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

基本信息

批准号：
10242209
负责人：
Landon H. Haslem
金额：
$ 5.1万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
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/10242209
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

项目摘要

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.

项目摘要： P66SHC是SHCA家族成员，其活性氧（ROS）产生和细胞色素C（Cyt C）相互作用会影响心血管病理，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影响了CAD和中风，足以预测其水平中风的严重程度或确定患者的CAD存在。虽然抑制或去除其他ROS生产蛋白质在小鼠中致命，p66SHC敲除无益，没有物理损害或增加补偿性ros。海斯实验室是第一个生产全长P66SHC的实验室，使我的项目陷入了独特位置为：1）定义ROS产生的机理2）验证体内的体外机械发现通过说明机械操作如何使病理学受益3）了解p66SHC结构的模型介导ROS产生和Cyt C相互作用。初步数据表明，P66SHC包含四个参与产生ROS的分子内二硫键，独立于金属或辅因子。要定义机制，我将：1）使用Danio Rerio（斑马鱼）人转基因P66SHC突变体增加或减少ROS产生或改变p66SHC定位信号体外以及P66SHC敲除，分析与ROS产生有关的MI后伤口愈合率 P66SHC定位（p66shc ROS或线粒体定位增加与较差的结果有关）2）确定特定半胱氨酸在硫醇二硫化物交换过程中通过域之间通过电子的顺序 3）确定可以产生ROS的物理相关的初始电子供体。我将通过用各种p66SHC基因型进行斑马鱼的冷冻夹，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 在无氧环境中。我的项目将直接通过识别来影响心血管疾病可能影响ROS介导的病理学的临床结果的机械和结构治疗靶标。先前对孤立的CH2结构域的研究，被认为是P66SHC ROS函数的原因，表明 CH2结构域将Cyt C氧化以产生ROS（热力学上不利）。我的全长P66SHC和分离的CH2结构域研究表明，它们减少了Cyt C，产生与Cyt C无关的ROS，并抑制Cyt C的Cyt C。心磷脂诱导的过氧化物酶活性。 Cyt C过氧化物酶抑制是一种热力学上可接受的 Priore物质研究中ROS增加的解释。我将定义结构性基础通过测试P66SHC及其CH2结构域的Cyt C结合，ROS的构象动力学活性，并通过进行氢居民交换分析。最后，我将优化当前的条件产生5Å衍射的晶体，以求解SHCA家族的第一个全长结构P66SHC。