Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases

Hobbs, G. Aaron; Gunawardena, Harsha; Campbell, Sharon L.

doi:10.1007/978-1-62703-791-4_6

Cited by 7 publications

(17 citation statements)

References 54 publications

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“…As Rac1 Cys 18 can be selectively modified by oxidized glutathione at physiological pH, we investigated whether the p K a of the Rac1 Cys 18 thiol is altered relative to a typical free cysteine thiol by measuring the cysteine reactivity of Rac1 WT and Rac1 C18S to ABD-f. We used the thiol-modifying reagent ABD-f, which preferentially reacts with the thiolate form of cysteine [59], to measure thiol reactivity over a wide pH range, as described previously by us for Ras [60]. ABD-f fluorescence was measured from pH 5.5 to 8.5 for Rac1 WT and Rac1 C18S (Fig.…”

Section: Resultsmentioning

confidence: 99%

Redox regulation of Rac1 by thiol oxidation

Hobbs

Mitchell

Arrington

et al. 2015

Free Radical Biology and Medicine

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The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys18) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys18 by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1C18D. We also evaluated Rac1C18S as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1WT but is resistant to thiol oxidation. In addition, Rac1C18D, but not Rac1C18S, shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1C18D in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1C18D in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1WT and the redox-insensitive Rac1C18S variant. Moreover, expression of Rac1C18D in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys18 is a novel posttranslational modification that upregulates Rac1 activity.

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Section: Resultsmentioning

confidence: 99%

Redox regulation of Rac1 by thiol oxidation

Hobbs

Mitchell

Arrington

et al. 2015

Free Radical Biology and Medicine

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“…NAC can activate the Ras-ERK (extracellular signal-regulated kinase) pathway in vitro through non-antioxidant mechanisms, protecting neuronal cells from death in the absence of trophic factors. Since Ras proteins contain essential reactive cysteines, it was suggested that NAC could trigger Ras by its reducing capability [ 385 , 386 ]. Some analyses showed that NAC could defend human neurons from the cerebral cortex against death induced by Aβ-amyloid 1−42 [ 187 ], causing p35/Cdk5 activation and decreasing phosphorylation/deactivation of the MLK3-MKK7-JNK3 signaling pathway [reviewed in 1].…”

Section: Modulation Of Cysteinet By N -Acetyl-cyst...mentioning

confidence: 99%

N-Acetyl-Cysteine: Modulating the Cysteine Redox Proteome in Neurodegenerative Diseases

Martínez-Banaclocha

2022

Antioxidants

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In the last twenty years, significant progress in understanding the pathophysiology of age-associated neurodegenerative diseases has been made. However, the prevention and treatment of these diseases remain without clinically significant therapeutic advancement. While we still hope for some potential genetic therapeutic approaches, the current reality is far from substantial progress. With this state of the issue, emphasis should be placed on early diagnosis and prompt intervention in patients with increased risk of neurodegenerative diseases to slow down their progression, poor prognosis, and decreasing quality of life. Accordingly, it is urgent to implement interventions addressing the psychosocial and biochemical disturbances we know are central in managing the evolution of these disorders. Genomic and proteomic studies have shown the high molecular intricacy in neurodegenerative diseases, involving a broad spectrum of cellular pathways underlying disease progression. Recent investigations indicate that the dysregulation of the sensitive-cysteine proteome may be a concurrent pathogenic mechanism contributing to the pathophysiology of major neurodegenerative diseases, opening new therapeutic opportunities. Considering the incidence and prevalence of these disorders and their already significant burden in Western societies, they will become a real pandemic in the following decades. Therefore, we propose large-scale investigations, in selected groups of people over 40 years of age with decreased blood glutathione levels, comorbidities, and/or mild cognitive impairment, to evaluate supplementation of the diet with low doses of N-acetyl-cysteine, a promising and well-tolerated therapeutic agent suitable for long-term use.

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“…Previous studies from our group and others have shown that several cysteine residues in a subset of RAS superfamily GTPases have a lower pKa relative to that of a free cysteine (12)(13)(14)(15).…”

Section: Kras G12c Exhibits a Perturbed Cysteine Pka By Chemical Modi...mentioning

confidence: 76%

“…KRAS and other RAS superfamily GTPases have previously been shown to be sensitive to cysteine oxidation both in vitro and in cellular contexts (12)(13)(14)(15). With the observed sensitivity of KRAS G12C to chemical ligation by acrylamides like AMG 510 and ARS-853 and our NMR data demonstrating the lowered pKa of the G12C cysteine, we hypothesized that this cysteine could also be sensitive to modification by intracellular oxidizing agents.…”

Section: Kras G12c Is Sensitive To Oxidative Modification In Vitro An...mentioning

confidence: 94%

Kinetic and Redox Characterization of KRAS G12C Inhibition

Huynh

Parsonage

Forshaw

et al. 2022

Preprint

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The development of mutant-selective inhibitors for the KRAS G12C allele has generated considerable excitement. These KRAS G12C inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain, motivating the present studies. Measurement of the C12 thiol pKa by NMR and an independent biochemical assay found a depressed pKa (relative to free cysteine) of 7.6 consistent with its susceptibility to chemical ligation. Using a novel and validated fluorescent KRAS Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that ARS-853 addition at 5°C elicited both a rapid first phase (attributed to binding, yielding a Kd of 36.0 ± 0.7 μM), and a second, slower pH-dependent phase taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRAS G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS G12C thiol to sulfinic acid alters RAS conformation and dynamics to be more similar to KRAS G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS G12C drug discovery efforts as well as identifying the occurrence of G12C oxidation with currently unknown biological ramifications.

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Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases

Cited by 7 publications

References 54 publications

Redox regulation of Rac1 by thiol oxidation

Redox regulation of Rac1 by thiol oxidation

N-Acetyl-Cysteine: Modulating the Cysteine Redox Proteome in Neurodegenerative Diseases

Kinetic and Redox Characterization of KRAS G12C Inhibition

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