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疾病发展的关键决定因素 并发症。SHCA家族蛋白是典型的信号转导蛋白 调节一系列不同的细胞反应通路(例如，酪氨酸激酶(TK)信号)的适配器。一 SHCA家族成员p66Shc也是ROS产生的主要来源(~30%)。 哺乳动物细胞。心肌缺血时SHCA介导的ROS产生是卒中大小的决定因素 糖尿病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酶功能的分子基础还不清楚或未知。这个项目是 重点是使用一系列生物物理方法在分子水平上定义SHCA蛋白质如何发挥作用。 SHCA蛋白已被证明很难在已定义的系统中进行研究，因为它们在标准系统中的低表达谱 实验室系统，它们的构象是可变的，容易进行翻译后修改，并且它们相互作用 体内的蛋白质种类繁多。这个项目的主要假设是SHCA蛋白的功能 作为分子变阻器，调节氧化还原状态、Cytc活性和某个位置、构象和代谢通量， 和绑定依赖的方式。我们将通过追求以下具体目标来研究这一假说：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)决定SHCA介导的功能的结构基础。 该项目的流出将为SHCA作为促进下游的一种手段提供分子基础 SHCA介导的ROS产生的异构体特异性抑制剂的研究进展。此外，SHCA蛋白 在细胞生存和凋亡的十字路口发挥作用，所以这个项目将进一步定义SHCA的作用 蛋白质在决定细胞应激反应中发挥作用。

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.