Radical-free biology of oxidative stress
Dean P. Jones



Free radical-induced macromolecular damage has been studied extensively as a mechanism of oxidative stress, but large-scale intervention trials with free radical scavenging antioxidant supplements show little benefit in humans. The present review summarizes data supporting a complementary hypothesis for oxidative stress in disease that can occur without free radicals. This hypothesis, which is termed the “redox hypothesis,” is that oxidative stress occurs as a consequence of disruption of thiol redox circuits, which normally function in cell signaling and physiological regulation. The redox states of thiol systems are sensitive to two-electron oxidants and controlled by the thioredoxins (Trx), glutathione (GSH), and cysteine (Cys). Trx and GSH systems are maintained under stable, but nonequilibrium conditions, due to a continuous oxidation of cell thiols at a rate of about 0.5% of the total thiol pool per minute. Redox-sensitive thiols are critical for signal transduction (e.g., H-Ras, PTP-1B), transcription factor binding to DNA (e.g., Nrf-2, nuclear factor-κB), receptor activation (e.g., αIIbβ3 integrin in platelet activation), and other processes. Nonradical oxidants, including peroxides, aldehydes, quinones, and epoxides, are generated enzymatically from both endogenous and exogenous precursors and do not require free radicals as intermediates to oxidize or modify these thiols. Because of the nonequilibrium conditions in the thiol pathways, aberrant generation of nonradical oxidants at rates comparable to normal oxidation may be sufficient to disrupt function. Considerable opportunity exists to elucidate specific thiol control pathways and develop interventional strategies to restore normal redox control and protect against oxidative stress in aging and age-related disease.
Keywords: thioredoxin, glutathione, cysteine, hydrogen peroxide, redox signaling, protein thiol

Glutathionine (GSH) redox network. A partial list of GSH-dependent proteins illustrates the need for research to understand the integrated function of these redox systems. 1) GSH is synthesized by a two-step pathway in which abundance of two enzymes, glutamate cysteine ligase (GSH0, GSH1) and GSH synthetase (GSHB), determine synthesis rate (97). GSH is degraded by γ-glutamyltransferase (GGT) at the surface of the brush border of the kidney, small intestine, and a number of other tissues, and probably also in the cisternae of the secretory pathway (142). 2) GSH is transported out of cells by several multidrug resistance proteins (MRP) (12). The chloride channel, which is mutated in cystic fibrosis (CFTR), also transports GSH (113), and GSH is transported into mitochondria by the dicarboxylate carrier (DIC) and a monocarboxylate carrier (OGCP) (103). GSH is transported into the cisternae of the endoplasmic reticulum (13), but the molecular nature of the transporter is not known. 3) GSH is used by a number of GSH transferases (GST), which include microsomal and nonmicrosomal locations, to modify electrophilic chemicals (9). These are thought to largely function in detoxification, but some also act on biosynthetic intermediates for prostaglandins and leukotrienes. A fraction of GSH is present as S-nitroso-GSH, a transnitrosylating agent generated from nitric oxide or its metabolites (168). 4) GSH functions in metabolism as a coenzyme for formaldehyde dehydrogenase, glyoxylase, and other metabolic reactions (4, 168). In these reactions, GSH is cyclically removed by one reaction and regenerated in a second reaction. 5) Several thiol transferases, also known as glutaredoxins, catalyze introduction and removal of GSH (110, 114). 5a) Several proteins are regulated by GS-ylation, and many others undergo GS-ylation under oxidative stress conditions (44, 93). 6) GSH is used as a reductant for selenium-dependent GSH peroxidases (GPX) and selenium-independent peroxiredoxin-6 (PRX6) and some GSH transferases (GST). 6a) The product of these oxidative reactions, GSSG, is reduced back to GSH by GSSG reductase (GSHR) in most tissues. In sperm, thioredoxin reductase-3 (TRXR3) has activity toward both Trx and GSH. The proteins included in this figure are present in multiple cellular compartments and are differentially expressed in cells so that development of functional maps will require tissue-specific measurements of individual reaction rates. Protein designations and common names are from the UniProtKB/Swiss-Prot database. Abbreviations are as follows: GSH0, Glu-Cys ligase, regulatory; GSH1, Glu-Cys ligase, catalytic; GSHB, GSH synthetase; GGT1,4, 5, 6, γ-glutamyltransferase; DIC, mitochondrial dicarboxylate carrier (SLC25A10); OGCP, mitochondrial 2-oxoglutarate/malate carrier; CFTR, cystic fibrosis transmembrane conductance protein; MRP, multidrug resistance-associated protein; MRP2, canalicular multispecific organic anion transporter 1; GST, GSH transferase; ADHX, alcohol dehydrogenase class-3; ESTD, S-formyl-GSH hydrolase; GLO2, Glyoxalase II; HAGHL, hydroxyacylGSH hydrolase-like; LGUL, lactoylGSH lyase; MAAI , maleylacetoacetate isomerase; PTGD2, GSH-requiring prostaglandin D synthase; PTGDS, prostaglandin-H2 D-isomerase; PTGES, prostaglandin E synthase; RBP1, RalA-binding protein 1 (RalBP1); GLRX, glutaredoxin and glutaredoxin-related proteins; YD286, glutaredoxin-like protein; GPX, GSH peroxidase; GSHR, GSSG reductase; TRXR3, thioredoxin reductase 3.

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