Redox Regulation of the rho-GTPases in the vasculature

Rho样GTP酶由于其调节肌动蛋白细胞骨架的能力而在血管功能中起重要作用。它们参与生理过程，例如平滑肌细胞收缩、内皮渗透性、血小板活化、白细胞迁移、血管生成和伤口愈合。此外，Rho GTP酶的失调促进与血管重塑、改变的细胞收缩性和细胞迁移相关的血管病症，例如血管通透性过高、肿瘤细胞侵袭、血小板聚集、动脉粥样硬化和再狭窄以及心脏肥大。 该建议的主要目的是研究巯基修饰在调节氧化还原活性Rho GTP酶中的作用。我们以前已经证明了Rho GTP酶的一个子集，即，Rac 1、RhoA和Cdc 42在鸟嘌呤核苷酸结合位点含有巯基，其与活性氧和氮物质（RNS、ROS）反应以调节Rho GT3活性。具体地，鸟嘌呤核苷酸结合通过氧化还原剂调节以促进巯基中间体的形成，所述巯基中间体促进鸟嘌呤核苷酸氧化和鸟嘌呤核苷酸底物的释放。这一机制与我们先前描述的Ras GTP酶的氧化还原调节机制相似。然而，与Ras相反，我们提供的证据表明，除了鸟嘌呤核苷酸解离的自由基介导的机制外，Rho GTP酶还受双电子氧化机制（离子）的调节，这是由于保守的磷酰基结合环中的反应性半胱氨酸的位置。在这个建议中，我们试图研究巯基氧化在调节Rac 1和RhoA GTPases的结构，生化和细胞活性中的作用。Rac 1和RhoA最近已被证明可以通过以ROS依赖性方式重塑肌动蛋白细胞骨架和粘附连接来调节内皮屏障功能，以响应缺氧和缺血/复氧。血管内皮衬里的主要功能是维持血液和组织之间的选择性渗透屏障，并且已经显示由缺氧诱导的内皮屏障功能的破坏有助于肺部疾病，例如急性呼吸窘迫综合征和缺血-再灌注损伤。因此，从这项工作中获得的信息可能有助于开发治疗方法，以对抗血管病理学，如呼吸窘迫综合征和缺血再灌注损伤。

Rho-like GTPases are important players in vascular function due to their ability to regulate the actin cytoskeleton. They are involved in physiological processes such as smooth muscle cell contraction, endothelial permeability, platelet activation, leukocyte migration, angiogenesis and wound healing. Moreover, deregulation of Rho GTPases promotes vascular disorders associated with vascular remodeling, altered cell contractility and cell migration such as vascular hyperpermeability, tumor cell invasion, platelet aggregation, atherosclerosis and restenosis and cardiac hypertrophy. The primary objective of this proposal is to investigate the role of thiol modification in regulation of redox active Rho GTPases. We have previously demonstrated that a subset of Rho GTPases, i.e., Rac1, RhoA and Cdc42, contain a thiol in the guanine nucleotide binding site that reacts with reactive oxygen and nitrogen species (RNS, ROS) to regulate Rho GTPase activity. Specifically, guanine nucleotide binding is modulated by redox agents to promote formation of thiol radical intermediates that facilitate guanine nucleotide oxidation and release of guanine nucleotide substrates. This mechanism is similar to that described by us previously for redox regulation of Ras GTPases. However, in contrast to Ras, we provide evidence that Rho GTPases are also regulated by two-electron oxidative mechanisms (ionic), in addition to the radical mediated mechanism of guanine nucleotide dissociation, due to the location of the reactive cysteine in the conserved phosphoryl binding loop. In this proposal, we seek to investigate the role of thiol oxidation in regulating the structure, biochemical and cellular activity of Rac1 and RhoA GTPases. Rac1 and RhoA have recently been shown to regulate endothelial barrier function in response to hypoxia and ischemia/reoxygenation via remodeling of the actin cytoskeleton and adherens junctions in a ROS-dependent manner. The primary function of the endothelial lining of blood vessels is to maintain a selective permeability barrier between blood and tissues, and breakdown of endothelial barrier function induced by hypoxia has been shown to contribute to lung diseases such as acute respiratory distress syndrome and ischemia-reperfusion injury. Thus, information derived from this effort may aid in developing therapies to combat vascular pathologies such as respiratory distress syndrome and ischemia-reperfusion injury.