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.

Public Health Relevance

Actin filaments form complex subcellular arrays that are essential for establishing cell shape, maintaining adhesion between neighboring cells, and generating connections between cells and their underlying connective tissue. Rho-like GTPase proteins 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. 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 their activity. The primary objective of this proposal is to delineate how thiol modification alters Rho GTPase activity and interactions with cellular regulators and effectors. Such studies are likely to yield insights into the cellular mechanisms that control both essential functions in normal cells, and dysfunctions that result in pathological cell behavior.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089614-09
Application #
8282951
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Knowlton, John R
Project Start
2000-12-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
9
Fiscal Year
2012
Total Cost
$288,272
Indirect Cost
$85,927
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Hobbs, G Aaron; Gunawardena, Harsha P; Campbell, Sharon L (2014) Biophysical and proteomic characterization strategies for cysteine modifications in Ras GTPases. Methods Mol Biol 1120:75-96
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Baker, Rachael; Lewis, Steven M; Sasaki, Atsuo T et al. (2013) Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Nat Struct Mol Biol 20:46-52
Hobbs, G Aaron; Bonini, Marcelo G; Gunawardena, Harsha P et al. (2013) Glutathiolated Ras: characterization and implications for Ras activation. Free Radic Biol Med 57:221-9
Davis, Michael F; Vigil, Dom; Campbell, Sharon L (2011) Regulation of Ras proteins by reactive nitrogen species. Free Radic Biol Med 51:565-75
Heo, Jongyun; Campbell, Sharon L (2006) Ras regulation by reactive oxygen and nitrogen species. Biochemistry 45:2200-10
Heo, Jongyun; Campbell, Sharon L (2005) Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide. J Biol Chem 280:12438-45
Heo, Jongyun; Thapar, Roopa; Campbell, Sharon L (2005) Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors. Biochemistry 44:6573-85
Heo, Jongyun; Prutzman, Kirk C; Mocanu, Viorel et al. (2005) Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation. J Mol Biol 346:1423-40
Heo, Jongyun; Campbell, Sharon L (2005) Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho GTPases. J Biol Chem 280:31003-10

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