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Review
. 2012 Apr;11(4):R111.013037.
doi: 10.1074/mcp.R111.013037. Epub 2011 Dec 8.

Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes

Jason M Held  1 , Bradford W Gibson
Affiliations
Review

Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes

Jason M Held et al. Mol Cell Proteomics. 2012 Apr.

Abstract

Oxidation is a double-edged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidation-reduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redox-sensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no one-size-fits-all analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role toward understanding the interplay between oxidative stress and redox signaling in health and disease.

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Figures

Fig. 1.
Fig. 1.
Crosstalk between catalytic activity and redox regulation. Caspase-3 is the terminal protease in the apoptosis cascade and cleaves numerous proteins to complete apoptosis. A, Under steady-state conditions the catalytic cysteine of caspase-3 is nitrosylated which inhibits its protease activity and prevents apoptosis (1). B, When tumor necrosis factor family member FasL binds to its cognate receptor FasR to trigger apoptosis, thioredoxin-2 transnitrosylates mitochondrial-associated caspase-3 derepressing its catalytic activity and promoting apoptosis (2).
Fig. 2.
Fig. 2.
Crosstalk between metal binding and redox regulation. A, The intron endonuclease I-TevI has two domains, a DNA-binding domain and a catalytic nuclease domain, separated by a linker region that uses a zinc finger (ZF) to stabilize the extended structure. Under steady-state conditions the linker is fully extended and the nuclease cleaves 23 and 25 nucleotides from the DNA-binding site. B, This allows maintenance of the endonuclease in an intron of the thymidylate synthase gene (TS intron) of the bacteriophage T4. C, Hydrogen peroxide-induced oxidation disrupts the ZF, shortening the linker between the DNA-binding domain and the nuclease domain leading to shorter, nonspecific DNA cleavage (3). D, Although I-TevI typically recombines within an intronless TS gene, the nonspecifically cleaved DNA sequences which result due to oxidation of I-TevI can homologously recombine at a new genomic site or host.
Fig. 3.
Fig. 3.
Reactivity of oxidants with cysteine and other amino acids. A, Second order rate constants (M−1 s−1) spanning nine orders of magnitude for radical and nonradical oxidants with cysteine. From (–17, 20, 21) with selected data from NDRL/NIST database (http://kinetics.nist.gov/solution/). Rate constants are in water at ∼pH 7.0 except where indicated. Rate constants for biologically relevant oxidants B, peroxynitrite at pH 7.4 (20), C, superoxide at pH 10.0 (8), D, hydroperoxyl radical at pH 1.6 (8), and E, hypochlorous acid at pH 7.4 (21) with all tested amino acids in each study to compare the relative reactivity of cysteine.
Fig. 4.
Fig. 4.
Common cysteine oxoforms and their chemical reversibility. Cysteines (orange) can be oxidized to a diverse set of oxidized species, including S-nitrosylation (SNO), glutathionylation (S-SG), disulfide (S-S), sulfenic acid (SOH), sulfinic acid (SO2H), and sulfonic acid (SO3H). Sulfenic acid is often an intermediate to other cysteine oxoforms. Oxidized cysteines in a yellow shade are chemically reversible by DTT and TCEP, whereas those shaded in red are chemically irreversible. In the case of peroxiredoxins, sulfinic acid is reducible through and ATP-dependent process catalyzed by sulfiredoxins (26, 27). There is no known repair process for sulfonic acids which are likely degraded.
Fig. 5.
Fig. 5.
Oxidation levels affect the redox status of cysteines in vivo and lead to divergent cellular responses. A, The redox continuum of oxidation in the cell ranges from reductive stress to oxidative stress with low levels of oxidation present in unstressed cells. B, C, Cysteines can be oxidized to different states depending on the oxidation level in the cell. While oxoforms essential to cell survival and proliferation (yellow) are the primary type of cysteine modification found in unstressed cells, a moderate increase in oxidation leads to an adaptive response and glutathionylation (S-GSH) of cysteines. Severe oxidative stress leads to senescence or cell death and is accompanied by overoxidized cysteine oxoforms such as sulfinic (orange) and sulfonic acid (red) which are dysfunctional. Hypoxia and reductive stress disrupts essential redox reactions (red X) and can result in cell death.
Fig. 6.
Fig. 6.
Differential alkylation indirectly detects oxidation and is modular. A, Structures of the maleimide and iodoacetamide functional groups. Bifunctional thiol specific reagents combine these two groups with fluorescent, stable isotope, or epitope tags (blue circles). B, The differential alkylation procedure labels nonoxidized cysteine thiols before (yellow ring) and after (purple ring) reduction with two different alkylation reagents to determine the percent of the cysteine that is reversibly alkylated. Fluorescent reagents include Cy3 and Cy5 tags and stable isotope labeled regents include d0 and d5 NEM or 12C9-ICAT and 13C9-ICAT reagents. Alkylation with an untagged alkylation reagent followed by a biotinylated thiol-specific reagent allows enrichment of reversibly oxidized peptides or proteins. Complete reduction of all reversibly oxidized cysteines can be achieved with TCEP or DTT. Selective reduction of specific cysteine oxoforms is possible using ascorbate plus CuCl to reduce S-nitrosylation, the enzyme glutaredoxin to reduce glutathionylation, and arsenite to reduce sulfenic acid.
Fig. 7.
Fig. 7.
Regulatory crosstalk between cysteine oxidation and other PTMs. A, Redox signals have been implicated in regulating the function of numerous enzymes which affect PTMs such as sumoylation, phosphorylation, acetylation, as well as others. Enzymes that both conjugate and remove PTMs can be affected with redox regulation typically repressing but occasionally enhancing enzyme activity. B, FoxO4 is a paradigm for the crosstalk between cysteine oxidation and other PTMs at the level of a single protein. Cysteine oxidation of FoxO4, via disulfide-mediated interaction with the acetyltransferase p300/CBP, increases acetylation and represses FoxO4 transcriptional activity in a redox-dependent manner (114). FoxO4 is phosphorylated and ubiquitinated as well, however it is not known if these PTMs are redox-regulated.
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