Redox Regulation of the rho-GTPases in the vasculature

Rho样GTP酶因其调节肌动蛋白细胞骨架的能力而在血管功能中发挥重要作用。它们参与了平滑肌细胞收缩、内皮通透性、血小板活化、白细胞迁移、血管生成和伤口愈合等生理过程。此外，Rho GTP酶的解除调控促进了与血管重塑、细胞收缩和细胞迁移相关的血管疾病，如血管高通透性、肿瘤细胞侵袭、血小板聚集、动脉粥样硬化和再狭窄以及心肌肥厚。 这项建议的主要目的是研究硫醇修饰在氧化还原活性Rho GTP酶调节中的作用。我们以前已经证明了Rho GTP酶的一个子集，即rac1，RhoA和CDC42，在鸟嘌呤核苷酸结合部位含有硫醇，它与活性氧和氮物种(RNS，ROS)反应来调节Rho GTP酶的活性。具体地说，鸟嘌呤核苷酸结合受到氧化还原试剂的调节，以促进硫醇自由基中间体的形成，从而促进鸟嘌呤核苷酸氧化和鸟嘌呤核苷酸底物的释放。这一机制类似于我们之前描述的Ras GTP酶的氧化还原调节机制。然而，与RAS相反，我们提供的证据表明，Rho GTP酶除了受到自由基介导的鸟核苷酸解离机制外，还受到双电子氧化机制(离子)的调控，这是因为反应性半胱氨酸位于保守的磷酸结合环中。在这个提案中，我们试图研究硫醇氧化在调节rac1和RhoA GTP酶的结构、生化和细胞活性中的作用。最近研究表明，rac1和RhoA通过重塑肌动蛋白细胞骨架和以ROS依赖的方式黏附连接来调节内皮屏障的功能，以应对缺氧和缺血/复氧。血管内皮细胞膜的主要功能是维持血液和组织之间的选择性通透性屏障，低氧引起的内皮屏障功能的破坏被证明与急性呼吸窘迫综合征和缺血再灌注损伤等肺部疾病有关。因此，从这一努力中获得的信息可能有助于开发治疗方法，以对抗呼吸窘迫综合征和缺血再灌注损伤等血管病理。

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.