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Poole-Nelson-COCB08.Pdf This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Available online at www.sciencedirect.com Discovering mechanisms of signaling-mediated cysteine oxidation Leslie B Poole and Kimberly J Nelson Accumulating evidence reveals hydrogen peroxide as a key transport chain in mitochondria, by lipoxygenases in the player both as a damaging agent and, from emerging evidence cytoplasm and by flavoprotein oxidases in peroxisomes. It over the past decade, as a second messenger in intracellular is increasingly appreciated that cytokines, growth factors, signaling. This rather mild oxidant acts upon downstream targets and integrins also activate multicomponent NADPH within signaling cascades to modulate the activity of a host of oxidase enzymes (Nox) as part of the cell’s receptor- enzymes (e.g. phosphatases and kinases) and transcriptional mediated signaling responses. Both superoxide and, regulators through chemoselective oxidation of cysteine hence, hydrogen peroxide are generated, with the latter residues. With the recent development of specific detection serving a particularly significant role as a second messen- reagents for hydrogen peroxide and new chemical tools to ger in signaling pathways. Targeting of Nox proteins to detect the generation of the initial oxidation product, sulfenic specific subcellular compartments, including lipid rafts acid, on reactive cysteines within target proteins, the scene is set within membranes, endosomal signaling vesicles, and to gain a better understanding of the mechanisms through which other cellular organelles, may serve to offset the highly hydrogen peroxide acts as a second messenger in cell signaling. diffusible nature of hydrogen peroxide and limit the oxidation signal to these microenvironments [1,2,3]. Addresses Department of Biochemistry, Center for Structural Biology, Wake Forest Consistent with this, evidence is accumulating that anti- University School of Medicine, Winston-Salem, NC 27157, United States oxidant enzymes that serve to control intracellular per- oxide levels, including glutathione peroxidases, catalases, Corresponding author: Poole, Leslie B ([email protected]) and and especially peroxiredoxins [4], may themselves be Nelson, Kimberly J ([email protected]) regulated by these oxidants, allowing a threshold concen- tration of peroxide to accumulate around the site of Nox Current Opinion in Chemical Biology 2008, 12:18–24 localization while preventing damage to cellular com- This review comes from a themed issue on ponents outside these regions (reviewed in reference [5]). Proteomics and Genomics Edited by Natalie Ahn and Andrew H.-J. Wang Transient generation of localized ROS is important in Available online 7th March 2008 receptor-mediated cell signaling, yet the ability to detect ROS with a high degree of spatial and temporal resolution 1367-5931/$ – see front matter remains a challenge [6]. Oxidation of widely used 20,70- # 2008 Elsevier Ltd. All rights reserved. dichlorodihydrofluorescein (DCFH) and dihydrorhoda- DOI 10.1016/j.cbpa.2008.01.021 mine 123 (DHR) reagents as detected by enhanced fluorescence gives a sense of the ROS burst, but these reagents exhibit a relative lack of specificity toward their Introduction oxidants and are also subject to auto-oxidation and photo- With the emerging understanding that hydrogen peroxide oxidation. The recent design and synthesis of new, highly generation is a crucial component of numerous receptor- selective chemical sensors of hydrogen peroxide, particu- mediated cell signaling events, investigators are seeking larly those that become fluorescent upon peroxide- to identify, within given signaling networks, the particular mediated removal of a boronate-based protecting group, oxidation-sensitive proteins, residues, and molecular is a very exciting development in this regard [7]. These events that lead to the modulation of cell signaling net- and other new ratiometric, protein- and/or nanoparticle- works by hydrogen peroxide. We discuss herein some of based reagents for peroxide sensing [8–10] are promising the rapidly evolving chemical tools and experimental tools for continuously monitoring intracellular peroxide approaches being developed both for detecting oxidant generation, yet will need to overcome limitations such as generation and for identifying the molecular targets modi- irreversibility and relatively slow rates of reaction to fied by the oxidants. We also summarize recent data achieve the level of sensitivity required for ‘instan- supporting cysteine sulfenic acid as a key intermediate taneous’ and nonperturbing detection of signaling- in the functional modulation of enzymes and transcription relevant hydrogen peroxide (for recent reviews, see refer- factors involved in peroxide-mediated cell signaling. ences [6,11,12]). Localization and control of hydrogen peroxide Cysteine redox chemistry for signaling It is increasingly clear that a major component of the Reactive oxygen species (ROS) are produced in cells as a ROS-linked modulation of cell signaling pathways is the result of the partial reduction of oxygen by the electron dynamic regulation of protein function by reversible thiol Current Opinion in Chemical Biology 2008, 12:18–24 www.sciencedirect.com Author's personal copy Signaling-mediated cysteine oxidation Poole and Nelson 19 modification. For many proteins, the first product of fications could, in spite of their typically irreversible cysteine oxidation by hydroperoxides, cysteine sulfenic status, also provide a signaling role analogous to a ‘fire acid (R-SOH) (Figure 1), undergoes rapid condensation alarm’ regarding cellular redox status [15]. either with another protein thiol (as an intramolecular or intermolecular interaction) or with a small molecule thiol Sulfenamide (sulfenyl-amide) has recently been recog- like glutathione or cysteine to form a disulfide bond. nized as another stabilized oxidation product generated Thiol-based reductants can then return this species to through condensation of the sulfenic acid with a neigh- the fully reduced thiol state (Figure 1). Disulfide bonds boring backbone amide nitrogen (Figure 1); this 5-mem- can stabilize extracellular proteins, protect against irre- bered ring structure appears to represent yet another versible inactivation, stabilize associations within protein form of oxidized cysteine resistant to hyperoxidation. complexes, modify structures to create, destroy or modu- The initial evidence for this species was crystallographic late functional sites, and ultimately regulate enzymatic or [16],leavingsomedoubtastotherelevanceofthis transcriptional activity of proteins. Under conditions species in a biological setting. However, a functional where reactive nitrogen species (RNS) are generated, role for the generation of the sulfenamide is suggested signaling through reversible S-nitrosation, perhaps in by the profound changes in structure imparted to the addition to the typical ROS-generated products, may also active site of protein tyrosine phosphatases with this play an important role [13]. The features of the protein modification, changes that may enhance access by reduc- microenvironment that control thiol reactivity and tants and/or modulate interactions with other domains stability of the resulting modifications are beginning to ([17]; reviewed in reference [18]). Studies using model be analyzed [14]. chemistry have also better defined the parameters that promote this cyclization reaction in the constrained set- In certain cases, the initial sulfenic acid may itself be ting of a protein active site [19]. Furthermore, recent stabilized within the protein microenvironment. This studies of the peroxide sensitive transcriptional regula- does, however, potentially leave the protein sulfenic acid tor, OhrR from Bacillus subtilis, support stable formation vulnerable to hyperoxidation to the largely irreversible of the sulfenamide species in solution and possibly in vivo sulfinic or sulfonic acids (Figure 1); these further modi- for this protein [20]. Figure 1 Biological modifications of cysteine thiols. Reactive cysteine thiols (green), typically in their ionized, thiolate form (R-SÀ), are oxidized by such oxidants as hydrogen peroxide, organic hydroperoxides, hypochlorous acid, and peroxynitrite to form sulfenic acids, which may be stabilized or go on to form other reversible (disulfides or sulfenamides, orange) or irreversible (sufinic and sulfonic acid, red) species. Both reactive oxygen species (ROS) and reactive nitrogen species (RNS) promote these oxidations. Note that the generation of sulfenamide (bottom) involves attack of a neighboring amino acid’s amide nitrogen (blue) on the sulfenic acid sulfur. Although sulfinic and sulfonic acids are shown here as irreversible modifications, recent discoveries show that some peroxiredoxins in this state can be recovered through action of specialized
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