Open access, freely available online Primer Exploiting Thiol Modifi cations Patricia J. Kiley*, Gisela Storz

s the premier biological species when they are released by and plays roles in the aging process electron acceptor, molecular animals, plants, and insects as a defense and the development of heart disease, oxygen (O ) serves a vital role against detrimental organisms such as diabetes, chronic infl ammatory diseases, A 2 in fundamental cellular functions, microbial pathogens. Reactive oxygen cancer, and several neurodegenerative including the process of aerobic species can damage cells in many ways: diseases (Halliwell 1999). respiration. Nevertheless, with the by inactivating proteins, damaging Many organisms have evolved benefi cial properties of O2 comes nucleic acids, and altering the fatty strategies to remove reactive oxygen the inadvertent formation of reactive acids of lipids, which leads in turn to species and repair damage, which oxygen species, including superoxide perturbations in membrane structure have enabled them to prosper − (O2 ), hydrogen peroxide (H2O2), and function. The accumulation of from the tremendous oxidizing • and hydroxyl radical ( OH); these this oxidative damage underlies the potential of O2 without succumbing differ from O2 in having one, two, and formation of many disease states in to oxidative damage. Bacteria, yeast, three additional electrons, respectively humans. It is postulated that tissue and mammalian cells all induce the (Figure 1). Cells also encounter injury by these reactive oxygen species synthesis of global regulatory responses elevated levels of these reactive oxygen accumulates over a long period of time to survive oxidative insults. The consequences of oxidative stress and the corresponding defense responses have been extensively studied in Escherichia coli. For ease of study in the laboratory, the stress responses are often provoked by the external addition of chemical oxidants that specifi cally elevate the levels of reactive oxygen species within cells, or by the use of mutant strains that disrupt the normal “homeostatic mechanisms” for removing reactive oxygen species or the damage they do. While this primer focuses on a particular set of protective and regulatory protein modifi cations induced by oxidative stress in E. coli, it should be noted that many of the same mechanisms are present in other organisms; some specifi c examples from other species will also be described. − The major target of O2 damage identifi ed in bacteria is a class of

Citation: Kiley PJ, Storz G (2004) Exploiting thiol modi- fi cations. PLoS Biol 2(11): e400.

Copyright: © 2004 Kiley and Storz. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abbreviations: H2O2, hydrogen peroxide; O2, molecular oxygen; O −, superoxide; •OH, hydroxyl radical DOI: 10.1371/journal.pbio.0020400.g001 2 Patricia J. Kiley is in the Department of Biomolecular Figure 1. Formation of Reactive Oxygen Species Chemistry, University of Wisconsin Medical School in Madison, Wisconsin, United States of America. The four-electron reduction of molecular O2 generates two molecules of H2O, which is O in its most reduced form. While this reduction normally occurs within the enzyme Gisela Storz is in the Cell Biology and Metabolism 2 Branch, National Institute of Child Health and Human cytochrome oxidase, one-electron transfers to O2 also occur outside of cytochrome oxidase via inadvertent reactions with other reduced electron carriers, resulting in Development, National Institutes of Health, Bethesda, Maryland, United States of America. partially reduced and reactive forms of O2. H2O2 is also produced by the enzymatic or − • spontaneous dismutation of O2 , and OH is generated by the reaction of iron with H2O2 *To whom correspondence should be addressed. (the Fenton reaction). In addition, the reactive oxygen intermediates are produced by a E-mail: [email protected] variety of organisms as a defense against microbial invasion. (Illustration: Rusty Howson, sososo design) DOI: 10.1371/journal.pbio.0020400

PLoS Biology | www.plosbiology.org 1714 November 2004 | Volume 2 | Issue 11 | e400 dehydratase enzymes that utilize [4Fe– 4S] clusters to bind their substrate (Imlay 2003; Djaman et al. 2004). Since some of these enzymes function in the citric acid cycle (also called the Krebs cycle) and in amino acid − biosynthesis, high levels of O2 lead to a requirement for certain amino acids in growth media (Imlay and

Fridovich 1991). H2O2 is well known for its role in oxidizing thiol (SH) groups of cysteinyl amino acid residues in proteins. Elevated levels of H2O2 also are associated with the oxidation of other amino acids, leading to the formation of methionine sulfoxide and a variety of carbonyls. Lastly, because DOI: 10.1371/journal.pbio.0020400.g002 of its extreme reactivity, •OH targets Figure 2. Thiol Modifi cations of Proteins all of the major macromolecules of Formation of sulfenic acid from the reaction of H2O2 with protein thiolates leads cells: RNA, DNA, protein, and lipids. to different protein modifi cations, depending on the protein. In proteins without a The extent to which membrane lipids second sulfhydryl, the sulfenic acid (–SOH) may be stabilized (e.g., OhrR) or may react with reactive oxygen species to generate the further oxidized sulfi nic (–SO H) are targets appears to depend on the 2 (e.g., thiolperoxidase; Tpx) and sulfonic acid (–SO3H) derivatives. Alternatively, if a presence of polyunsaturated fatty acids second cysteinyl residue is in proximity within the same polypeptide (e.g., OxyR) or an in lipids, which are not as prevalent in associated protein (e.g., Yap1 and Orp1), a disulfi de bond can form between the two bacteria as they are in mammals. sulfur atoms (–S–S–). Lastly, the sulfenated cysteinyl residue can react with glutathione Many enzymes that protect against (GSH), leading to a mixed disulfi de (e.g., MetE). (Illustration: Rusty Howson, sososo design) oxidative damage have been identifi ed in E. coli (Imlay 2002, 2003). Three Reduced thioredoxin is regenerated no cysteinyl residue is nearby, the superoxide dismutases, each of which by thioredoxin reductase and NADPH. sulfenated cysteine can be further contain a different metal center and The fact that NADPH is required oxidized to sulfi nic or sulfonic acid, or show different expression patterns and to maintain the reduced state of it can remain in the sulfenic acid state. subcellular localization, catalyze the glutathione and thioredoxin indicates All but the sulfi nic and sulfonic acid − dismutation of O2 to H2O2. While the that the response to oxidative stress is modifi cations are readily reversible − superoxide dismutases eliminate O2 , coupled to the physiological status of by reduction, using proteins such as they also are a source of endogenously core pathways that generate NADPH. thioredoxin or glutaredoxin; though produced H2O2 in E. coli. The major sulfi nic acid reductase activities have enzymes involved in reducing H O Regulatory Roles of Thiol recently been identifi ed in yeast and 2 2 Modifi cations to H2O and O2 in E. coli are catalase mammalian cells (denoted sulfi redoxin and alkyl hydroperoxide reductase. As mentioned above, proteins—in and sestrin, respectively) (Biteau et al. There is no enzymatic mechanism for particular, the thiols of cysteines—are 2003; Budanov et al. 2004). • decreasing levels of OH, produced the major targets of H2O2. The reaction Given the reversible nature of most • of cysteinyl thiolates with H O can from H2O2. Thus, levels of OH will be 2 2 forms of thiol oxidation, it has been directly proportional to levels of H2O2, lead to the formation of different suggested that thiol modifi cations can and accordingly, catalase and alkyl modifi cations, such as sulfenic acid play roles in signal transduction that hydroperoxide reductase activities are (–SOH), sulfi nic acid (–SO2H), and are similar to protein phosphorylation/ critical to oxidative stress survival. sulfonic acid (–SO3H), as well as dephosphorylation (Sitia and Molteni Another component to the oxidative disulfi de bond formation (–S–S–) and 2004). In support of this model, there stress response is the reduction of glutathione conjugation (–S–GSH) are several examples of proteins oxidized thiols that arises through one (Jacob et al. 2004; Poole et al. 2004) whose activities are modulated by thiol of the mechanisms described below. (Figure 2). These modifi cations often oxidation and reduction. The tripeptide glutathione and the alter the structure and function of the The fi rst of these examples is the thiol reductants glutaredoxin and protein. Recent progress in this fi eld OxyR transcription factor, which thioredoxin are key to the restoration points to a common chemistry in the upregulates peroxide defenses in E. coli of thiols to their reduced state (SH) reaction of H2O2 with thiolates through and a variety of other bacteria. OxyR (Fernandes and Holmgren 2004). E. coli the initial formation of sulfenic acid. In contains two critical cysteines that are contains three glutaredoxins that utilize the case of proteins that have a nearby oxidized to form an intramolecular the reducing power of glutathione to cysteinyl residue, a disulfi de bond disulfi de bond when cells encounter catalyze the reduction of disulfi de bonds forms between the two sulfur atoms. peroxide stress (Zheng et al. 1998; (–S–S–) in the presence of NADPH The sulfenated cysteinyl residue also Aslund et al. 1999). Disulfi de bond and glutathione reductase. There are can react with a cysteinyl residue on formation is associated with a two thioredoxins in E. coli that also another protein or with glutathione, conformational change that alters function to reduce disulfi de bonds. leading to a mixed disulfi de. If OxyR binding to DNA and allows the

PLoS Biology | www.plosbiology.org 1715 November 2004 | Volume 2 | Issue 11 | e400 protein to activate the transcription stable sulfenic acid upon its reaction raises questions about the function of of genes encoding enzymes, such as with H2O2 or organic peroxides the zinc. Perhaps Zn binding provides catalase and the alkylhydroperoxide (Fuangthong and Helmann 2002). some additional control over the reductase, that destroy H2O2. Once the Oxidation of the single cysteine leads reactivity of the cysteine thiols, or

H2O2 concentration is decreased, OxyR to the dissociation of OhrR from its perhaps the loss of the zinc facilitates is reduced and the system is reset. The DNA binding site and the derepression conformational changes. Recently, the unusually reactive cysteine in OxyR of the gene encoding an organic oxidative, stress-induced thioredoxin- that is oxidized by H2O2 to form the hydroperoxidase that eliminates the 2 from E. coli has also been shown to sulfenic acid intermediate can clearly initial oxidizing insult. contain a H2O2-labile zinc site, although be nitrosylated and glutathionylated in In this issue, Hondorp and Matthews the loss of zinc does not change its vitro (Hausladen et al. 1996; Kim et al. (2004) provide an example of a thiol reductase activity (Collet et al. 2003). 2002), but the in vivo relevance of these modifi cation that protects an enzyme Thus, the way this oxidatively labile Zn other modifi cations is questionable activity during oxidative stress. Their site affects thioredoxin function has yet (Mukhopadhyay et al. 2004). data suggest that when cells encounter to be established. Two other examples of redox- oxidative stress, a key cysteinyl residue The extent of thiol oxidation regulated proteins are the E. coli near the active site of methionine within the cell remains another open chaperone protein Hsp33 (Jakob et synthase (MetE) is glutathionylated. question. The variety of modifi cations al. 2000) and the Streptomyces coelicolor This modifi cation blocks access of that arise from treatment with H2O2 anti-sigma factor, RsrA (Li et al. 2003; the substrate and prevents further and the experimental challenges Paget and Buttner 2003; Bae et al. synthesis of methionine. This fi nding associated with their detection has 2004). For these proteins, the cysteine is signifi cant in that it presents a made it diffi cult to catalog all the residues, which form intramolecular mechanism to reversibly preserve the proteins that are modifi ed and all the disulfi de bonds, are in a reduced state function of a protein during oxidative types of modifi cations that exist. In this when coordinated to a zinc ion (Zn2+), challenge. By glutathionylating a issue, Leichert and Jakob (2004) report and zinc is released upon oxidation of single cysteinyl residue, the protein is a general method for detecting cellular the thiols. For both proteins, oxidation protected from further oxidation of proteins whose cysteinyl residues were and zinc release are associated with that cysteinyl residue to the irreversible modifi ed after imposing an oxidative an opening of the protein structure. sulfi nic and sulfonic acid forms. Once stress. Such an approach will greatly For Hsp33, this structural change the stress is removed, the mixed enhance our understanding of targets allows for dimerization and activates its disulfi de bond will be readily reduced, of oxidative stress. The method chaperone activity (Graf et al. 2004). and access to the substrate restored. described by Leichert and Jakob also RsrA, on the other hand, dissociates will be useful in detecting transient from a promoter specifi city factor of Prevalence of Regulatory Thiol cysteine modifi cations. RNA polymerase (an extracytoplasmic- Modifi cations? The importance of monitoring function-type alternative sigma factor) As illustrated by the examples above, transient changes in cysteines is allowing the transcription of genes that an array of chemical modifi cations highlighted by the recent fi nding permit recovery from the stress (Li et obtained by oxidizing cysteinyl residues that oxidation of the Yap1 activator al. 2003; Bae et al. 2004). Among the has been exploited in combating of antioxidant genes in the yeast target gene products is a thioredoxin, oxidative stress. Yet it is important Saccharomyces cerevisiae requires a which reduces the disulfi de bonds that to note that not all cysteinyl residues peroxidase denoted Gpx3 or Orp1 form within oxidized RsrA. Presumably, of proteins are readily oxidized by (Delaunay et al. 2002). In this case, reduction of the disulfi de restores oxidants such as H2O2. We do not H2O2 reacts with a cysteine in Orp1, the binding of zinc and its inhibitory currently understand all of the features forming an unstable sulfenic acid association with the sigma factor. that determine the reactivity of a intermediate that then reacts with

Thus, the RsrA regulatory circuit particular thiol to H2O2 (Poole et al. a cysteinyl residue of Yap1 to form provides another example, comparable 2004). The pKa of the thiolates clearly an intermolecular disulfi de. The to OxyR, in which the modifi cation plays an important role, as thiolates are disulfi de undergoes an exchange with of a regulatory protein thiol group more reactive than their protonated a second cysteine within Yap1 to form can be linked to a change in the counterparts. In addition, the an intramolecular disulfi de that locks transcriptional output of genes that contribution of protein environment Yap1 in a confi rmation that masks the remediate stress. to the stability of the oxidized products nuclear export signal (Wood et al. The peroxide-sensing repressor is also known to be a factor, but is not 2004). Thus, methods that allow the OhrR from Xanthomonas campestris well understood. Given that many of appearance of thiol modifi cations in pv. phaseoli (Panmanee et al. 2002) the thiol modifi cations do not appear cells to be monitored kinetically will and Bacillus subtilus (Fuangthong and to be in equilibrium with the redox greatly enhance our understanding of Helmann 2002) can be inactivated by state of the cell, the features of the how cysteine residues become oxidized.

H2O2 or by organic peroxides (ROOH) protein that determine the rate at The examples mentioned here formed by the oxidation of a variety which the modifi cations are formed are illustrate the versatile potential of thiol of organic molecules in the cell or in another important parameter. modifi cations. Given the reversibility the environment. The B. subtilis OhrR The added complexity of the cysteine of thiol oxidations and the wide transcription regulator contains only targets that compose part of a Zn range of structural constraints that a single cysteine that forms a relatively binding site found for Hsp33 and RsrA can be imposed by the formation of a

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