Radical-free biology of oxidative stress

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-kappaB), receptor activation (e.g., alphaIIbbeta3 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.

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).

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.

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.

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 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.

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)-κB, Nrf-2, AP-1, P53, glucocorticoid receptor (GR), estrogen receptor (ER), and HIF-1α (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.

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-κB 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).

Fig. 7.

Steady-state redox potential (E_h) values for thiol-disulfide couples within different subcellular compartments. Approximate steady-state E_h 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 E_o equal to −210 mV. This comparison shows that the range of E_h 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).

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 Pr_A, Pr_B, 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.

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-κB activation involving IκB phosphorylation and degradation (182); 7) NF-κB 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).