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REVIEW

Radical-free biology of oxidative stress

Division of Pulmonary, Allergy and Critical Care Medicine, Clinical Biomarkers Laboratory, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia

Submitted 31 May 2008 ; accepted in final form 31 July 2008

ABSTRACT

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-B), receptor activation (e.g., IIbβ3 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.

thioredoxin; glutathione; cysteine; hydrogen peroxide; redox signaling; protein thiol

Many proteins contain redox-sensitive thiols, and reactions of thiol systems occur largely by nonradical two-electron transfers. Accumulating data show that central thiol-disulfide couples are maintained under nonequilibrium conditions in biological systems. This presents a condition wherein changes in abundance and distribution of redox catalysts and changes in rates of generation of relevant oxidants (e.g., peroxides) and precursors for NADPH supply can account for pathological effects of oxidative stress through altered functions of enzymes, receptors, transporters, transcription factors, and structural elements, without free radicals.

In this review, I summarize free radical and nonradical mechanisms in the redefinition of oxidative stress (82, 163) and develop the redox hypothesis based on recent data showing that major thiol systems exist under stable nonequilibrium conditions. In this development, I focus on H₂O₂ as a nonradical oxidant (see Table 1) and do not address details of radical and nonradical oxidants because they have been addressed previously (95, 177). An overview of distribution and function of thiol systems, with a focus on GSH- and thioredoxin (Trx), is presented to illustrate that reversible oxidation/reduction of thiols controls enzymes, receptors, and transcription factors and regulates cell signaling. Evidence is also available to show that protein trafficking, protein synthesis and degradation, and cytoskeletal structure are redox sensitive, but these are not addressed due to space limitations. Accumulating experimental evidence for nonequilibrium conditions of thiol-disulfide couples (88) is summarized, with discussion of the methods and potential artifacts. Finally, available evidence to support nonradical mechanisms of oxidative stress is reviewed. These data indicate that two-electron oxidants are likely to be quantitatively important determinants of oxidative stress. The results identify important opportunities for cell physiology research to define critical thiol targets and novel strategies for therapeutic interventions to prevent nonradical mechanisms of oxidative stress in aging and disease.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Two mechanisms of oxidative stress. Oxidative stress is defined as an imbalance in prooxidants and antioxidants, which results in macromolecular damage and disruption of redox signaling and control (163). Considerable research has focused on free radical (1-electron) mechanisms in macromolecular damage (left). The present review focuses on an alternative mechanism that involves 2-electron oxidants (right). Based on the xanthine oxidase kinetics, which show that univalent reduction of O2 to superoxide anion radical is always a minor fraction of the bivalent reduction to H2O2 (45), one can anticipate that 2-electron oxidants predominate during oxidative stress. Thus for an arbitrary rate of oxidant generation equal to 1,000, perhaps 900 would go directly to 2-electron oxidants and only 100 would be 1-electron oxidants. Free radical scavenging mechanisms are efficient and convert free radicals into nonradical oxidants. The rate of oxidant production would be partitioned; if the radical scavengers are >99% effective, then there would be a rate of macromolecular damage of <1 and a rate of 2-electron oxidant production equal to >999. Thus if nonradical oxidants contribute to disease pathology by disruption of redox signaling and control mechanisms, the pathology can correlate with macromolecular damage and yet be relatively insensitive to free radical scavengers. Nonradical oxidants (e.g., conjugated aldehydes) also cause macromolecular damage, which can disrupt redox signaling and control pathways (not shown).

This distinction between free radical chemistry in purified systems and in biological systems may be critical in understanding oxidative stress because biological systems generate more nonradical oxidants than free radicals (Fig. 1). For instance, in the well-studied xanthine oxidase reaction, which produces the free radical superoxide anion, the predominant product is H₂O₂ (45). A comparison of the univalent flux to the divalent flux showed that under optimal conditions, superoxide represented only 30% of the flux (45); under most conditions the univalent flux was considerably less, consistent with partitioning of radical and nonradical oxidant delivery in cells as depicted in Fig. 1. Sequential univalent transfer occurs with many redox proteins that generate superoxide so that H₂O₂ is a common product of these reactions. Moreover, dismutation of superoxide anion to H₂O₂ and O₂ occurs at a rapid rate (46), and reaction of radicals with free radical scavengers can result in a highly efficient conversion of free radicals to nonradical oxidants. The free radicals nitric oxide and superoxide react to generate the nonradical oxidant peroxynitrite (179, 180). The two-electron, nonradical oxidant H₂O₂ is produced at 1 to 4% of the rate of O₂ consumption and therefore represents a major oxidant burden. Importantly, if oxidative stress produces radicals and nonradical oxidants as described in Fig. 1 and nonradical oxidants are quantitatively important in disease, then free radical-dependent macromolecular damage can correlate with pathological insult but, none the less, be largely irrelevant to the disease process. Hence, the impetus of the current review is the need to consider two-electron oxidation as a component of oxidative stress that is distinct from free radical-mediated macromolecular damage.

In certain regards, this parallels and overlaps concepts elaborated as the "mitohormesis hypothesis" (176). In mitohormesis, sublethal mitochondrial stress is proposed to produce beneficial outcomes through mitochondrial generation of reactive oxygen species, which serve as signaling elements for cytoprotection. The concept is supported by studies in Caenorhabditis elegans, which show that glucose restriction stimulated mitochondrial respiration and 2,7-dichlorodihydrofluoroscein diacetate oxidation and increased life span (155). A recent review of these concepts proposes novel therapeutic approaches to enhance tolerance to cardiac ischemia by activating the relevant signaling mechanisms (156). However, the mitohormesis hypothesis does not discriminate between free radical and nonradical mechanisms, and a better understanding of the distinct signaling and toxicological mechanisms of the radical and nonradical species will facilitate development of effective therapies.

Thiol-containing proteins that function in redox signaling and physiological regulation are susceptible to two-electron oxidation by nonradical oxidants, including H₂O₂, lipid hydroperoxides, aldehydes, quinones, and disulfides. Either abnormal oxidation or irreversible modification can interfere with reversible oxidation-reduction reactions of thiols that physiologically function in receptor signaling, transcriptional regulation, cell proliferation, angiogenesis, and apoptosis. Thus the contemporary refinement in definition of oxidative stress (82, 163) represents a shift to include both nonradical oxidants and reversible oxidative reactions of redox signaling and control as key components of oxidative stress.

The Redox Hypothesis In an attempt to clearly delineate the nonradical complement to free radical theories that have dominated oxidative stress research, I have formulated a "redox hypothesis" with four postulates: 1) All biological systems contain redox elements [e.g., redox-sensitive cysteine, Cys, residues] that function in cell signaling, macromolecular trafficking, and physiological regulation. 2) Organization and coordination of the redox activity of these elements occurs through redox circuits dependent on common control nodes (e.g., thioredoxin, GSH). 3) The redox-sensitive elements are spatially and kinetically insulated so that "gated" redox circuits can be activated by translocation/aggregation and/or catalytic mechanisms. 4) Oxidative stress is a disruption of the function of these redox circuits caused by specific reaction with the redox-sensitive thiol elements, altered pathways of electron transfer, or interruption of the gating mechanisms controlling the flux through these pathways.

The subsequent sections of this review address the experimental basis for these postulates and discuss their potential relevance to oxidative stress as a causal mechanism in disease.

Thiols as critical components in redox systems biology. Proteins contain two common functional groups [thiol of Cys and thioether of methionine, Met] that undergo reversible oxidation-reduction. The present review focuses on Cys because of the more abundant data on Cys oxidation. The relevant oxidation states are the thiol (-SH), disulfide (-SS-), sulfenate (-SO^–), sulfinate (-SO₂^–), and sulfonate (-SO₃^–), of which the thiol and disulfide are most common. Thiyl radicals (-RS·) generated from thiols in the presence of oxygen-centered radicals (197), as well as other reactive sulfur species (50), have also been considered as toxic species involved in oxidative stress. Thiyl radicals rapidly react to form disulfides (171), and this may serve as a convergence point between one-electron and two-electron events. Sulfenates are relatively unstable and are converted to disulfides in the presence of thiols. In certain protein structures, they can be stabilized as sulfenamides (150, 185). The higher oxidation states (sulfinates and sulfonates) are typically not reversible in mammalian systems, although a yeast enzyme (sulfiredoxin) was recently discovered, which reduces sulfinate in peroxiredoxin (15).

Reversible oxidations of Met residues and the less common amino acid selenocysteine (Sec) also occur. Oxidation of Met to methionine sulfoxide occurs in association with oxidative stress and aging (169, 170). Such oxidation affects biological activity as shown by loss of ₁-antitrypsin inhibitor activity upon Met oxidation (22). Two types of methionine sulfoxide reductases act on the structurally distinct S- and R-sulfoxides (65, 89, 196); methionine sulfoxide reductases activities are dependent on thioredoxins (Trx) (196) and have been associated with longevity (123, 130, 147, 152). The less common selenol of Sec undergoes reversible oxidation-reduction during catalytic functions of Trx reductases and Se-dependent GSH peroxidases (52, 96). These Se-dependent enzymes are present at key positions in both Trx and GSH pathways. Although the present review is focused on Cys oxidation, both Met and Sec oxidation are relevant to the redox hypothesis because of their relationships to the thiol systems and because of their individual functions in control of protein structure and activity.

Regulation of biological functions by thiols. Reversible oxidation of thiols to disulfides or sulfenic acid residues controls biological functions in three general ways, by chemically altering active site cysteines, by altering macromolecular interactions, and by regulating activity through modification of allosteric Cys (Fig. 2). Although these are not mutually exclusive, classification by these criteria provides an easy way to view the different aspects of redox regulation. Reversible chemical modification of active site Cys residues yields an "on-off" mechanism for control. Crosslinking of proteins through disulfides provides a mechanism for aggregation and trafficking. Oxidation of Cys distal to active sites provides an allosteric mechanism. Because proteins often contain multiple Cys residues that can undergo reversible modification to intramolecular and intermolecular disulfides, sulfenic acids, sulfonic acid, S-glutathione derivatives, and S-nitroso derivatives, a broad range of thiol-dependent regulation, can occur.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Sulfur switches (SH) provide flexible control mechanisms for protein function. Cysteine (Cys) is found in the active site of many proteins, and reversible oxidation or modification provides an "on-off" mechanism for protein function (left). Cys residues are also important regulatory elements that control the macromolecular interaction of proteins (center). Reversible modification of nonactive site Cys residues also provides an allosteric type mechanism to regulate activity.

REGULATION OF MACROMOLECULAR INTERACTIONS. Thiol oxidation also controls macromolecular interactions of proteins. Early research showed that thiols prevented aggregation during protein purification, and subsequent studies of protein structure revealed that formation of specific disulfide crosslinks between Cys residues was important for correct three-dimensional structure and activity of proteins. The mechanisms for introduction of disulfides have been studied extensively in protein processing and contribute to stability of secondary, tertiary and quaternary structure. β-Actin contains a conserved Cys, which results in reversible binding of proteins, S-GS-ylation, and crosslinking of actin filaments upon oxidation (33, 112, 140). Oxidation functions in glucocorticoid receptor translocation into nuclei (116, 136), and oxidation controls export of yeast AP-1 (Yap-1) from nuclei (38, 98). Disulfide crosslinks control fluidity of mucus (165). Such changes in protein structure and interaction due to reversible oxidation can provide a central mechanism for specificity in redox signaling (34).

ALLOSTERIC REGULATION. In addition to containing active site and/or structural thiols, many proteins contain Cys which regulate activity by an allosteric mechanism. This type of regulation can provide a "rheostat" rather than an "on-off" switch (88), thereby providing a means to throttle processes by GS-ylation or S-nitrosylation (Fig. 2). Interaction with a metal ion, especially the non-oxidizable Zn²⁺, provides a further level of control through allosteric Cys residues. GS-ylation regulates activity of NADH dehydrogenase (178). S-Nitrosylation of Cys69 in Trx-1 (62) and Cys118 of Ras (102) regulate respective activities. NMDA-receptor activity is modulated by Zn²⁺ ions which bind Cys residues and change receptor activity (31).

Orthogonal regulation in proteins with multiple Cys residues. Individual proteins often contain multiple Cys residues. This allows for a single protein to be controlled in different ways by selective redox mechanisms. These different types of Cys residues can be viewed as orthogonal control elements because they allow a single protein-dependent biological function to be simultaneously regulated by multiple independent mechanisms. Orthogonal regulation is illustrated by known modifications of thioredoxin-1 (Trx-1) (Fig. 3). The active site dithiol motif undergoes oxidation-reduction during the reaction cycle and is important as a regulatory factor through structural interactions with apoptosis signal-regulating kinase-1 (Ask-1). Binding of the C32,C35 dithiol form of Trx-1 inhibits Ask-1 activity. Upon oxidation, release and activation of Ask-1 activates apoptosis. Oxidation of the active site therefore functions as an "on-off" switch for apoptosis. In addition to these active-site thiols, the three nonactive site Cys residues are also subject to redox regulation and provide orthogonal mechanisms for control. Modification of these residues can result in allosteric changes that affect the active site or structural changes, which affect macromolecular interactions. Irreversible modification of C73 with acrolein or 4-hydroxynonenal creates a form that inhibits Trx reductase-1 (TrxR1) and induces pathogenic phenotype (54). This Cys is also a site for GS-ylation and protein-protein disulfide formation, suggesting that physiological regulation could occur through this Cys (23). The remaining Cys residues, C62 and C69, form a second disulfide under oxidative stress conditions (189). Formation of this C62-C69 disulfide inhibits TrxR1 (189), perhaps by disrupting a surface -helix.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Orthogonal regulation can occur through multiple Cys residues on a single protein. Thioredoxin-1 (Trx-1) illustrates how different redox-sensitive elements can be used to simultaneously transmit independent redox signals. The active site (C32,35) undergoes reversible oxidation during catalytic turnover. Oxidation of the active site dithiol results in loss of binding to Ask1 and activation of apoptosis. C73 is an oxidizable amino acid which can undergo glutathionylation (GS-ylation) and also can bind to other cell proteins. C62 undergoes S-nitrosylation and also can form an intramolecular disulfide between C62 and C69 which blocks reduction by TR1.

Together, the data show that there are a large number of redox-sensitive thiols within the Cys proteome. These are widely distributed among signaling, structural, and regulatory proteins. Thus there is a secure basis for the first postulate of the redox hypothesis.

GSH and Trxs as Common Control Nodes for Protein Thiol Redox Circuits The second postulate is that the redox-sensitive elements are organized into redox pathways and control networks. Two major thiol-containing systems function in redox control of protein thiols. GSH, the most abundant thiol in mammals, was discovered over a century ago, and its central function in detoxification and protection against oxidants was recognized 50 years ago (124). Trx was discovered 45 years ago as a reductant for ribonucleotide reductase (106, 126), but its central protective function has only become recognized over the past two decades (74). Both GSH and Trx support enzyme systems for elimination of peroxides, but each system has other distinct functions.

GSH pathways. GSH is a high abundance component present at millimolar concentrations in cells; it serves as a short-term storage form of the amino acid Cys, as a nucleophile for efficient detoxification of reactive electrophiles, and as an antioxidant. It is well suited for redox control of monothiols in proteins (153); as described above, GS-ylation can serve as an "on-off" or a "rheostat" mechanism for control of protein function. GS-ylation reactions are catalyzed by thiol transferases known as glutaredoxins (Grx). These reactions are reversible so that a highly reduced GSH/GSSG redox potential drives reduction of protein disulfides while an oxidizing potential drives protein GS-ylation (171, 187). GSH is also used to protect against two-electron oxidants, such as peroxide reduction catalyzed by GSH peroxidases, and aldehydes and quinones catalyzed by GSH transferases.

An overview of the reactions of the GSH-dependent redox network is illustrated in Fig. 4. An important goal of redox systems biology (88) is to integrate these reactions into quantitative models to describe and test biological activities. However, the number and diversity of processes involving GSH present a challenge for development of such models, especially given the heterogeneous distribution of the proteins and difficulty in study at the subcellular level in intact cells and tissues. Furthermore, the wealth of knowledge on GSH systems under toxicological conditions may have obscured a general function of GSH in physiological regulation. There is a long history of research on GS-ylation as a covalent modification (49, 93, 181, 208), but the facile formation of protein oxidation products during oxidative stress and cell fractionation has led to perhaps an overly conservative point of view concerning the biological importance of regulation by thiol modifications.

View larger version (19K): [in this window] [in a new window]   Fig. 4. 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 -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, -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.

Trx pathways. Trxs differ from GSH in that they are small proteins and present at several orders of magnitude lower concentrations. GSH is ideally suited to react with monothiols (at the sulfenic acid level) to generate S-glutathione derivatives (mixed disulfides) of proteins and then with S-glutathionyl derivatives to generate GSSG and reduced protein. In contrast, Trx is a two-electron reductant that can directly reduce sulfenic acids in proteins and also control redox state of dithiol-disulfide motifs. The active site dithiol of Trx is highly conserved in evolution and widely distributed among Trx superfamily proteins.

Similar to GSH, Trx supports a large network of redox reactions (Fig. 5), including those catalyzed by ribonucleotide reductase, peroxiredoxins, redox factor-1 (1), and methionine sulfoxide reductases. A global antioxidant function of Trx has been inferred from studies showing that Trx can reduce many oxidized proteins. Kinetic studies with different proteins, however, show that there is an underlying specificity for target proteins in terms of the rates of catalysis (14, 74, 110). C35 mutants of Trx function as dominant-negative forms when expressed in cells and have been used as bait to trap putative targets as stable disulfides (119). Such experiments suggest that Trxs could support individual redox pathways based upon this specificity. In addition, other Trx-like proteins probably exist in networks whereby changes in abundance or oxidation of one component regulates function of multiple target proteins (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5. A partial list of proteins in the Trx redox network. 1) Thioredoxins are reduced by thioredoxin reductases (TRXR1,2,3). 2) Trx functions as a reductant for ribonucleotide reductase (RNR). 3) Reduced Trx-1 or Trx-2 binds to apoptosis signal-regulating kinase-1 (ASK-1), inhibiting its function. 4) Trx-1 also binds to other proteins, including the vitamin D3-binding protein (VDUP1; TXNIP) (133). Interaction with such proteins regulates activity and may determine distribution between cytoplasm and nuclei. 5) In cell nuclei, Trx-1 reduces redox factor-1 (REF-1), which is the DNA repair/redox enzyme, AP endonuclease (200). REF-1 maintains conserved Cys residues of transcription factors in the reduced form required for DNA binding. These transcription factors include nuclear factor (NF)-B, Nrf-2, AP-1, P53, glucocorticoid receptor (GR), estrogen receptor (ER), and HIF-1 (1, 17, 72, 75, 116, 182, 184). 6) Trx-1 is a reductant for methionine sulfoxide reductases (MSR) and also for 7) a pathway for MSRB2 reduction mediated by metallothionein in the metal-free thionein form (148). 8) Trx supports six peroxiredoxins (PDRX) that have heterogeneous subcellular distributions but a common activity to eliminate peroxides (146). Redox functions can be provided by Trx-related proteins (TRP14, TRP32, nucleeoredoxin and Trx-related transmembrane protein), which contain a Trx motif and may have evolved to recognize distinctive groups of proteins from those recognized by Trx-1 (105, 107, 122, 198). A large number of Trx-like proteins are known (below dotted line), and many of these are likely to be redox active, either in pathways dependent on Trx or in parallel pathways, which evolved to provide additional specificity beyond that provided by the Trx proteins. Protein designations and common names are from the UniProtKB/Swiss-Prot database. TXN4A, Trx-like protein 4A (spliceosomal U5 snRNP 15 kDa; Dim-1); TXN4B, Trx-like protein 4B (Dim1-like protein); TXND9, ATP-binding protein associated with cell differentiation (APACD); TXNL1, Trx-like protein 1 (32 kDa); NXNL1, nucleoredoxin-like protein 1 (Trx-like protein 6); GLRX3, glutaredoxin-3, PKC-theta-interacting protein; TXND3, spermatid-specific Trx-2; TXND8, spermatid-specific thioredoxin-3 (Trx-6); PDIA6 Protein disulfide-isomerase A6; TXD10, Trx-related transmembrane protein 3 (PDI); TXND1, transmembrane Trx-related protein; DJC10, ER-resident protein; ERdj5, microthioredoxin; TXND4, endoplasmic reticulum protein ERp44; TXND5, endoplasmic reticulum protein ERp46; TXD12, Trx domain-containing protein 12 (ERp18); TXND, Trx domain-containing proteins.

Critical issues for integrating Trx and GSH systems into redox network models have been identified (88). Effective control requires both insulation of redox elements and mechanisms to coordinate functions. In this context, an accurate definition of redox pathways is difficult because the pathways are dependent on rate constants for individual redox elements and sensitive to abundance, distribution, and steady-state redox potentials. With a large number of redox-sensitive elements and central GSH- and Trx-dependent systems, it appears likely that there is an underlying organization, i.e., a "redox code" that allows efficient coordination of biological redox functions (88). However, organizing principles for GS-ylation, S-nitrosylation, thiol-disulfide control, and other functions are not yet fully defined (121). Moreover, the concept of a pathway, as applied to glycolysis or other metabolic pathway, may be inaccurate for a redox pathway. In metabolism, a metabolic pathway is defined by the product of one catalytic step being a substrate for a subsequent catalytic step. In redox pathways, specificity may be defined, at least in part, by the proximity of thiols that undergo oxidation. Thus the spatial distribution may determine the substrate-product relationship so that the meaning of "redox pathway" has both spatial and kinetic components.

To develop redox system models, quantification of electron flow is needed for the overall systems and for individual components. Relatively little information is available on physiological reaction rates for any redox signaling or control mechanism. However, the abundant knowledge of GSH-dependent and Trx-dependent control of signaling, structural, and physiological processes supports the postulate that redox elements are organized through pathways dependent on these central redox couples. Initial information described below further indicates that the networks supported by the GSH and Trx systems are kinetically and functionally distinct.

Nonequilibrium Conditions of Thiol-Disulfide Couples in Biological Systems The third postulate is that redox-sensitive elements are spatially and kinetically insulated so that "gated" redox circuits can be activated by translocation/aggregation and/or catalytic mechanisms. This represents a departure from traditional views that Cys residues in cellular proteins are fully reduced and that GSH provides a "redox buffer" to protect against oxidation of protein thiols. Accumulating data show that critical Cys residues, which function in redox signaling and control, are not in equilibrium with each other or with the NADPH/NADP⁺ couple. Some Cys residues are present in proximity to cationic amino acids, which lowers their pKa values and provides an autocatalytic means to facilitate oxidation-reduction reactions (166). Kinetic control of the redox-sensitive Cys residues allows the systems to be rapidly responsive and dynamically regulated. At the same time, specificity in redox signaling mechanisms requires that individual signaling elements be insulated from other redox-active components. Such specificity is possible because noncatalyzed oxidation and thiol-disulfide exchange reactions are slow at prevailing conditions in cells. This can occur through spatial and kinetic insulation of redox elements, allowing "gated" redox circuits to be activated by translocation/aggregation of redox components and/or regulated activation of redox catalysts. Importantly, as outlined below, the current through such circuits only needs to be a small fraction of total electron transfer in cells. A low rate of oxidation is often observed during protein purification, and this raises the possibility that what has been traditionally considered nonphysiological "autooxidation" of proteins during purification may be characteristics of unrecognized regulatory mechanisms.

Several lines of evidence support the interpretation that critical thiol-disulfide redox couples are maintained under stable, nonequilibrium conditions in biological systems. One of the most unequivocal pieces of evidence is the disequilibrium of the cysteine/cystine (Cys/CySS) and GSH/GSSG couples in human plasma (84, 87). Steady-state redox potential (E_h) values provide a convenient way to compare the tendency of redox couples to accept or donate electrons (83). The difference in E_h values (E_h) between two couples is proportional to the free energy for electron transfer (G = nFE_h, where n is the number of electrons transferred and F is Faraday's constant). E_h is dependent on both the inherent tendency of the chemical to accept or donate electrons, expressed in the standard potential (E_o), and also the concentrations of the acceptor (oxidized) and donor (reduced) species of the couple, as defined by the Nernst equation {E_h = E_o + RT/nF ln([oxidized]/[reduced])}. The mean E_h of GSH/GSSG in the plasma of 24 healthy, young adults was –137 ± 9 mV, whereas the concurrent value for the E_h of Cys/CySS was –80 ± 9 mV (84). These values are displaced from equilibrium by approximately two orders of magnitude. Because 1) human plasma is a homogeneous fluid with no subcellular compartments, 2) no significant noncovalent binding occurs with proteins, and 3) the same analytic procedures were used for simultaneous measurements of all of the chemical species involved; the results show that the major low molecular weight thiol-disulfide couples in human plasma are maintained in a nonequilibrium state (83, 84).

This supports an earlier conclusion that plasma thiol-disulfide couples are not at equilibrium based on reactions of Cys and GSH with plasma proteins (104). The conclusion is further supported by calculations using the second-order rate constant for thiol-disulfide exchange reactions [20 M^–1min^–1; (49)]. At prevailing concentrations of GSH (2 µM) and CySS (40 µM) in plasma (84), the calculated rate of reaction of GSH with CySS is 0.0016 µmol·l^–1·min^–1. This is much lower than the rate of Cys turnover in humans, which was determined by stable isotopic tracer methods to be about 1 µmol·kg^–1·min^–1 (47, 115, 144). Thus the calculated rates are consistent with measured steady-state redox values showing that the rates of thiol-disulfide exchange are too slow to allow redox couples to equilibrate in human plasma.

Steady-state redox potential of the GSH/GSSG couple in cells and tissues. Earlier calculations of the E_h in the liver (49) indicated that this value (–255 mV) is highly displaced from the E_h value for NADPH/NADP⁺ [–380 to –405 mV (161)]. Sies and Summer (164) and a later systematic review by Gilbert (49) concluded that the kinetic characteristics of GSSG reductase were incapable of maintaining GSH/GSSG in equilibrium with NADPH/NADP⁺ because of a relatively high K_M for GSSG. Studies with a number of cell lines and tissues are consistent with the E_h for GSH/GSSG being in the range of –260 to –200 mV (82), i.e., highly displaced from equilibrium with NADPH/NADP⁺.

Studies performed with HT29 cells evaluated possible artifacts in estimation of E_h due to pH, concentration measurements, and inhomogeneous distribution of GSH and GSSG between compartments. Cell volume was measured by distribution of ³H₂O with extracellular volume determined by distribution of [¹⁴C]inulin (92). Cytoplasmic pH was determined by distribution of the pH indicator [¹⁴C]dimethadione (92). Errors due to sequestration by mitochondrial and secretory compartments were estimated by digitonin fractionation (83, 86). The results showed that the E_h of the cytoplasmic GSH/GSSG couple was about –260 mV under proliferative conditions, about –220 mV under differentiated conditions, transiently oxidized and then reduced following treatment with a GSH synthesis inducer (6), and oxidized to about –170 mV during apoptosis (21, 82, 91). In contrast, E_h of the Cys/CySS couple was –145 ± 3 mV under proliferative conditions and oxidized by about 30 mV by either the differentiation agent, sodium butyrate, or the GSH synthesis inducer benzyl isothiocyanate (92). Results showed that errors due to volume and pH were negligible (92). Contamination due to contents of subcellular compartments was estimated to be no more than 10 mV (82, 86). These results confirmed that E_h for cellular GSH/GSSG is in the range of –260 to –200 mV, i.e., considerably oxidized relative to the NADPH/NADP⁺ couple (82).

Steady-state E_h of Cys residues in proteins. Development of methods to evaluate redox potential of individual thiol-disulfide couples in proteins (see Perspectives) has allowed determination of E_h values for Trx-1 and Trx-2 in cells and tissues (64, 69, 70, 188, 189). Results showed that the steady-state E_h for Trx-1 was –300 to –270 mV, a range that is more negative (reduced) than E_h for GSH/GSSG in CaCo-2 cells (135), THP-1 cells (188), HeLa cells (57), and keratinocytes (64). Thus the E_h for cytoplasmic Trx-1 is not in redox equilibrium with E_h for cellular GSH/GSSG. Studies of the mitochondrial compartment are more limited, but in HeLa, HT29, THP-1 cells, and isolated liver mitochondria, E_h for Trx-2 was –360 to –330 mV, whereas estimates for E_h GSH/GSSG were approximately –300 mV (21, 57, 64, 66, 70). Together, these results show that major thiol-disulfide redox components within subcellular and extracellular compartments are not equilibrated.

Trx and GSH redox systems are functionally distinct. In addition to the biochemical evidence as illustrated in Figs. 4 and 5, several studies show that the Trx and GSH systems are functionally distinct in cells. For instance, mitochondrial Trx-2 was found to be more sensitive to oxidation than cytoplasmic Trx-1 by exogenously added peroxides (27), certain metals (arsenic and cadmium) (69), and TNF- (70). The nuclear compartment was more resistant to oxidation than cytoplasm as indicated by Trx-1 oxidation (57) and the extent of nuclear protein GS-ylation (57, 63) in cells with targeted nuclear generation of H₂O₂ or limited NADPH generation. The secretory pathway has also been shown to be relatively oxidized compared with the cytoplasm (76).

The nonequilibrium steady-state E_h values for Trx-1, GSH/GSSG, and Cys/CySS couples show that in biological systems, proteins that rapidly interact with Trx-1 do not rapidly interact with GSH/GSSG or Cys/CySS (88). This explicitly means that Trx-1 and glutaredoxin (Grx-1) do not function as reversible reductants/oxidants for the same protein substrates because such reactivity would equilibrate the couples. Thus the nonequilibrium conditions support the concept that GSH and Trx function as control nodes for different redox networks.

Electron transfer within a redox pathway is kinetically limited. In a study of electron flow within a thiol-disulfide pathway, Go et al. (57) used redox Western blot analysis to measure Trx-1 redox state and BIAM-blot to measure fractional reduction of TrxR1, Ref-1 (redox factor-1), and the p50 subunit of NF-B. Under control conditions in HT29 cells, the fractional reduction in the cytosol was consistent with a redox gradient for the sequence NADPHTrxR1Trx-1Ref1NF-B(p50) (Fig. 6). In contrast, the fractional reduction in the nuclei indicated that the transfer of electrons was blocked between Trx-1 and Ref-1 (Fig. 6). When cells were transferred to glucose- and glutamine-deficient media, the cellular NADPH pool became oxidized. Under this condition, each component in the cytoplasmic pathway became oxidized while the nuclear pathway was more uniformly reduced (Fig. 6). The results showed that even without an oxidative challenge, this pathway was kinetically limited both in the cytoplasm and in the nuclei, thereby supporting the interpretation that thiol-disulfide redox circuits are kinetically limited.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Kinetic limitation in a thiol-disulfide electron transfer pathway. Measurement of the fractional reduction of proteins in the pathway from NADPH through TrxR1, Trx-1, and redox factor-1 (Ref-1) to NF-B p50 subunit under control conditions showed that the pathway is kinetically limited in both the cytoplasmic and nuclear compartments. In the nuclear compartment, the characteristics suggested that electron transfer was blocked between Trx-1 and Ref-1. Under conditions where NADPH supply was limited by removal of Glc and Gln from the culture media, the cytoplasmic pathway was selectively oxidized while the nuclear pathway showed a loss of the kinetic limitation. The results show that metabolic conditions determine sites of rate limitation, supporting the concept that rate control in redox pathways is important in cell signaling and regulation. Scheme is based upon data of Go et al. (57).

Partitioning of H₂O₂ metabolism between enzyme systems. The knowledge of peroxide metabolizing systems has progressed with sequential discovery of catalase, Sel-dependent GSH peroxidase, Sel-independent GSH peroxidases (glutathione transferases), and peroxiredoxins. Several lines of evidence indicate that in mammalian cells, catalase has little contribution to peroxide metabolism outside the peroxisomes (32, 85, 164). Before the recognition of peroxiredoxins, peroxide metabolism was assumed to be largely catalyzed by the Se-dependent and Se-independent GSH peroxidases (20). Partitioning of metabolism between these systems was inferred from GSSG efflux in perfused liver and heart preparations (20, 201). Protein thiol oxidation was not measured in these studies, and it is unclear how efficiently GSSG was reduced back to GSH, as opposed to being transported out of cells. A similar study with steady-state infusion of diamide in hepatocytes showed that protein thiol oxidation constituted 73% of the net thiol oxidation while GSH oxidation was only 27% of the total (183). Thus substantial protein oxidation can occur without depletion of GSH, indicating that protein and GSH oxidation occur concurrently.

If metabolism of endogenously produced H₂O₂ is partitioned among all 214,000 Cys residues in proteins, this could mean that about 1,070 specific protein thiols (i.e., 0.5% of total) undergo reversible oxidation/reduction at a rate of 1 per minute (see Table 2). Because thiols are present in redox pathways where thiols of one protein donate electrons to disulfides of other proteins [e.g., NADPH-TrxR1-Trx-1-Ref1-NF-B(p50)], the actual number could be three- to fivefold higher. Thus the emergent picture is that there are hundreds to thousands of dynamic redox control elements (out of the estimated 21,000 to 42,000 readily oxidizable thiols) that support redox signaling and integration of metabolic functions through low-current redox circuits. In other words, the classes of redox elements described in Fig. 2 can be controlled by redox systems as described in Figs. 4 and 5 to coordinate and regulate cell functions through kinetically controlled pathways.

E_h values are sufficient to provide an energetic driving force for low current redox control pathways. The E_h values for thiol-disulfide couples range from –360 mV for Trx-2 in mitochondria to a mean value of –60 mV for Cys/CySS in plasma. According to the Nernst equation where n = 2, E_h becomes 30 mV more positive with a 10-fold increase in the ratio of oxidized:reduced forms. This means that a E_h of 60 mV between two couples is sufficient to drive a 100-fold change in dithiol-to-disulfide ratio. The 300-mV span between mitochondrial Trx-2 and Cys/CySS is therefore sufficient to control different redox functions through relatively shallow redox gradients provided that spatial or catalytic mechanisms are available to control rates. Because H₂O₂ and O₂ are always present under aerobic conditions, coupling of electron transfer to peroxidase or oxidase reactions can further provide an energetic driving force to maintain function of low-current redox control pathways.

The span of redox potentials for known thiol-disulfide couples in different subcellular compartments is summarized in Fig. 7 to illustrate the magnitude of possible effects on protein thiol-disulfide redox state. If a hypothetical protein with a dithiol-disulfide motif with E_o value of –210 mV were equilibrated with cytoplasmic GSH/GSSG at –210 mV, the dithiol-to-disulfide ratio would be 1. If the same protein were equilibrated with cytoplasmic Trx-1 at –270 mV, the ratio would be 100:1. If the protein were equilibrated with mitochondrial Trx-2 at –360 mV, the ratio would be 100,000:1. If the protein were equilibrated with cytoplasmic Cys/CySS at –150 mV, the ratio would be 1:100. Equilibration with plasma Cys/CySS at –60 mV would yield 1:100,000. Thus, if catalysts or autocatalytic structures are present to allow selective interaction of thiols with the central redox control nodes, the range of redox potentials is sufficient to provide a considerable organization of protein structures and functions.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Steady-state redox potential (Eh) values for thiol-disulfide couples within different subcellular compartments. Approximate steady-state Eh values for mitochondrial Trx-2, mitochondrial GSH/GSSG, nuclear Trx-1, cytoplasmic Trx-1, cytoplasmic GSH/GSSG, cytoplasmic cysteine/cystine (Cys/CySS), endoplasmic reticular GSH/GSSG, plasma GSH/GSSG, and plasma Cys/CySS are listed along with different dithiol-disulfide ratios (PrSH/PrSS) for a hypothetical protein couple with Eo equal to –210 mV. This comparison shows that the range of Eh values is sufficient for relatively shallow redox gradients between couples to control protein functions based on catalyzed redox circuits between redox couples or between the listed couples and the H2O2/H2O couple (not shown).

where Trx controls a series of sulfur switches. Each switch has an autocatalytic character due to proximal cationic residues or has a specific enzyme catalyst such as one of the peroxiredoxins or other Trx family members. Thus the oxidation of the switch is determined by either proximity of the elements to the H₂O₂ generation or activation of the catalyst. Similar pathways can be written for GSH-dependent pathways (Fig. 8) in which GS-ylation can be catalyzed by glutathione transferases in the presence of H₂O₂ and countered by Grx-dependent removal of the GSH moiety. Alternatively, GS-ylation could occur as a consequence of local Gpx-dependent generation of GSSG in the presence of H₂O₂. In this case, a transient burst of H₂O₂ could generate a local GS-ylation with an autorecovery after the peroxide burst. Other proteins, such as the Trx reductases (10), thioneins (148), and redox factor-1 [Ref. 1; (200)] may also serve as control nodes supporting additional redox control networks.

View larger version (12K): [in this window] [in a new window]   Fig. 8. Scheme depicting possible organization of Trx- and GSH-dependent pathways into redox control networks. Both Trx and glutaredoxins (Grx) catalyze oxidation-reduction reactions with multiple protein substrates. Both are dependent on electron transport pathways with NADPH as the electron donor. Left, Trx is reduced by TrxR and in turn reduces sulfur switches, which are protein disulfide or sulfenic acids [designated Pr1(SS), Pr2(SS), etc.]. The sulfur switches are depicted as either 1) cysteine residues present in autocatalytic structures that are directly oxidized by H2O2 or 2) substrates for catalysts that are oxidized by H2O2. Right, Grx uses reducing equivalents from GSH, which is maintained by GSSG reductase, to reduce sulfur switches designated as PrA, PrB, etc. The precise mechanism for introduction of GSH moieties into proteins is not clear. In this figure, GS-ylation of these sulfur switches is depicted (hypothetically) as being catalyzed by 3) glutathione transferase (GST), in the presence of H2O2. Alternatively, 4) local generation of H2O2 can generate GSSG by Gpx, and the local high GSSG concentration could be used by Grx to form the S-glutathionyl derivative of the sulfur switch. Protein substrates for Trx and Grx are discussed in the text, but no studies are available to test interactions of pathways in functional Trx or GSH/Grx networks.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Redox-sensitive steps in hypothetical redox signaling pathways. Accumulated research from multiple investigators provides evidence for complex spatial and temporal redox events that can be combined in hypothetical signaling pathways with multiple redox sensitive steps. Evidence is collected from experiments using different cell lines and under different conditions so that the combined reactions should not be considered validated reaction pathways, but rather, as evidence that redox signaling involves so many redox-sensitive sites that the regulation is likely to have evolved a functional design that is coordinated through a smaller number of control nodes. Sites sensitive to changes in thiol-disulfide redox state are indicated by an "-SH/-SS-" balance. A: in early proinflammatory signaling in endothelial cells, redox-dependent signaling includes 1) extracellular Cys/CySS redox potential (55); 2) cell surface thiol sensor (55); 3) kinase/phosphatase regulation (39); 4) mitochondrial oxidant generation from mitochondrial electron transport (Mito ET) (Go and Jones, unpublished); 5) H2O2 (55); 6) NF-B activation involving IB phosphorylation and degradation (182); 7) NF-B binding to DNA (160); 8) translation (109); 9) processing of proteins in the secretory pathway (7, 24); and 10) cytoskeletal/surface structure (33, 140, 187). B: in receptor-mediated signaling, redox-sensitive steps include 1) metalloprotease-sensitive growth factor release (134); 2) metalloprotease-sensitive degradation of growth factor inhibitor (51); 3) redox-dependent activation of receptors (31, 94); 4) H2O2-dependent Ca2+ influx and Nox-5 activation (40); 5) active-site Cys residues required for phosphatase activity [PTP1B, SHP2, PTen; (39)]; 6) Ras activity (3); 7) Src activity (48); 8) H2O2 metabolism; 9) lipoxygenase activity (42); 10) LPS activation of cytoplasmic and mitochondrial H2O2 production through Toll-like receptor 4 [TLR4 (77, 139)] (42, 77, 139).

The extensive literature on redox signaling clearly supports the concept that multiple redox elements are present. Although integrated models with experimental support for simultaneous involvement of multiple redox sites are not available, the large number of redox-sensitive steps identified indicates that underlying organizational principles must exist and that these probably involve GSH- and Trx-dependent redox networks. Thus there is a considerable need to extend research efforts beyond experimental models with nonspecific reagents [such as N-acetylcysteine (NAC) and high concentrations of H₂O₂] to ones in which the specificity of redox signaling and control can be elucidated. The available evidence supports the third postulate of the redox hypothesis, namely, that thiol redox pathways are kinetically limited. Studies to identify kinetically limiting sites of redox control will provide sites that are subject to disruption during oxidative stress.

Oxidative Stress as a Disruption of Redox Signaling and Control The above conceptualization that redox mechanisms contribute to structural and functional organization of cells through a continuous, reversible oxidation/reduction of about 0.5% of the Cys proteome provides a basis to consider the fourth postulate of the redox hypothesis, namely that oxidative stress is a condition where there is a disruption in the normal function of the redox networks. Because the networks involve reversible two-electron oxidations, disruption can occur with or without concomitant free radical-mediated macromolecular damage. Organization of redox elements in insulated redox circuits allows for specificity in oxidative disruption at low levels of oxidants due to type and source of oxidant. In contrast, at high levels, a more global disruption would occur. Thus, in the context of the first three postulates, the fourth postulate predicts specificity in oxidant-dependent mechanisms in disease under usual in vivo conditions and a loss of such specificity when excessive oxidants are introduced.

H₂O₂ as a constitutively produced nonradical oxidant in cells. H₂O₂ is a normal cellular metabolite that is continuously generated and maintained at low concentrations by a number of peroxidases. Techniques based on the spectral and kinetic properties of catalase (137, 162) provided the first reasonable estimates of H₂O₂ concentration in mammalian cells. Importantly, these studies showed that steady-state H₂O₂ production occurs at rates that maintain nanomolar concentrations; during detoxification, rates can be stimulated in the liver to >10% of the total O₂ consumption rate (25, 85, 164). Measurements by this approach largely reflect concentrations and metabolism in peroxisomes. Peroxisomes are abundant only in some organs, e.g., liver and kidney, and may contribute little to H₂O₂ metabolism in other tissues (201). Thus quantitative evaluation of peroxide metabolism in other compartments is indirect.

Mitochondria continuously release peroxide at rates that are estimated to be 1% to 4% of the mitochondrial O₂ consumption rate (25). Consequently, mitochondria are a major constitutive source of H₂O₂ outside of peroxisomes (25). Other sources include H₂O₂-producing enzymes distributed in subcellular compartments, e.g., Nox family enzymes (cytoplasm, plasma membrane, nuclei), xanthine oxidase (cytoplasm, extracellular), endoplasmic reticulum oxidases, sulfhydryl oxidases (nuclei, extracellular), thiol oxidases (plasma membrane, plasma), and monoamine oxidases (mitochondria outer membrane). Data are available, therefore, to show that H₂O₂ is produced throughout the cell. Furthermore, increased H₂O₂ has been implicated in disease and/or toxicity from peroxisomal proliferators (100), mitochondrial respiratory inhibitors (186), activators of Nox (59, 60), ER stress (71), precursors of xanthine oxidase (58), and substrates of monoamine oxidases (167). While previously considered in terms of a global imbalance of pro-oxidants and anti-oxidants that cause macromolecular damage, these data could alternatively mean that there is a constitutive peroxide tone in subcellular compartments that provides a counterbalance to the Trx and GSH systems to maintain nonequilibrium steady states for redox control circuits (88).

In vitro studies support the interpretation that H₂O₂ is a signaling molecule. The discoveries that low levels of peroxides stimulate cell growth (19), that cancer cells produce increased H₂O₂ (174), and that a family of NADPH oxidases (Nox) is associated with cell proliferation (101, 172), led to the general recognition that redox signaling pathways have an important function in growth control. Superoxide is an immediate product of Nox, but sequential one-electron transfer and/or rapid dismutation of superoxide result in substantial H₂O₂ generation. Importantly, catalase and peroxidases affect redox signaling (55, 67, 101) showing that H₂O₂ is a quantitatively important signaling species.

Studies with targeted increases in abundance of Trx, peroxiredoxins, enzymes of GSH biosynthesis, and GSH peroxidases provide evidence for compartment-specific redox regulation involving H₂O₂. Cytoplasm- and nuclei-specific redox control in transcription factor activation (1) has been supported by recent evidence that H₂O₂ functions in both cytoplasmic activation of NF-B and Nrf-2 and nuclear inhibition of transcription factor binding to DNA (67, 68). For instance, a nuclear export signal (NES)-containing fusion protein (NES-Prx-1) of the antioxidant Prx-1 blocked oxidant-induced activation of NF-B in the cytoplasm (67). A different fusion protein (NLS-Prx-1) containing Prx1 linked to a nuclear localization sequence (NLS) enhanced nuclear NF-B activity (67). Thus the data suggest that a constitutive H₂O₂ concentration exists, which can be decreased in the cytoplasm to block activation while a similar decrease in the nuclei results in increased transcription.

In vitro studies support interpretation that H₂O₂ is a toxic species. Elevated peroxide concentrations can contribute to death signaling without macromolecular damage because oxidation of either Trx-1 or Trx-2 stimulates release of Ask-1 and activation of apoptosis (149, 206). Protection against oxidant-induced apoptosis was observed in 143B osteosarcoma cells and SH-SY5Y human neuroblastoma cells with the nonradical reductant mitochondrial Trx-2 (28, 29). Decreased mitochondrial Trx-2 in DT40 chicken B cells increased oxidants and caused spontaneous apoptosis (175). Similarly, decrease in the Trx-2-dependent peroxidase Prx3 increased H₂O₂ and sensitized HeLa cells to staurosporine- or TNF--induced apoptosis (26). Increased abundance of Prx3 also protected against apoptosis caused by H₂O₂ (205). Depletion of mitochondrial GSH also potentiated peroxide accumulation and sensitized cells to cell death (8, 157). Small interferring RNA (siRNA) for Grx-2 increased sensitivity to doxorubicin, whereas overexpression of mitochondrial Grx-2 inhibited cardiolipin loss and prevented apoptosis induced by doxorubicin (41, 111). Because Trx and GSH protect against two-electron oxidants, this in vitro research points to the plausibility of oxidant-induce cell death mechanisms signaled by nonradical reactions, which disrupt redox control.

In vivo mouse models support nonradical mechanisms of oxidative stress. Genetic mouse models with altered peroxide-metabolizing enzymes also support the interpretation that two-electron oxidants have a central causative role in oxidative stress. Expression of catalase in mitochondria increased longevity in transgenic mice (154). Decreased Trx-2 in hemizygous Trx-2^+/– mice had increased sensitivity to oxidants (141), and Trx-1 transgenic mice were protected against cardiotoxicity from doxorubicin (159). Gpx1 knockout mice demonstrated increased susceptibility to H₂O₂ (36). Gpx4 (–/–) knockouts are embryonic lethal, but cells isolated from Gpx4-deficient mice (+/–) showed an increased susceptibility to peroxides (202). Similarly, the GSH synthesis inhibitor buthionine sulfoximine (BSO) potentiated MPTP toxicity in mice (199). Furthermore, MPTP treatment depleted brainstem GSH (204), and mice deficient in Gpx-1 exhibited increased vulnerability to MPTP (37) .

Other nonradical oxidative chemicals are produced in tissues. In this review, I limited discussion to H₂O₂, but other nonradical oxidants are also likely to contribute to disease. These include lipid hydroperoxides and epoxides generated from polyunsaturated fatty acids by cyclooxygenases, lipoxygenases, and cytochromes P450, aldehydes generated from oxidation of ethanol and other alcohols, and quinones generated from catechols and polyphenols. However, some of these chemicals, such as conjugated aldehydes, are also electrophilic and can disrupt redox circuits by chemical modification of protein thiols. Importantly, thiols are among the most sensitive functional groups for modification by reactive chemicals. Acrolein and 4-hydroxynonenal selectively modify C73 of Trx-1 when added at low concentrations (54). Microinjection of C73-Acr-Trx-1 into endothelial cells showed that this single modification was sufficient to activate pro-inflammatory signaling and monocyte adhesion (54). Specificity in quinone toxicity has also been implicated in the opposing roles of NADPH:quinone reductase (NQO1) and the related quinone reductase-2 (QR2) in quinone toxicity in the retina (18). Metal ions, such as mercury, cadmium, lead, and arsenic also can cause selective disruption of redox circuits based on the specificity of thiol interactions. For instance, 10 µM Fe³⁺ or Cu²⁺ oxidized GSH/GSSG but not Trx-1 or Trx-2, whereas 10 µM As³⁺ or Hg²⁺ oxidized Trx-1 and Trx-2 without oxidation of GSH/GSSG (69).

Nonradical mechanisms of oxidative stress in human disease. Disruption of thiol-disulfide redox pathways as a contributing mechanism to human disease has not been explicitly tested, although several correlative studies support this possibility. The most direct studies are based on cellular research showing that signaling pathways that contribute to disease mechanisms are dependent on extracellular thiol-disulfide redox state. For instance, microinjection of C-73-modified Trx-1 into endothelial cells activated signaling for monocyte adhesion (54). This elicitation of pathogenic signaling by modification of a single amino acid on a protein shows that specificity occurs in oxidative stress effects on redox signaling pathways.

Another example involves the death receptor Fas, which has been found in regions of the retina affected by age-related macular degeneration (AMD). Fas ligand stimulates apoptosis in retinal pigment epithelial (RPE) cells, the initial cells to be lost in AMD (78, 80). Cell studies showed that peroxide-induced cell death in RPE is stimulated by oxidized extracellular E_h (79). Extracellular E_h was oxidized in association with age in AMD patients (128), and soluble Fas ligand increased in human plasma with increasing age (78). A relevant double-blind interventional trial with antioxidants (vitamins C and E; β-carotene) and Zn²⁺ showed that the antioxidants and Zn²⁺ protected against progression of AMD and also against age-related increase in plasma CySS and oxidation of plasma Cys/CySS redox state (128, 129). Thus the results suggest a causal pathway in which antioxidants and Zn²⁺ control plasma Cys/CySS redox state and plasma Cys/CySS redox state controls redox-dependent signaling of apoptosis in RPE, which contributes to AMD.

Other studies relevant to cardiovascular disease show that monocyte adhesion to endothelial cells, an early proinflammatory step in atherogenesis, is stimulated by an oxidized Cys/CySS redox state. The specific mechanism involves intracellular generation of H₂O₂, activation of NF-B, and transcriptional activation and increased cell surface expression of cell adhesion molecules (55). Both GSH/GSSG and Cys/CySS redox states are oxidized in association with cardiovascular disease risk factors, including cigarette smoking (127) and chronic alcohol abuse (203). Oxidized plasma GSH/GSSG redox state has also been associated with Type 2 diabetes (151) and increased carotid intima media thickness, an early risk factor for cardiovascular disease (11). Other in vivo studies show that an oxidized Cys/CySS redox state is associated with increased persistent atrial fibrillation (132) and with myocardial perfusion defects (2). Together, with in vitro mechanistic studies, these clinical studies indicate that redox-dependent changes in peroxide are likely to be important components of CVD risk.

Other in vitro studies also support mechanistic links between thiol-disulfide redox signaling at cell membranes and human disease. For instance, profibrotic signaling by TGF-β1 in pulmonary fibroblasts is stimulated by oxidized extracellular E_h (145), and this suggests that conditions which oxidize plasma Cys/CySS could contribute to fibrotic disease. Platelet activation has an optimal extracellular redox potential mediated through redox changes of Cys residues in IIIbβ3 (43) and could be susceptible to aberrant function due to altered plasma redox states. Proliferative signaling in a colon cancer (CaCo-2) cell line is dependent on redox activation by a metalloproteinase, apparently providing redox-dependent growth factor release (134) or cleavage of growth factor inhibitors (51). Plasma E_h values for both GSH/GSSG and Cys/CySS are oxidized following chemotherapy (81), suggesting that antiproliferative effects may, in part, be mediated by altered signaling dependent on extracellular thiol-disulfide couples.

These circumstantial data point to the need for clinical studies to determine whether disruption of thiol-disulfide redox pathways by nonradical oxidants represents a quantitatively important mechanism for oxidative stress in human disease. Reanalysis of data from previous trials could provide information, but experimental designs developed to observe free radical-derived products may be inadequate to measure nonradical perturbation of redox pathways or discriminate relative importance of one- and two-electron reaction schemes. The numerous trials with NAC, a precursor to increase GSH, have provided inconsistent evidence for benefit in vivo (5) and evidence for potential toxicity in vitro (131). However, the extent to which NAC affects tissue GSH in humans is difficult to assess. Recent data showing that GSH/GSSG and Cys/CySS redox states undergo a diurnal variation linked to dietary intake of sulfur amino acids (16) suggest careful timing and dosing may be needed because NAC absorption and clearance are rapid. Furthermore, homeostatic regulation of Cys and GSH declines with age, and this occurs at a younger age in men than in women (16). Thus dosing regimens may be critical in development of antioxidant strategies designed to protect or correct disruption of thiol redox systems by nonradical oxidants.

Perspectives The available evidence supporting the redox hypothesis indicates both opportunities and challenges for future development. There is considerable need to elucidate the thiol redox signaling and control pathways and identify critical sites where interventions could protect against disruption of function and/or restore normal signaling and control. The recent development of methods to measure redox states of specific proteins in different organelles (56) will allow more complete delineation of compartment-specific disruption of redox pathways in disease. These methods include detection of reduced and oxidized forms of proteins by redox Western blot analysis (64, 189). The redox Western blot analyses of Trx-1 and Trx-2 have recently been found to be suitable for use with human biopsy samples. Results with this approach (Y. Mannery, L. Hao, D. P. Jones, T. R. Ziegler, unpublished observations) show that redox states in normal intestinal biopsies are similar to values obtained in cell culture and that redox states are oxidized in inflamed tissues. An alternate BIAM blot uses an initial labeling of thiols with biotinylated maleimide, followed by immunoprecipitation and Western blot with detection by streptavidin (90).

New discovery-based redox proteomic methods with mass spectrometry use Isotope-Coded Affinity Tag (ICAT) reagent to label thiols with heavy (¹³C) and light (¹²C) forms of biotin-labeled iodoacetamide to quantify redox states of specific Cys residues in proteins (56, 108). Sequential addition of the heavy isotopomer before reduction and then addition of light isotopomer after reduction provides a measure of the reduced fraction of specific Cys residues in terms of the ratio of labeling. This method is also sensitive enough to allow measurement of redox states in proteins in blood cells and biopsies (Y.-M. Go, D. P. Jones, unpublished observations). Application of this approach could considerably enhance understanding of physiological and pathological variations in redox signaling and control in humans. Imaging approaches using magnetic resonance spectroscopy for GSH measurement in the brain (207), fluorescence imaging of GSH/GSSG redox potential (61), and thiol-dependent redox-sensitive contrast agents are also being developed and provide means to evaluate redox signaling and control in vivo.

In summary, research literature on thiol-disulfide systems provides the basis for four postulates in the development of a redox hypothesis of oxidative stress. This hypothesis is that critical mechanisms of oxidative stress that contribute to human aging and disease involve nonradical two-electron oxidations rather than free radical reactions. A central observation is that redox-sensitive Cys residues, which undergo reversible two-electron oxidation-reduction reactions, are common in proteins. These Cys residues have important functions as active sites residues, as regulators of macromolecular interactions and as allosteric regulators. The number of distinct redox-sensitive elements, which includes sites undergoing oxidation to disulfides and sulfenic acids, S-nitrosylation, and GS-ylation, appears to be in the range of a few hundred to several thousand distinct Cys residues, with total oxidative turnover of about 0.5% of cell thiols per minute.

Arguments are developed to suggest that these elements exist in redox pathways dependent on controlled generation of oxidant, such as H₂O₂, balanced by NADPH-dependent reduction pathways. Available evidence indicates that these pathways exist in networks controlled by Trxs and GSH. However, these networks currently are poorly defined and could include other control nodes as well as unidentified catalysts for transnitrosylation, GS-ylation, or other redox reactions.

In the limited number of pathways studied to date, oxidation and reduction of thiol elements are kinetically limited. Noncatalyzed thiol-disulfide exchange rates are slow at prevailing concentrations of thiols and disulfides in cells. This provides a kinetic insulation that is essential for dynamic control of the low-current redox circuits. Under these conditions, efficient control of reactive thiols is obtained by spatial reorganization and regulation of activity of redox catalysts.

Available evidence indicates that redox-sensitive thiol elements function in signaling and control of virtually all aspects of life, including energy regulation, cytoskeletal structure, transport, proliferation, differentiation, and apoptosis. Consequently, disruption of thiol redox signaling and control by nonradical, two-electron oxidants could underlie much of the pathology linked to oxidative stress. To test this hypothesis, research is needed to identify critical redox elements and understand their physiology. Such knowledge can be expected to provide the basis for novel therapeutic approaches to restore normal signaling and control and, thereby, prevent pathological and toxicological consequences of oxidative stress.

GRANTS

Support for research in the author's laboratory was provided by National Institute of Environmental Health Sciences Grants ES-009047 and ES-011195.

ACKNOWLEDGMENTS

The author gratefully acknowledges the estimation of the number of unique Cys and methionine residues in the human proteome provided by Scott Devine and the critical review of the manuscript by Young-Mi Go, Jennifer M. Johnson, and Lou Ann S. Brown.

FOOTNOTES

Address for reprint requests and other correspondence: D. P. Jones, Div. Pulmonary Medicine/Dept. of Medicine, Emory Univ. School of Medicine, 615 Michael St., Suite 205P, Atlanta, GA 30322 (e-mail: dpjones{at}emory.edu)

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