Detection of Ras GTPase protein radicals through immuno-spin trapping

Abstract

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward non-metalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of ·NO-mediated protein radical formation in the HRas GTPase. HRas is a non-metalloprotein that plays a critical role in regulating cell growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states where Ras GTPases play an important role.

Redox regulation of Rac1 by thiol oxidation

“…We have previously reported that thiyl radical formation at Ras Cys 118 can promote guanine nucleotide exchange and Ras activation [72,73], whereas non-radical-mediated nitrosation and glutathiolation does not alter Ras nucleotide binding [75,76]. In contrast, we show herein that Rac1 can be activated by nonradical oxidation at Cys 18 .…”

“…In addition, p38 MAPK can be activated by reversible cysteine oxidation [70], whereas a number of protein tyrosine phosphatases become inactivated by oxidation of a catalytic cysteine [71]. In addition to these signaling proteins, we have previously reported that thiyl radical formation at Cys 118 of the small GTPase Ras can regulate its activity [72,73]. This mechanism of activation appears to play a key role in Ras-mediated tumorigenesis [74].…”

Rho GTPases, oxidation, and cell redox control

“…39,40 Given the >10-fold higher levels of cellular GTP compared with GDP, this enhanced exchange likely activates Ras. 41 In support of these findings, radical-mediated activation of Ras at Cys 118 contributes to tumor maintenance in a pancreatic cancer cell line and in a severe combined immunodeficiency/ beige mouse model.…”

“…50 However, an altered pK a is not always a prerequisite for redox sensitivity, especially for radical-mediated reactions, as Ras Cys 118 does not have an altered pK a despite its regulation by free radical-mediated oxidants. 51 Furthermore, as oxidants are generally short-lived and confined to the compartment in which they are generated, localization of the enzymes that generate oxidants with their target proteins is often critical for regulation by ROS and RNS.…”

Immuno-spin trapping from biochemistry to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals

“…Since the production of the anti-DMPO antibody [23], a number of protein radicals have been detected by DMPO-based IST [21, 32], including myoglobin [23, 34–36], hemoglobin [22, 35, 37–38], cytochrome c [39], neuroglobin [40], Cu, Zn-superoxide dismutase (SOD1) [41–43] and Mn-SOD [44], catalase [33], myeloperoxidase [45–46], eosinophil peroxidase [47], lactoperoxidase [48–49], microglobulin [50], albumin [43, 51], thyroid peroxidase (TPO)[25], α-lactalbumin [52] and Ras GTPase [53]. Oxidants used to generate these radicals included H 2 O 2 , peroxynitrite, Cu 2+ /H 2 O 2 , hypochlorous acid, tert -butylhydroperoxide, peroxymonocarbonate, and reactive species generated from ketoprofen plus UV-A, riboflavin plus UV-A irradiation and nano-TiO 2 plus UV-A irradiation.…”