University of North Carolina At Chapel Hill
Grant: $281,806 - Department of Health and Human Services - Jul. 17, 2009
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Award Description: Redox Regulation of Rho-GTPases in the Vasculature - ARRA 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, understanding the molecular basis for Rac1 and RhoA cysteine oxidation will aid in the design and interpretation of studies, conducted in collaboration with the Burridge laboratory, to investigate ischemia/reperfusion in pulmonary endothelial cells, human dermal microvascular endothelial cells as well as the lung. Information derived from this effort may aid in developing therapies to combat vascular pathologies such as respiratory distress syndrome and ischemia-reperfusion injury. Specific Aims Aim 1. We propose that selective oxidation of GXXXXGK(S/T)C motif Cys in Rho GTPases can alter GTP/GDP cycling and thus Rho GTPase activity. We further propose that Rho GTPases (i.e., RhoA, RhoB and RhoC) possessing a variation of this motif, a GXXXCGK(S/T)C motif containing a second Cys, can undergo a distinct type of regulation by thiol oxidants, giving rise to differential redoxmediated regulation relative to Rac1. Aim 2. We propose that oxidation of the GXXXXGK(S/T)C motif Cys in Rac1 and RhoA will perturb the phosphoryl binding loop and disrupt interactions with guanine nucleotide substrates. To test this hypothesis, we will characterize the structural features of select oxidized forms of Rho GTPases using NMR and spectroscopic approaches. Aim 3. To investigate the hypothesis that Rac1 and RhoA are regulated by cellular oxidants, we will characterize oxidative modification(s), the activation state and phenotype, of Rho GTPases in ECs, in response to both endogenous and exogenous sources of ROS and RNS.
Project Description: As per the aim 1 objectives, we have further characterized the biochemical properties of Rac1 and RhoA P-loop cysteine variants to determine which variants show the most similar biochemical properties to wt GTPases. GTPase variants which lack the redox active cysteine, yet retain similar biochemical properties of wt Rac1 and RhoA, should prove useful for the in vitro and cell-based studies proposed in the grant. Preliminary in vitro biochemical results indicate that both cysteine and serine substitutions increase the rate of GDP dissociation (3-10X), but that the serine substitution is most similar to wt RhoA. As our earlier cell-based studies were conducted using at C20AC16A mutant of RhoA, we also initiated characterization studies of this variant. However, our results indicate that this variant shows significant defects in guanine nucleotide binding relative to wt RhoA, compared to the C20S, C16A variant. These results indicate that there is some coupled effect of alanine mutations at the two cysteines in the RhoA phosphoryl binding loop. Therefore, in collaboration with the Burridge laboratory, additional RhoA variants were expressed in endothelial cells and the activation state (percentage of GTP-bound RhoA) of the various cysteine mutants was assessed. Consistent with our in vitro studies, the activation state of the C20S variant was most similar to that of wt RhoA, whereas the C20A, C16A variant showed much higher levels of bound GTP. While we still need to characterize the how these cysteine variants respond to GEF and GAP stimulation, our preliminary cell-based results suggest that the C20S variants of Rac1 and RhoA might be the best redox resistant variants to use for future studies.
Jobs Summary: This report shows a modified FTE jobs created number, which has been calculated based on payroll expenditures as of 9/30/2009. The narrative below provides an estimate/description of jobs that will be created/retained once the project is fully underway. Original jobs created/retained estimate: 2 Narrative: This grant provides funding for retention of two graduate students, who are working on redox regulation of Rac1, RhoA and RhoG GTPases. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
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