4 3. RasGTPase Regulation by ROS Although several studies implicate RasGTPases in the pro- duction and regulation of intracellular ROS, many evidences indicate that Ras proteins can also be direct targets of ROS. Similar to the action of GEFs, various redox agents, including both ROS and RNS, have been shown to stimulate Ras guanine nucleotide dissociation in vitro and upregulate Ras function in vivo. Lander and coworkers showed for the ﬁrst time that NO is able to activate Ras by promoting RasGDP dissociation in vitro, GTP binding to Ras in vivo, and stimulation of pathways downstream to Ras [47–54]. The target site of NO-mediated guanine nucleotide dissociation on Ras is Cys118, which is located in the nucleotide-binding NKCD motif [49, 50, 54, 55]. Further studies indicated that NO2, a reaction product of NO with O2, reacts with the Ras Cys118 thiol to induce a radical-based process leading to stimulation of nucleotide exchange on Ras [56, 57]. In addition to NO, O2 − showed to be able to facilitate guanine nucleotide dissociation from Ras as well as the Ras- related GTPase Rap1A. The molecular mechanism of O2 −- mediated guanine nucleotide dissociation is similar to that of the NO/O2-mediated guanine nucleotide dissociation [58]. The redox-sensitive NKCD motif has been found within the Ras subfamily of GTPases such as H, N, K, and E-Ras as well as in Rap1A [7]. Redox-active motifs were afterwards found to be present in other Ras superfamily GTPases, suggesting that redox reg- ulation of GTPase signaling is more widespread that previ- ously envisioned [59]. The GXXXXGK(S/T)C redox-sensitive motif, located in the phosphoryl-binding loop important for redox-mediated regulation of guanine nucleotide exchange activity in vitro, was identiﬁed and characterized in the Rho family GTPases. This motif contains a redox-sensitive cysteine (Cys18, Rac1 numbering) at the C-terminus and it is conserved in almost half of Rho family GTPases such as Rac1 (and its isoforms Rac2 and 3), Cdc42, and RhoA (and its isoforms RhoB and C) [59, 60]. The radical-based molecular mechanism of Rho GTPase guanine nucleotide exchange appears similar in nature to the mechanism characterized for Ras GTPases. An in vivo study aimed to analyze the eﬀect of exogenous and endogenous ROS on the activation of RhoA in ﬁbroblasts was performed by Aghajanian and coworkers [61]. This study showed that RhoA can be directly activated by ROS in cells by oxidative modiﬁcation of critical Cys residues within the redox-active motif, and that ROS-mediated activation of RhoA can induce cytoskeletal rearrangement, thus support- ing the existence of a novel mechanism of regulating GTPase signaling cascades, independent to classical regulation by GEFs and GAPs, that can aﬀect cytoskeletal dynamics [61]. A number of Rab proteins also have the GXXXXGK(S/T)C motif (Rab1B, Rab2A/B, Rab4A/B, Rab14, Rab15, Rab19, and Sec4). Intriguingly, many Rab GTPases (Rab1A, Rab8A/B, Rab10, and RAb13) possess both the NKCD and GXXXXGK(S/T)C motifs, whereas some Rab proteins (Rab3A/B/C/D, Rab 7, Rab22, and Rab38) possess only the NKCD motif [7]. Journal of Signal Transduction A CGNKXD redox-sensitive motif was found in Ran pro- tein [62]; this motif contains a redox-sensitive cysteine, Cys120, at the N-terminal. In addition to this CGNKXD motif, Ran possesses an additional redox-sensitive cysteine Cys85 (Ran numbering). This type of redox center is also conserved in Dexras1 and Rhe proteins as well as in some Rab GTPases [62]. Although redox regulation of the members of Rab and Ran families has been recently discovered, its physiological relevance and pathological consequences linked to the mis- regulation of redox signaling associated with these redox sensitive small GTPases have not yet been explored [7]. 4. Pathological Implications of the Interplay between Small GTPases and ROS Over the past several years, it has become clear that ROSs play an important role in physiological processes like cell diﬀerentiation, proliferation, migration, and vasodilatation. On the other hand, production of ROS “in the wrong place at the wrong time” results in oxidative stress leading to cellular dysfunction and apoptosis, which contributes to diﬀerent pathologies like atherosclerosis, heart failure, hypertension, ischemia/reperfusion injury, cancer, aging, and neurodegen- eration [40]. There is a vast body of literature that links vascular ROS production to cardiovascular disease [63]. Vascular ROS production as well as Rac1 activation has been associated with hypertrophy and smooth muscle cell proliferation, endothelial dysfunction as well as endothelial cell migration, hypertension inﬂammation, and atherosclerosis [64–67]. Vascular hypertrophy has been ascribed to the eﬀects of various receptor agonists, including Angiotensin II (Ang II), which induces ROS production in VSMCs in a Rac1- dependent fashion [68]. Recent studies showed that this Ang II-induced ROS production also requires the membrane adapter caveolin, which is involved in Rac1 activation [69, 70], and the lipid kinase PI3K-γ [68, 71]. Ischemia/reperfusion (I/R) injury is also associated with ROS production. This is a clinically relevant problem oc- curring as damage to the myocardium following blood res- toration after a critical period of coronary occlusion. It is well known that immediately following the reinstitution of oxygenated blood into ischemic tissue, there is a rapid burst of ROS, but the molecular basis and source of this process are not yet convincingly identiﬁed [14, 72]. However, both in vitro and in vivo experiments [73, 74] have suggested that Rac1 plays a dominant role in ROS generation after I/R, and it activates the nuclear factor NF-κB and stimulates mRNA expression of several inﬂammatory genes, such as TNF-α and iNOS in the liver, leading to massive hepatocyte necrosis. Thus, eﬀorts aimed at inhibiting Rac protein function could be useful therapeutic strategies in a variety of clinical settings in which there is concern about the potential harmful eﬀects of I/R injury [73, 74]. Data from the literature suggest that ROS and RhoA activation are associated to airway smooth muscle contrac- tility [75–77]; it has been shown that oxidative stress with