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Deciphering ShcA-mediated ROS Production as a Novel Intervention Strategy in Diabetes Therapy

解读 ShcA 介导的 ROS 产生作为糖尿病治疗的新型干预策略

基本信息

批准号：
9753262
负责人：
FRANKLIN Alan HAYS
金额：
$ 28.25万
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：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9753262
关键词：
Adaptor Signaling Protein Apoptosis Apoptotic Binding Biophysics Cardiovascular system Caspase Cell Culture Techniques Cell Survival Cell physiology Cells Cellular Stress Response Clinical Treatment Collagen Coupled Cutaneous Cytochrome c Peroxidase Data Deuterium Development Diabetes Mellitus Diabetes prevention Disease Disulfides Electron Transport Energy Metabolism Expression Profiling Family Family member Fibroblasts Foundations Future Human Hydrogen Hypertension Intervention Lipids Location Mammalian Cell Mass Spectrum Analysis Mediating Metabolic Metal Ion Binding Mitochondria Molecular Molecular Conformation Molecular Structure Myocardial Ischemia N-terminal Oxidation-Reduction Oxidative Stress Oxidoreductase Oxygen PTB Domain Pathway interactions Peripheral Peroxidases Play Post-Translational Protein Processing Production Protein C Protein Family Protein Isoforms Protein Tyrosine Kinase Proteins Reactive Oxygen Species Reperfusion Injury Role Severities Signal Transduction Source Stroke Structure Sulfhydryl Compounds Superoxides System Therapeutic Wound Healing X-Ray Crystallography base biophysical techniques cytochrome c design diabetes mellitus therapy disulfide bond endothelial dysfunction human disease in vivo inhibitor/antagonist insight mouse ShcA protein novel p66(ShcA) protein protein function receptor response small molecule therapeutic target tissue culture

项目摘要

Oxidative stress is a key determinant in the development of diabetes related microvascular and cardiovascular complications. ShcA (Src homology and collagen homology) family proteins are prototypical signaling adaptors that regulate a diverse array of cellular response pathways (e.g., tyrosine kinase (TK) signaling). One ShcA family member, p66Shc, is also a major source (~30%) of reactive oxygen species (ROS) production in mammalian cells. ShcA-mediated ROS production is a determinant of stroke size in cardiac ischemia reperfusion injury and endothelial dysfunction in diabetes (e.g., delayed cutaneous wound healing) and hypertension. The molecular basis for ShcA enzymatic function is poorly defined or unknown. This project is focused on defining how ShcA proteins function at the molecular level using a range of biophysical methods. ShcA proteins have proven difficult to study in defined systems due to their low expression profile in standard lab systems, they are conformationally variable and prone to posttranslational modification, and they interact with a broad range of proteins in vivo. The overarching hypothesis of this project is that ShcA proteins function as molecular rheostats to modulate redox state, cyt c activity, and metabolic flux in a location, conformation, and binding-dependent manner. We will investigate this hypothesis by pursuing the following Specific Aims: 1) define the enzymatic mechanism of ShcA-mediated ROS production, 2) define the functional interactions between ShcA proteins and cytochrome c, and 3) determine the structural basis for ShcA-mediated function. Outflow from this project will provide a molecular basis for ShcA function as a means to facilitate downstream development of isoform specific inhibitors of ShcA-mediated ROS production. In addition, ShcA proteins function at the crossroads of cell survival and apoptosis so this project will further define the role that ShcA proteins play in determining cellular stress response.

氧化应激是糖尿病相关微血管和心血管发展的关键决定因素并发症ShcA（Src同源性和胶原同源性）家族蛋白是典型的信号传导蛋白，调节多种细胞应答途径的衔接子（例如，酪氨酸激酶（TK）信号传导）。一 ShcA家族成员p66Shc也是大肠杆菌中活性氧（ROS）产生的主要来源（约30%）。哺乳动物细胞ShcA介导的ROS产生是心肌缺血时卒中大小的决定因素糖尿病中的再灌注损伤和内皮功能障碍（例如，皮肤伤口愈合延迟）和高血压ShcA酶功能的分子基础定义不清或未知。这个项目是专注于使用一系列生物物理方法在分子水平上定义ShcA蛋白质的功能。 ShcA蛋白已被证明难以在确定的系统中研究，这是由于它们在标准环境中的低表达谱。在实验室系统中，它们是构象可变的，易于翻译后修饰，并且它们相互作用与多种蛋白质结合。该项目的首要假设是ShcA蛋白的功能作为分子变阻器来调节氧化还原状态、细胞色素C活性和代谢通量，和依赖绑定的方式。我们将通过追求以下具体目标来研究这一假设：1）确定ShcA介导的ROS产生的酶机制，2）确定功能相互作用 ShcA蛋白和细胞色素c之间的关系，以及3）确定ShcA介导的功能的结构基础。该项目的流出物将为ShcA功能提供分子基础，作为促进下游开发ShcA介导的ROS产生的同种型特异性抑制剂。此外，ShcA蛋白 ShcA在细胞生存和细胞凋亡的十字路口发挥作用，因此该项目将进一步定义ShcA的作用蛋白质在决定细胞应激反应中起作用。