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

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 在细胞存活和凋亡的十字路口发挥作用，因此本项目将进一步确定ShcA的作用， 蛋白质在决定细胞应激反应中起作用。

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.