Redox Regulation of Ras and Ras-Related GTPases

描述（由申请人提供）：自由基一氧化氮（NO）已在许多研究中被证明可以激活Ras原癌蛋白。NO对Ras的作用类似于鸟嘌呤核苷酸交换因子（GEFs）， GEFs和NO都通过促进鸟嘌呤核苷酸交换和Ras在活性Ras- gtp状态下的填充来调节Ras的活性。一旦被NO激活，Ras可以通过多种途径转导信号，导致细胞生长控制的变化。除NO外，活性氧（ROS）(即超氧阴离子自由基（O2-）和羟基自由基（OH.）已被证明在体外可提高Ras鸟嘌呤核苷酸解离率，并在体内调节Ras信号传导。虽然NO和ROS被认为是Ras活性的重要调节因子，但对NO和ROS介导鸟嘌呤的机制知之甚少。Ras上的核苷酸交换（GNE）我们最近假设了一种新的机制，通过这种机制，NO和O2-。促进Ras激活，这可能与其他具有氧化还原活性的Ras超家族GTPases相同。本文的主要目的是研究no -和O2-的结构和生化细节。-介导的GNE对Ras和Ras相关蛋白的影响，以评估并进一步描述与所提出的机制相关的结构和动力学细节。为了实现这些目标，将采用采用结构、生化和生物物理方法的多学科方法。

DESCRIPTION (provided by applicant): The free radical nitric oxide (NO) has been shown in numerous studies, to activate the Ras protooncoprotein. The action of NO on Ras is similar to that of guanine nucleotide exchange factors (GEFs) in that both GEFs and NO modulate Ras activity by promoting guanine nucleotide exchange and populating of Ras in its active Ras-GTP state. Once activated by NO, Ras can transduce signals through a multitude of pathways, leading to changes in cellular growth control. In addition to NO, reactive oxygen species (ROS) (i.e., superoxide anion radical (O2-.) and hydroxyl radical (OH.) have been shown to enhance the rate of Ras guanine nucleotide dissociation in vitro and modulate Ras signaling in vivo. Although NO and ROS are believed to be important regulators of Ras activity, little to known about the mechanism of NO- and ROS-mediated guanine. nucleotide exchange (GNE) on Ras. We have recently postulated a novel mechanism by which NO and O2-. promote Ras activation, which is likely to be common to other redox-active Ras superfamily GTPases. The main goal of this proposal is to investigate the structural and biochemical details of NO-and O2-.-mediated GNE on Ras and Ras-related proteins to both evaluate and further delineate structural and kinetic details associated with the proposed mechanism. To achieve these goals, a multidisciplinary approach employing structural, biochemical and, biophysical methods will be employed. Three main specific aims are proposed: Investigate the structural basis of NO- and O2-.-mediated GNE on Ras and Ras-related GTPases using Nuclear Magnetic Resonance (NMR) spectroscopy. Investigate the structural-basis and kinetic mechanism(s) of NO and O2-.-mediated GNE on Ras and Ras-related GTPases in the presence and absence of GTPase GNE factors using kinetic, biochemical and spectroscopic approaches. Delineate the mechanism of the NO and O2-.-mediated Ras and Ras-related GTPase GNE using Electron Paramagnetic Resonance (EPR) spectroscopy. Understanding the structural, kinetic, and spectroscopic features of the GTP/GDP-binding cycle in the presence and absence of the Ras-activity regulators will aid in understanding how Ras is regulated in the cell and may provide a molecular and mechanistic basis for developing Ras-mediated tumor therapies.