shc含有四个参与产生ROS的分子内二硫键， 不依赖于金属或辅因子。为了定义该机制，我将：1）使用斑马鱼（Danio rerio）人类 转基因p66 Shc突变体增加或减少ROS产生，或改变p66 Shc定位信号， 体外以及p66 Shc敲除，以分析MI后伤口愈合率与ROS产生的关系， p66 Shc定位（p66 Shc ROS或线粒体定位增加与更差的结局相关）2） 确定巯基二硫键交换过程中特定半胱氨酸在结构域之间传递电子的顺序 3)鉴定能够产生ROS的生理学相关的初始电子供体。我将通过以下方式实现这一目标： 对不同p66 Shc基因型的斑马鱼进行冷冻损伤，半胱氨酸诱变结合 超氧化物敏感质谱研究，并测量潜在电子供体产生的ROS 分别在无氧环境中。我的项目将通过识别 可能影响ROS介导的病理学的临床结果的机制和结构治疗靶点。 先前对分离的CH 2结构域（被认为负责p66 Shc ROS功能）的研究表明， CH 2结构域氧化细胞色素c产生ROS（在药理学上是不利的）。我的全长p66 Shc和 分离的CH 2结构域研究表明，它们减少cyt c，产生不依赖于cyt c的ROS，并抑制cyt c's 心磷脂诱导的过氧化物酶活性。细胞色素c过氧化物酶抑制剂是一种药物学上可接受的 在上述研究中增加ROS的解释。我将定义结构基础， 这些相互作用的构象动力学通过测试p66 Shc及其CH 2结构域的细胞色素c结合，ROS 活性，并通过进行氢-氘交换分析。最后，我将优化目前的条件， 产生5 μ m衍射晶体以解析ShcA家族的第一个全长结构p66 Shc。