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Published December 4, 2014 Redox regulation of cysteine-dependent enzymes1 R. P. Guttmann2 Departments of Gerontology and Physiology, and the Sanders-Brown Center on Aging, University of Kentucky, Lexington 40536 ABSTRACT: It is well-established that maintenance of the intracellular redox (i.e., reduction-oxidation) state is critical for cell survival and that prolonged or abnormal perturbations toward oxidation result in cell dysfunction. This is exemplified by the widespread observation of oxidative stress in many pathological conditions, as well as the positive effects of antioxidants in treating certain conditions or extending the life span itself. In addition to the effects of oxidation on the lipid bilayer and modification of DNA in the nucleus, proteins are also modulated by the redox state. One of the primary targets of oxidation within a protein is the AA cysteine, whose thiol side chain is highly sensitive to all types of oxidizing agents. Although this sensitivity is used to prevent oxidation within the cell by potent defense mechanisms, such as glutathione, the use of cysteine in the active site of enzymes leaves them open to oxidant-mediated damage. Whether the damage is due to a pathological condition or to postmortem mediated loss of redox homeostasis, cysteine-dependent enzymes are targets of all forms of reactive oxygen, nitrogen, and sulfur species. A greater understanding of the redox-mediated control of cysteine-dependent enzymes opens the door to the selective use of antioxidants to prevent or reverse the cellular damage their inhibition causes. Key words: aging, antioxidant, diet, methionine, oxidative stress, postmortem ©2010 American Society of Animal Science. All rights reserved. INTRODUCTION In addition to many well-known posttranslational modifications, such as phosphorylation, that regulate protein function, modifications mediated by reductionoxidation (redox) are also recognized as a significant source of enzymatic control with general importance in normal cell function (Janssen-Heininger et al., 2008; Ottaviano et al., 2008; Trachootham et al., 2008; Bonham and Vacratsis, 2009; Figtree et al., 2009; Pervaiz et al., 2009). Primarily, protein sensitivity to oxidation is mediated by a reactive sulfur species within either methionine or cysteine, although other side chains are also susceptible. Of relevance here, enzymes that utilize cysteine either as an important structural component or within their active site are often discovered to be regulated by the redox state of their environment (Bon- 1 Based on a presentation at the Cell Biology Symposium titled “Redox regulation of cell function” at the Joint Annual Meeting, July 12 to 16, 2009, Montreal, Canada. Manuscript publication sponsored by the Journal of Animal Science and the American Society of Animal Science. Supported by National Institutes of Health grants AG026521 and AG05144. 2 Corresponding author: [email protected] Received August 7, 2009. Accepted September 29, 2009. J. Anim. Sci. 2010. 88:1297–1306 doi:10.2527/jas.2009-2381 ham and Vacratsis, 2009; Figtree et al., 2009; Leonard et al., 2009; Nadeau et al., 2009). Changes in the redox microenvironment occur because of various reactive oxygen, nitrogen, and sulfur species as well as because of modifications by cysteine-containing proteins or peptides. Although pathological states are easiest to understand with regard to redox imbalance, increased reactive species are generated under physiological states as well. Physiologically, thiol-reactive species are generated through several mechanisms. One prominent means by which reactive species are created is as a by-product of energy production in mitochondria that produces the oxidant superoxide. Oxidases, such as xanthine oxidase, in addition to receptor-mediated peroxide production, such as through the insulin receptor, are other examples of physiological pathways that alter the redox balance. Pathologically, increased oxidative stress has been associated with many conditions because loss of cellular membrane integrity allows the highly oxidizing extracellular compartment to permeate. Although hotly debated, there remains good evidence supporting a positive role for prevention of oxidative stress (Parkes et al., 1998; Ferreira and Reid, 2008; Jang et al., 2009; Perez et al., 2009; Zhang et al., 2009), and maintenance of cysteine homeostasis is likely to be vital in this regard. For example, as discussed subsequently, many key en- 1297 1298 Guttmann zymes linked to transcription, energy production, apoptosis, and general cell signaling have critical cysteines in their active sites, which, once oxidized, result in cellular dysfunction. In addition to endogenous production of oxidants, changes in redox are also produced by exogenous sources, such as by the use of various interventions used to treat animals. Either by design or because of a side effect, treatments to prevent or cure disease in animals may have a significant impact on the balance of oxidants and antioxidants. Obviously, mitochondria are one such drug target that can have a dramatic impact on the production of oxidants because of the oxidizing chemistry involved in energy production. Finally, understanding the potential effects of thiol oxidation is significant because of its importance in the production and storage of quality food products. It is well known that postmortem changes result in increased oxidation, leading to decreased initial quality as well as spoilage of food (Koohmaraie, 1992; Boehm et al., 1998; Chen et al., 2008). Because of the highly sensitive nature of the thiol group within cysteine, an understanding of the role of oxidation of key cysteinedependent enzymes will assist researchers in developing improved techniques to combat the undesired outcomes of such oxidation-mediated damage more effectively. REDOX HOMEOSTASIS Redox refers to the loss or gain of electrons, resulting in a change in the oxidation number of an atom. This review focuses on redox changes with respect to the AA cysteine, because cysteine is one of the major sources of redox balance in biological systems (Claiborne et al., 2001; Barford, 2004; Jacob et al., 2006; Jones, 2008). This includes a variety of cysteine-dependent enzymatic and nonenzymatic constituents as parts of an intricate system that works to maintain redox homeostasis. Homeostasis is a critical factor in the long-term survival of a cell, and all cellular systems function with a balance of constituents that are needed to work together. There are times, however, when that balance must shift, such as when kinase activity in a cell is increased to support specific functions. Because prolonged increases in kinase activity would likely lead to cellular dysfunction, this increase cannot last and homeostasis is eventually restored by phosphatase activity. Thus, although homeostatic set points may change with time or events, there is always a balance to be reached, although this may be arguable in some instances, such as in the case of normal aging mechanisms, which may purposefully move in one direction or another with time. However, similar to the kinase-phosphatase system, redox regulation is maintained by an array of oxidant and antioxidant systems working together to achieve proper balance of needed oxidation for respiration, signaling, and reducing potential to prevent undesirable oxidation of cysteine-dependent enzymes or other redox-sensitive proteins. CYSTEINE OXIDATION Cysteines are 1 of 2 AA that are naturally incorporated in proteins that contain sulfur, with the other being methionine. Although oxidation of sulfur on both AA occurs, the thiol group (-SH) of cysteine is more reactive than the thioether (-CH2-S-CH3) of methionine. Cysteines are able to participate in a variety of reactions, in large part because of the ability to exist in many different oxidation states, including thiol, disulfide, sulfenate, sulfinate, sulfonate, and the thiyl radical, among others (Figure 1). This flexibility has allowed cysteines to have a variety of roles, such as conformation-stability, metal-binding, and catalytic activities. The major reason for cysteine sensitivity to oxidation is the ability to generate anionic sulfur at physiological pH. Most cysteines are not reactive to oxidants because their microenvironment makes them less nucleophilic or they are found to be buried within the tertiary or quaternary structure. The typical pKa of a cysteine residue thiol is approximately 8.5, which is too great to be reactive at physiological pH and must therefore be reduced. For example, recent results have shown that the reactivity of the functional cysteine (Cys-106) within the human protein, DJ-1 or Park-7, is due to its acceptance of a hydrogen bond from a protonated glutamic acid side chain that reduces the pKa of the thiol group to approximately 5.4 (Witt et al., 2008). In the case of many cysteine proteases, the formation of the anionic sulfur is generated by the action of a participating histidine within the catalytic dyad or triad. In cases in which the cysteine is buried and inaccessible, they generally remain nonreactive. It should be noted, however, that oxidation of buried sulfur groups is possible, but it is both rate and oxidant dependent. For example, Keck (1996) observed that although the oxidizing agent t-butyl hydroperoxide was not able to oxidize buried methionines within interferon-γ, hydrogen peroxide (H2O2) was able to penetrate and oxidize all 5 methionines within this protein. CYSTEINE REACTIVE SPECIES Although the oxidants discussed subsequently are able to modify other AA, resulting in the modification of enzymatic function, this discussion is focused on those aspects that involve cysteines. Three major subdivisions of oxidizing species can modify cysteines: reactive oxygen, nitrogen, and sulfur species. It should also be noted that not all oxidants are equivalent, with some being free radicals (i.e., containing an unpaired electron) and others being 2-electron oxidants. Each oxidant will also have different reaction rates with thiols and be differentially reduced by the antioxidant systems, as discussed below. Oxidants elicit different responses, depending on where they are generated and with what targets they are able to react. Within these subdivisions, numerous types of biologically relevant oxidizing molecules result Redox regulation of cysteine-dependent enzymes 1299 Figure 1. Schematic of the major forms of oxidized cysteines observed in biological systems. Peroxide oxidation generates sulfenic acid, which is an initial step in the formation of other forms of oxidation, including sulfinic and sulfonic acids as well as disulfide bonds. Nitric oxide will oxidize susceptible cysteines, resulting in S-nitrosylation. S-Glutathionylation is a commonly observed modification caused by reaction with oxidized glutathione. Free radicals that interact with sulfhydryl will generate a reactive sulfur species in the form of a thiyl radical. With the exception of sulfonic acid, all are reversible, although only peroxiredoxin has been found to be reversible by sulfiredoxins to reduce sulfinic acid. In general, there is overlap among the antioxidant systems that allows them to reverse several different forms of cysteine oxidation, with a few exceptions in free radical removal. In some cases, it has also been found that thioredoxin or glutaredoxins are more specific for removal of oxidants from specific cysteines, which must be determined experimentally. Black ovals represent protein, G represents glutathione, R represents any protein, and GSSG represents glutathione disulfide. in cysteine oxidation, including H2O2, superoxide, nitric oxide (NO), peroxynitrite, and others. These modifications are most often reversible and result in different cysteine modifications, including disulfide bonds, S-glutathionylation, S-nitrosylation, thiyl radical formation, and formation of sulfenic, sulfinic, and sulfonic acids. The most common oxidants and their activities are described briefly below. Hydrogen peroxide is a nonradical oxidant that is generated in various compartments throughout the cell by a variety of sources, such as during the dismutation of superoxide, as a receptor-signaling molecule, or in immune-response cells. Peroxide is membrane permeable and is an important physiological redox regulator with relatively low reactivity, resulting in a long halflife. Peroxide can also react with metals, such as cop- per or iron, to generate a hydroxyl radical. However, because the hydroxyl radical is highly reactive, with a short half-life, and the conditions under which it can be generated are relatively rare in biological systems, its significance is unclear (Pacher et al., 2007). Peroxide oxidation contributes to disulfide bond formation, as well as to formation of sulfenic, sulfinic, and sulfonic acids. Peroxide-mediated oxidation is typically reducible, making it an excellent cell-signaling molecule. Interestingly, until recently it was thought that both sulfinic and sulfonic acids were nonreducible. However, Park et al. (2009) showed that enzymes (i.e., sulfiredoxins) are present in both yeast and mammals that are capable of reducing sulfinic acid to sulfenic acid, at least within the antioxidant enzyme family of peroxiredoxins. 1300 Guttmann − The superoxide anion (e.g., ∙O2 ) is a radical oxidizing species that is generated by respiration in the mitochondria and by the immune system. Superoxides will, either spontaneously or through a catalyzed reaction with superoxide dismutase, decompose to peroxide (Pacher et al., 2007). Although debatable, it is through the formation of peroxide and other metabolic products that superoxide likely exerts its effects on the cell. Nitric oxide, also known as epithelium-derived relaxation factor, is a vasodilator that is enzymatically produced by various isoforms of NO synthase, using arginine as a substrate. Although its main function appears to be as a signaling molecule through the activation of guanylate cyclase, this free radical has emerged as a significant molecule in the study of redox regulation through multiple actions (Pacher et al., 2007). Although NO can be converted to higher orders of reactive nitrogen species, the 2 major reactive forms found in cells are those of S-nitrosylation and peroxynitrite. S-Nitrosylation is the covalent addition of an NO group to critical anionic sulfur within cysteine to form an Snitrosothiol derivative. Nitric oxide also reacts with superoxide to form the more reactive peroxynitrite, which has a half-life sufficient to cross biological membranes (Pacher et al., 2007). Peroxynitrite can oxidize thiols directly, resulting in the generation of a sulfenic acid that may subsequently form a disulfide. Alternatively, the decomposition products of peroxynitrite, the hydroxyl radical and nitrogen dioxide (NO2), will result in the generation of a thiyl radical. Although cysteines are a significant target of peroxynitrite, other AA, such as tyrosine, methionine, tryptophan, and histidine, are modified. As a biological oxidant, however, peroxynitrite is a significant factor in the regulation of cysteinedependent enzymes. ANTIOXIDANT SYSTEMS For a protein to be reduced, the reducing agent or antioxidant must be oxidized during the reaction. Thus, the general goal of a cell, to remove oxidants, will be to shuttle the electron(s) from the oxidized species to generate water or oxygen. This reduction reaction chain is a multistep process utilizing several cofactors, including flavin adenine dinucleotide (FAD), NADH, or reduced NAD phosphate (NADPH). Without a final electron acceptor with the ability to eventually remove the electron pair (note the difference between 2-electron nonradical oxidants and 1-electron free radicals), there would be no net change in redox state within the cell. The cell has an array of defensive mechanisms in its arsenal that deal with the formation of oxidized species. These include enzymes [e.g., superoxide dismutase (SOD) and catalase], in addition to nonenzymatic pathways, such as cysteine, glutathione, and vitamins (e.g., vitamins C and E). In addition to those factors that are most directly involved in the reduction of oxidized species, numerous cofactors, such as FADH/H2, NADH, and NADPH, must also be maintained. There are 2 main forms of SOD in mammalian cells, MnSOD (SOD2; localized to mitochondria) and CuZnSOD, with 2 types of CuZnSOD that are found either intracellularly (SOD1) or extracellularly (SOD3). These enzymes are responsible for the conversion of superoxide to H2O2, although this occurs slowly and spontaneously as well. Although superoxide is an oxidizing radical, its direct action is limited, and it would seem that the main reason for maintaining increased SOD activity is to prevent the formation of peroxynitrite, the reaction between superoxide and NO (Pacher et al., 2007). Catalase and peroxidases are enzymes that remove H2O2. Hydrogen peroxide is a product of various oxidases, such as those found in peroxisomes or in the endoplasmic reticulum, and the dismutation of superoxide. Removal of H2O2 is important because peroxide is converted, through the Fenton reaction (i.e., the reaction of peroxide with ferrous iron), into a hydroxyl radical that is a highly reactive but short-lived oxidant. Thioredoxin (TRx) is a ubiquitous small protein, approximately 12 kDa in size, consisting of 105 AA with a conserved Cyx-Xaa-Xaa-Cys motif in its active site, that reduces target proteins by exchanging disulfide to dithiol. Its reducing activity is maintained by NADPH and TRx reductase (TRxR; Ren et al., 1993). Mammalian cells contain 2 TRx systems, the cytosolic TRx and TRx reductase (TRxR1) and mitochondrial TRx2TRxR2 (Damdimopoulos et al., 2002). In mammals the TRxR exist as homodimeric selenoenzymes containing an FAD and a C-terminal selenocysteine residue in the active site. Thioredoxin reductase 1 is cytosolic but can translocate to the nucleus or be exported from the cell even though it lacks a targeting sequence. Targets of TRx include hypoxia-inducible factor 1, nuclear factorκB, activator protein 1, and the tumor suppressor p53. In addition, TRx have been shown to scavenge hydroxyl radicals. Glutaredoxins (GRx) are also small proteins of 10 to 16 kDa in size that have 2 classes: the dithiol GRx, which contain CxxC, and the more recently discovered monothiol, which lack the C-terminal cysteine (Lillig et al., 2008). The GRx system is composed of glutathione, glutathione reductase, NADPH, and GRx. There are several GRx isoforms that do not completely overlap in activities with each other or with the TRx, although many are shared. Some localization differences exist because GRx1 is localized mainly in the cytosol and the intermembrane space of the mitochondria, whereas GRx2 localizes to either the matrix of the mitochondria or the nucleus (Pai et al., 2007). Glutaredoxin 2 can also be reduced by TRxR, which is unique among the GRx. Glutaredoxin 5, a monothiol GRx, is found in the mitochondria that are required for the activity of ironsulfur enzymes. Glutathione peroxidases (GPx) are selenocysteinecontaining proteins primarily involved in the reduction of H2O2 and lipid peroxides (Lu and Holmgren, 2009). Several isoforms (GPx1 through GPx6) exist, with at least one mammalian isoform that does not utilize sele- Redox regulation of cysteine-dependent enzymes nocysteine. Similar to the other enzymatic systems, these enzymes are found throughout the cell with cytosolic, nuclear, mitochondrial, and secreted forms. Some are tissue specific, such as GPx5, which plays a role in protecting DNA in sperm from oxidative damage, and GPx6, which is localized to the olfactory system. Peroxiredoxins (Prx) are ubiquitously expressed and catalyze the removal of H2O2 and lipid hydroperoxides as well as peroxynitrite. At least 6 Prx are found in mammalian cells and can be divided into 3 subclasses: 1) typical 2-Cys-Prxs (Prx I through Prx IV homodi mers); 2) atypical 2-Cys-Prx (Prx V); and 3) 1-Cys-Prxs (Prx VI). For the typical Prx, the active-site cysteine is oxidized to sulfenic acid, which then reacts to form an intermoleculer disulfide, which is reduced by TRx. It is possible for the sulfenic acid to be further oxidized to sulfinic acid, which can be reduced by sulfiredoxin (Park et al., 2009). It has also been found that ascorbate may be used by some Prx to regenerate the active site thiol (Monteiro et al., 2007). Glutathione (GSH) is a tripeptide (γ-glutamylcysteinyl-glycine) that, along with glutathione disulfide (GSSG), plays a significant role in antioxidant defense. This redox couple (GSH:GSSG) is a major determinant of the redox state inside the cell, where they are found at millimolar concentrations (Jones, 2008). In addition to being able to regenerate various antioxidant systems and serve directly as an antioxidant, GSH also acts as a source for the AA cysteine. Once oxidized, GSSG is reduced in a NADPH-dependent process by GSH reductase. Lack of NADPH leads to increased GSSG abundance, which is important in red blood cells that lack mitochondria and that therefore rely on the glucose-6-phosphate dehydrogenase pathway. A result of insufficient glucose-6-phosphate dehydrogenase activity is hemolytic anemia, which is observed in millions worldwide and can be caused by certain drugs, such as antimalarial compounds (Aliciguzel and Aslan, 2004). Cysteine is the major extracellular antioxidant (Moriarty-Craige and Jones, 2004). Once oxidized, it forms the low molecular weight disulfide, CySS, or other forms of oxidized thiols, as described previously. However, it is primarily found as CySS because the extracellular environment is relatively oxidizing. Although this pool is integrated with the other major intracellular antioxidant, GSH, the 2 are not in equilibrium (Go and Jones, 2008); however, they influence each other because GSH formation is dependent on the presence of cysteine and vice versa. Although cysteine is not considered an essential AA, it is derived from methionine, which is essential. Because very young or very old animals may have difficulty absorbing sufficient quantities from the diet to produce adequate concentrations of cysteine, sufficient methionine and cysteine in the diet is vital (Rutherfurd and Moughan, 2008). Vitamin C (ascorbate) is an essential nutrient in humans, but not in most animals. It serves as a watersoluble antioxidant and cofactor for the regeneration of some enzymatically important thiols (Mandl et al., 1301 2009). As an antioxidant, ascorbate is typically involved in nonenzymatic 1-electron (i.e., radical) transfers and is therefore important in the removal of superoxide, hydroxyl, and peroxyl radicals. The monodehydroascorbate produced by the scavenging of radicals is ultimately reconverted to ascorbate by enzymatic conversions that use NADH, NADPH, or GSH. In the presence of heavy metals, ascorbate can become pro-oxidant, although the frequency of this occurrence in biological systems is unclear (Mandl et al., 2009). Vitamin E refers to a group of 8 related compounds, among which α-tocopherol shows the greatest biological activity. It is a lipid-soluble antioxidant whose main role is the prevention of membrane lipid peroxidation. Once oxidized, vitamin E is likely regenerated by either ascorbate or β-carotene. EFFECTS OF REDOX ON CELLULAR FUNCTION The oxidants and antioxidants work as a complete system, which has implications for the physiological regulation and pathological impact of numerous proteins. The following section highlights, using several examples, the manner in which oxidative stress contributes to various cellular pathways of major significance to all researchers interested in redox biology. Redox Regulation of Transcription Heat shock transcription factor 1 (HSF1), an important regulator of inducible heat shock proteins (Hsp) in mammals, is one example of a transcription factor modulated by redox state. In its monomeric form, HSF1 is located in the cytosolic fraction. When cells are placed under stress, HSF1 forms a homotrimer that is translocated to the nucleus, where it binds to a heat shock element within DNA and activates transcription of Hsp, such as Hsp70 and Hsp90. Lu et al. (2008) recently observed that disulfide bridges control the formation of the homotrimer in vitro. They found that the 2 intermolecular disulfide bonds had differential sensitivities to redox state, which they manipulated by the addition of different concentrations of the thiol-specific reducing agent dithiothreitol. In the presence of stress and oxidizing conditions, an intramolecular disulfide bond predominated, which prevented activation of HSF1. Under stress and mildly reducing conditions, the intramolecular bond was broken and HSF1 could not form the required trimer. However, under more highly reducing conditions, the homotrimer disulfide bridges were broken. This example illustrates the fine sensitivity of the redox state that, in this case, controlled protein function through disulfide bond formation. Nuclear factor E2-related factor 2 (Nrf2) is a ubiquitously expressed master transcription factor that regulates antioxidant response elements, which in turn mediate expression of antioxidant proteins. Kelch-like 1302 Guttmann ECH-associated protein (Keap1) is a cysteine-rich, zinc-binding protein that is a negative regulator of Nrf2. After many years of study, a unique mechanism was found in which Keap1 targets Nrf2 for rapid turnover by proteolytic degradation by the proteasome (Sekhar et al., 2002). As a major factor in the inducible upregulation of antioxidant enzymes, the Nrf2-Keap1 interaction is responsive to changes in the redox state, which are now believed to be mediated not by Nrf2 itself, but rather by a mechanism associated with Keap1. Although there is some debate over the precise mechanism and elements involved in the redox regulation in vivo, it is clear that modifications of one or more of the key cysteines within Keap1 are involved and likely result in the dissociation of Keap1 from Nrf2 (Piccirillo et al., 2009). Examples of Redox-Regulated Enzymes Although several of the key antioxidant systems are cysteine-dependent enzymes, this section focuses on a select group of thiol-dependent enzymes that are not directly related to maintaining the redox state. Evidence for their oxidation is presented first, followed by an integrated discussion of the impact of their oxidation on cellular function to illustrate the importance of recognizing the influence of oxidation in data interpretation and experimental design considerations. Protein tyrosine kinases (PTK) are a family of kinases that contain a conserved active-site cysteine (His-Cys-X-X-Gly-X-X-Arg-Ser/Thr). Because of its microenvironment, this active-site cysteine has a low pKa, which makes it susceptible to redox regulation by forming the highly sensitive thiolate anion at physiological pH. A reduced cysteine within the active site acts as a nucleophile, which is required to phosphorylate PTK substrates. Oxidation of cysteine results in a less nucleophilic center, which cannot participate in the reaction. Protein tyrosine phosphatase 1b is a classical example of such PTK redox regulation. Protein tyrosine phosphatase 1b has been found to be reversibly oxidized by several oxidizing agents, notably H2O2, to form sulfenic acid (Denu and Tanner, 1998), as well as by glutathionylation (Barrett et al., 1999a,b). The net result of oxidative stress in this case is an increase in tyrosine phosphorylation that occurs under physiological conditions, such as during insulin signaling through the insulin receptor. Creatine kinase is an important enzyme in energy transfer because it catalyzes the transfer of high-energy phosphate from ATP to creatine. Multiple isoforms exist that are tissue specific, as well as being found in both the cytosol and mitochondria. Creatine kinase isoforms contain up to 4 cysteines, of which only one appears to be necessary and sufficient for full creatine kinase activity and is the only one that appears to be physiologically relevant (Konorev et al., 2000; Liu et al., 2008). Modifications to creatine kinase include modification by reactive nitrogen species, disulfide formation, and S-glutathionylation (Konorev et al., 2000; Reddy et al., 2000; Liu et al., 2008). Calpains are cysteine-dependent proteases that are involved in several physiological pathways and are thought to contribute to a variety of pathological states (Goll et al., 2003). They have a catalytic triad, related to papain, consisting of a cysteine, histidine, and asparagine. Calpains exist as multiple isoforms with ubiquitous and tissue-specific isoforms that are primarily found in various muscle types, which has led to their study in meat tenderization, as discussed below. Similar to other thiol-dependent enzymes, the active-site cysteine generates an anionic sulfur group during catalysis that is subject to oxidative attack. The various members of this family of enzymes have been found to be inhibited by a variety of oxidants, most notably peroxide and NO, resulting in reversible enzyme inactivation (Guttmann et al., 1997; Guttmann and Johnson, 1998; Koh and Tidball, 2000). The importance of recognizing the groups discussed above is that they represent the typical types of oxidative modifications that are observed and the different outcomes that result. It should be easily appreciated that changes in redox balance can have both focused and broad effects, even though only a single cysteine may be modified within a given protein. For example, in cases in which redox state is used as a primary modulator of enzyme activity, such as with protein tyrosine phosphatase 1b and its regulation by insulin, a change in redox has a controlled and relatively localized influence. However, when more systematic changes in redox occur, the changes can be cell-wide, resulting in modifications of many thiol-containing proteins because of transcription-mediated responses. The sensitivity and importance of the AA cysteine should also be evident because in each case, only 1 or 2 cysteines within a quarternary structure that can be targeted by oxidative stress will have a significant influence on the function of a protein. That is, not all cysteines that are oxidized within a given protein will contribute to any change in activity. In addition, it should be clear that virtually all the types of oxidation that are chemically possible are observed. However, the possible oxidations observed in vitro may not have physiological meaning, and it is therefore important to have a good understanding of the redox compartment in which a protein under study is likely to be found and to verify any in vitro studies with in vivo experimentation. REDOX-RELEVANT ISSUES TO ANIMAL SCIENTISTS The previous examples can illustrate several broad areas for the animal scientist to consider. These include understanding the effects of 1) experimental conditions on results that may be related to oxidative stress, 2) nutrition on redox-regulated biological pathways, 3) oxidative stress on aged animals, and 4) postmortem oxidation of food. Redox regulation of cysteine-dependent enzymes Experimental Considerations A common reagent added to reactions is a thiolreducing agent, usually dithiothreitol or the related β-mercaptoethanol. Often it is added to prepare samples for analysis, such as with gel electrophoresis, and is needed to fully unfold proteins by removing disulfide bridges. However, when added to cell or tissue homog enates that will be used for bioassays, this also results in adjusting the redox state from what it was at the time the samples were taken. In many instances, this may not have a significant impact on the results or data interpretation. However, if there is an active thiol group within the protein of interest or a regulator of this protein (e.g., as observed with the Keap1-Nrf2dithiothreitol experiments described previously), then the addition of increased concentrations of reducing agents may lead to an over- or underestimation of activity. Recently, we demonstrated this phenomenon in brain homogenates, showing that the estimation of calpain activity was clearly affected by the presence oxidation related to the disease (Marcum et al., 2005). This conclusion was reached by examining calpain activity in homogenates that were treated with or without dithiothreitol. We found that although earlier estimations of calpain activity appeared elevated in disease vs. control states, this was likely due to the addition of dithiothreitol to the assay, which negated any influence of the oxidative stress known to be present in the disease state. This approach itself adds a layer of complexity because the homogenization or other manipulations that must be taken to prepare samples will change the redox state. However, with proper controls, an examination of samples under normoxic and reducing conditions would yield useful results with respect to the potential impact of oxidative stress. A second consideration is the use of specific antioxidant systems as measures of oxidative stress. As indicated previously, these systems are in some ways interdependent because they share some common pathways or cofactors or because they exist within the same compartment. However, an indication of the redox level of one system does not necessarily translate into an accurate picture within each compartment. As reviewed by Jones (2008), the GSH:GSSG is only one measure of the redox state of the cell, so care must be taken when attempting to extrapolate changes in the levels or ratio of this redox couple. As in the examples above, several enzymatic processes play vital roles in the reduction of oxidized thiols, and although they are regenerated in large part by the glutathione system (directly or indirectly), a decrease in reduced glutathione does not necessarily significantly modulate the capacity of other redox compartments. Additionally, alterations in the GSH:GSSG redox couple would not necessarily indicate the redox state of a protein of interest. Fortunately, with the increased availability of highly sensitive thioldetecting reagents and equipment, it is now possible 1303 to monitor specific cysteines to determine the extent to which they are oxidized or reduced. Thus, an understanding of the global redox state with GSH:GSSG or Cys:CySS measures is valuable, but care should be taken when interpreting these values in the context of a particular activity of interest. A third area to be evaluated with respect to the role of oxidative stress in experimentation is the influence of drug treatments that may perturb the redox balance. Of particular note are compounds that alter mitochondrial energetics. As an important source of superoxide, mitochondrial efficiency can have a significant impact on the redox state of the inner and outer mitochondrial spaces, as well as on the entire cell. As one example, doxorubicin, a chemical used in the treatment of solid tumors, is restricted in its use in part because of its cardiotoxicity. There is strong evidence that toxicity is caused by the interaction of doxorubicin with mitochondrial proteins, resulting in increased production of superoxide. One example of this has been observed with creatine kinase. It was found that in mitochondria isolated from animals treated with doxorubicin, creatine kinase was S-glutathionylated, which was reversible by overexpression of glutaredoxin (Diotte et al., 2009). These results clearly show that care must be taken to consider the impact of therapeutics that may have unexpected outcomes on redox-regulated enzymes. The influence of oxidants and reductants, including dithiothreitol, on electrophoresis and fluorescent-based assays also needs to be considered. Gel electrophoresis is a common analytical approach, and we have found that while conducting in vitro experiments with increased concentrations of dithiothreitol, this and other reductants and oxidants alter the ability of proteins to be visualized by Western blotting (R. P. Guttmann, unpublished data). Although we have not attempted a systematic study of this phenomenon, we have found that the more reducing agent is used, the stronger will be the signal observed by Western blot. To overcome this potential limitation, the use of freshly prepared and equal concentrations of reducing agents added to the final SDS-sample buffer used in preparation for the loading of protein is mandatory and has been successful in preventing this artifact. In attempting to determine the extent to which oxidants or reductants affect activities, highly sensitive fluorescent-based activity assays have become more common. However, using such assays under highly oxidizing or reducing conditions must be done with caution. We have found that reductants can have direct effects on the fluorescence of coumarinbased moieties, although we have not evaluated other types of fluorescent probes. To address this issue, it is imperative that testing of all control and treatment conditions be done in the presence or absence of the enzyme or molecule under study to evaluate the extent to which the oxidant or reductant may be affecting the fluorescent signal. Alternatively, measurements of the fluorescent moiety in the buffers and conditions being 1304 Guttmann tested can be taken to confirm that its fluorescence is not being directly affected by the presence of increased concentrations of oxidizing or reducing agents. Nutrition The impact of proper nutrition on the redox state of the intracellular and extracellular environment is another important factor to be considered by animal scientists. First, there must be an awareness of the metabolic pathways utilized by a particular species. As noted previously, bioavailability of methionine and cysteine is critical to maintaining a proper physiological environment and adequate concentrations of cysteine. In addition, knowledge of the proper supplementation of vitamins, such as ascorbate, is vital because not all animals have the same biochemical pathways available to process nutrients in feed, including minerals and vitamins. Selenium is another potential supplement for which abundance should be evaluated because of the importance of selenium in redox biology. Because increasing the quantity of antioxidants has general positive outcomes, the use of natural or synthetic compounds to support the natural antioxidant systems is a tantalizing approach. One well-studied example is Nacetylcysteine and its related compounds. These compounds have been tested in both animals and humans and appear to be well tolerated and effective at decreasing oxidant-mediated damage (Ferreira and Reid, 2008; Banerjee et al., 2009; Ciftci et al., 2009; Lu et al., 2009; Tunc et al., 2009). Whether these positive outcomes are caused by providing an additional source of cysteine or whether N-acetylcysteine is acting directly as an antioxidant is still under investigation. However, this is strong evidence that N-acetylcysteine or other compounds that have potent antioxidant activity are good candidates for study in the prevention or treatment of oxidative stress. In addition to N-acetylcysteine, other compounds, such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Mitchell et al., 1991), are available and being developed that seek to improve health by decreasing oxidative stress. Studies of the importance of redox state on the thiol groups in seeds have also been under investigation. In a study to evaluate the role of disulfides in the assembly of 11S storage globulins, it was found that manipulation of the redox state in vitro using various concentrations and ratios of GSH:GSSG resulted in an increase in the rate of 11S trimer assembly and hexamer formation (Jung et al., 1997). Proper regulation of thiols in seeds was also studied in experiments showing the importance of regulating the formation of disulfide bonds during germination. In that study, the authors concluded that disulfide bonds of seed storage proteins were required to be reduced, likely by TRx, to be degraded and mobilized during germination. More recent work has extended these findings to more fully evaluate the contribution of TRx in seed germination, in addition to discovery of the role of a cysteine-protease in the processing of the embryo-specific protein 2 (Yano et al., 2001). Aging Animals It is clear that oxidative stress is closely linked with the aging process. Although it remains to be determined whether it is cause or effect, results have consistently shown that as an animal ages, markers of oxidation increase. There is good evidence that increases in oxidative stress with age are detrimental, although the extent to which increasing antioxidant concentrations prolong life span remains hotly debated. Much of the work that laid the foundation for evaluating oxidative stress and life span has naturally been done in lower order organisms. Nevertheless, these initial observations, along with animal and human data, demonstrate the potential power of redox regulation in improving the quantity and quality of life. Although still under intense investigation, other work has shown that caloric restriction results in decreased oxidative stress and has been proposed as one mechanism to explain the increase in longevity associated with a restricted diet (Sohal and Weindruch, 1996; Lass et al., 1998; Zainal et al., 2000; Martin et al., 2007). As more information has been accumulating about the importance of proper nutrition, it would seem clear that proper nutrition related to maintaining sufficient raw materials (such as cysteine, methionine, selenium, and vitamins C and E) to allow for optimal antioxidant synthesis is an important area for animal scientists. It would be expected that improving the redox balance in newborn and young animals would positively affect the overall health of animals. In addition, maintaining redox balance could have more profound influences on aged-related changes, such as fertility, muscle fatigue, stamina, and other important characteristics that are desirable. Postmortem Changes and the Role of Oxidative Stress Postmortem changes affect many forms of research by creating artifacts and are thus a constant area of concern. However, in the case of food sciences, understanding the impact of postmortem changes is vital. As discussed previously, calpains were observed to be sensitive to a variety of oxidants, resulting in enzyme inactivation. Because several calpain substrates are cytoskeletal components, as well as because of the increased muscle concentration of calpains, evaluation of the postmortem activity of calpain was undertaken. It was found that postmortem calpain activity was a significant factor contributing to meat tenderness (Kretchmar et al., 1990; Koohmaraie, 1992; Wheeler et al., 1992; Whipple and Koohmaraie, 1992). More recent studies have found that meat tenderness is decreased by oxidative inactivation of calpains and that treatment with antioxidants can increase meat tenderness, Redox regulation of cysteine-dependent enzymes which is at least partially attributable to preventing oxidative inactivation of calpains (Rowe et al., 2004). Prevention of oxidative stress postmortem is also important in packaging and storage because the impact of oxidative stress makes food less appealing, less palatable, and, from a food safety standpoint, potentially dangerous. The understanding of oxidation prevention in the market is observed by noting the use of techniques that prevent oxidation to store and package foods for human consumption. In addition to inhibiting the growth of bacteria and other pathogens, decreasing oxidation of the food product will prevent cysteine oxidation, which could contribute to protein aggregation through disulfide bonds. It would seem that a balance is required because high-thiol-dependent enzymatic activities postmortem could also be a factor contributing to food spoilage through the actions of cysteine-dependent proteases (Chen et al., 2008). Conclusions In summary, this review has attempted to provide a sense of the scope of the potential roles of thiol oxidation in cellular function and has instructed the reader on the importance of considering the influence of redox potential in experimentation with animals, animal products, and food. The reader is also directed to some of the outstanding reviews of the details of each oxidant and antioxidant system, which contain more specific information regarding the chemistry of the redox reactions and enzymatic pathways related to cysteine oxidation (Giles et al., 2003; Moriarty-Craige and Jones, 2004; Pacher et al., 2007; Go and Jones, 2008; Jones, 2008; Winterbourn and Hampton, 2008; Brandes et al., 2009). LITERATURE CITED Aliciguzel, Y., and M. Aslan. 2004. N-Acetyl cysteine, l-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes. Clin. Exp. Med. 4:50–55. Banerjee, A., M. B. 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