The Cysteine Proteome 13 N Q114 Young-Mi Go, Joshua D

Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect 2 3 4 Free Radical Biology and Medicine 5 6 journal homepage: www.elsevier.com/locate/freeradbiomed 7 8 9 Review Article 10 11 12 The cysteine proteome 13 n Q114 Young-Mi Go, Joshua D. Chandler, Dean P. Jones 15 Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University, Atlanta, GA 30322, USA 16 17 18 article info abstract 19 20 Article history: The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and 21 Received 17 November 2014 the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological 22 Received in revised form switching of structure and reactivity. These biological modifications include sulfenylation and disulfide 23 17 March 2015 formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metalation, and other Accepted 22 March 2015 24 modifications. Extensive knowledge about these systems and their compartmentalization now provides 25 a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed Keywords: 26 understanding of redox signaling pathways and sensing networks is becoming available to allow the Cysteine proteome discrimination of network structures. This research focuses attention on the need for atlases of Cys 27 Redox proteome modifications to develop systems biology models. Such atlases will be especially useful for integrative 28 Redox signaling studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved 29 Functional network Thiol redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to 30 Free radicals the quantitative proteome in the omics continuum connecting the genome to the exposome. 31 & 2015 the autors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license 32 (http://creativecommons.org/licenses/by-nc-nd/4.0/). 33 67 34 68 Contents 35 69 36 70 Introduction...... 2 37 71 Lessons from the redox proteome ...... 2 38 72 Centraldogma...... 2 39 Protein thiols exist in a dynamic steady state ...... 3 73 40 Selective pressure against Cys in protein ...... 4 74 41 Redox compartmentalization ...... 4 75 42 Functional networks within the Cys proteome ...... 5 76 43 Cys proteome chemistry and functions ...... 6 77 44 Chemical properties of cysteinyl sulfur ...... 6 78 45 Reactions of protein Cys thiol...... 6 79 46 Factors affecting protein thiol reactivity ...... 6 80 47 The role of Cys in catalytic sites of enzymes ...... 7 81 Protein Cys redox regulation ...... 8 48 82 TrxandGSHsystems...... 8 49 83 Regulation of actin cytoskeleton proteins ...... 9 50 Cys proteome in nuclear protein trafficking ...... 10 84 51 Nuclear functions of the Cys proteome ...... 10 85 52 Oxidation of the Cys proteome by H2O2 and derivative species ...... 10 86 53 H2O2 ...... 10 87

54 Oxidant species derived from H2O2 ...... 12 88 55 S-nitrosylation ...... 12 89 56 Persulﬁdation ...... 12 90 57 Alkylation ...... 13 91 58 Integrated function of the Cys proteome ...... 13 92 59 93 60 94 61 95 n 62 Corresponding author. Fax: þ404 712 2974. 96 63 E-mail address: [email protected] (D.P. Jones). 97 64 http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.022 98 65 0891-5849/& 2015 the autors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 99 66

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 2 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Perspective and future directions...... 13 67 2 Acknowledgments...... 14 68 3 References...... 14 69 4 70 5 71 6 72 7 73 8 Introduction challenge also provided sensing and signaling systems for tem- 74 9 poral and spatial execution of differentiation and development in 75 10 The sulfur atom of cysteine (Cys) provides a considerable range multicellular organisms. 76 fl 11 of chemical reactivity and structural exibility in the proteome. In the present review of the Cys proteome, we start with a 77 12 The presence of conserved Cys in motifs of proteins found in summary of lessons learned from study of the redox proteome, 78 13 essentially all life forms indicates that these chemical character- that portion of reversible and irreversible covalent modifications 79 14 istics were harnessed in early evolution to support enzyme that link redox metabolism to biologic structure and function [1]. 80 15 catalysis, transcriptional regulation, protein folding, and three- We follow this with a more specific consideration of the chemistry 81 16 dimensional structure. A remarkable range of biologic functions is of sulfur and the spectrum of oxidative reactions that defines Cys 82 17 supported by Cys because sulfur is stable in multiple coordinate proteomic functions. In a third section, we address the redox 83 18 covalent bonds with the major atoms of living organisms (C, H, O, regulation systems, especially focusing on major reducing systems 84 19 N, P), forms stable coordinate complexes with transition metal that balance the oxidative reactions and support a rapidly evolving 85 – – 20 ions (Zn, Fe, Cu), and is stable at a range of oxidation states ( SH, understanding of the spatiotemporal nature of redox regulation. In 86 – – 1 – 1 21 SS , SO2 , SO3 ). Additionally, Cys thiols differentially undergo this, we provide specific examples illustrating the organizational 87 22 reversible ionization to the negatively charged thiolate form over structure of stable, kinetically controlled functional redox net- 88 fl 23 the physiologic range of pH to exibly optimize the functions of works and summarize other modifications such as S-nitrosylation 89 fi 24 speci c peptidyl Cys. and persulfidation. Finally, we discuss the Cys proteome within the 90 25 The human genome encodes about 214,000 Cys. Cys is widely context of integrated omics approaches, highlighting the ongoing 91 26 distributed among proteins, with most expressing at least one and need for development of quantitative redox biology models. 92 27 many having multiple coordinated Cys and Cys-rich domains. The 93 28 reactivity and diverse functions of Cys are mirrored by a spectrum 94 29 of susceptibilities and dysfunctions of their respective proteins, Lessons from the redox proteome 95 30 resulting in central roles of the Cys proteome in development, 96 31 signal transduction, biologic defenses, aging, and disease. Con- Central dogma 97 32 siderable knowledge has accumulated for the symptoms and 98 33 etiology of major human diseases, such as cardiovascular disease, Cys is incorporated into protein as the thiol (RSH) form, with 99 34 Alzheimer disease, lung disease, metabolic syndrome, eye disease, apparently no exception. Thiols are oxidized to sulfenic acids 100 35 and diseases of other organ systems. These often share related (RSO) intermediate to formation of disulfides (RSSR) and higher 101 oxidative phenotypes and mechanisms, and progress in redox 36 oxidation states (e.g., RSO2 ). Thiols and disulfides also undergo 102 37 systems biology is beginning to provide an understanding of the exchange reactions in which the thiol reacts to form a new 103 integration of the underlying redox systems. 38 disulfide and liberates a different thiol (RSH þ R1SSR1 2 RSSR1 104 Early photosynthetic organisms transformed the earth by fixing 39 þ R1SH) [5]. Disulfide is a common posttranslational modification 105 40 the carbon atom of CO2 into hydrocarbons and releasing bound for three-dimensional structure, as a component of multistep 106 41 atomic oxygen as gaseous molecular O2. This changed the atmo- vectorial processing and transport, as a switching mechanism in 107 o 42 sphere from 1% to about 21% O2 and had a profound effect on regulation or signaling, and as a consequence of oxidative stress. 108 43 life forms by creating a diversity of external environments with Research with redox Western- and mass spectrometry-based 109 44 different oxidative conditions. Contemporary redox proteomic redox proteomics methods shows that partial oxidation is com- 110 45 systems have evolved as a result of the selective pressures of an mon for Cys residues throughout the proteome of mammalian 111 46 oxygen environment and aerobic respiration, with extant genomes systems [6–10]. This contradicts earlier interpretations that Cys 112 47 having selectively advantageous redox proteomic structures that thiol oxidation represents only an artifact of extraction [11] and 113 48 protect the genome and ensure its replication despite environ- supports the hypothesis that significant speciation of different 114 49 mental, organismal, and developmental variation in oxidative and peptidyl Cys in basal oxidation, organization, and function occurs 115 fl 50 reductive challenges. Although oxidative pressure in uences mul- within the Cys proteome. 116 51 tiple critical loci of the redox proteome, such as methionine (Met) Discussions of procedures and pitfalls in measurements of 117 52 and selenocysteine (Sec), the Cys proteome is central within this thiols and thiol/disulfide have been reviewed [12–14]. Measure- 118 53 spectrum of biological plasticity and is the focus of this review. ments involving extraction and chemical modification reflect the 119 54 The Cys proteome (Fig. 1) serves as a central adaptive interface efficiency of alkylation relative to oxidation in trapping the thiol 120 55 between the functional genome and the external environment of form (see Hansen and Winther [13] for detailed discussion). 121 56 an organism [1]. The external environment is a critical determi- Importantly, processing must be rapid under various conditions 122 57 nant of individual survival; in human health, this is now discussed to minimize both oxidative and reductive artifacts. Artifacts are 123 “ ” 58 in terms of an individual's exposome [2]. The exposome is more likely in experimental studies with added oxidants, metal 124 fi fl 59 de ned as the cumulative measure of environmental in uences ions, or electrophiles, especially if these are not removed before 125 60 and associated biological responses throughout the life span, processing biologic materials for thiol analysis. Extraction at 0 1C 126 including exposures from the environment, diet, behavior, and 61 slows reaction rates about 10-fold relative to 37 1C, and higher O2 127 62 endogenous processes [3]. In addition to utility in contemporary in air-equilibrated solution increases rates about 5-fold that of 128 63 environmental health research [3,4], this provides a useful way to tissues in vivo in mammals. Thus, in the absence of added oxidants 129 64 think about the evolution of complex multicellular organisms as and metals, oxidation of Cys during assay occurs at relatively slow 130 fi 65 the content of O2 increased in the atmosphere. Speci cally, the rates, similar to the ongoing autoxidation rates in vivo. High 131 66 redox sensing and signaling mechanisms supporting responses to concentrations of thiol-reactive chemicals can be readily achieved 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

1 under most experimental conditions to minimize significant basal conditions, including Cys374 of actin in A431 cells [17] (more 67 2 artifact [13]. An alternate reductive artifact can also occur with discussion of Cys modification below). However, these targeted 68 3 addition of thiol-trapping reagents under conditions where studies mostly involve proteins that are regulatory or signaling in 69 4 NADPH-dependent reduction continues. nature, leaving open the possibility that steady-state oxidation is 70 5 Discussion of the redox activities of the Cys proteome requires an uncommon event. An important breakthrough during the past 71 6 care in consideration of assumptions upon which interpretations are decade is derived from mass spectrometry-based proteomic 72 7 based. For instance, redox potential refers to equilibrium conditions, methods, which provide much greater coverage of oxidation of 73 8 which can be approximated experimentally in dilute solution with the Cys proteome. 74 9 sufficient equilibration time. However, total protein concentration in Sethuraman et al. [18] showed that isotope-coded affinity tag 75 10 biological systems is very high, many proteins are continuously (ICAT) reagents [19] could be used to measure oxidation of 76 11 undergoing reversible interaction with other proteins, and many individual Cys-containing peptides, specifically comparing differ- 77

12 processes are kinetically limited and displaced from equil- ences between control and H2O2-treated samples. The design in 78 13 ibrium. Local pH and metal ion concentrations may not be uniform, this study did not permit evaluation of steady-state oxidation 79 14 and dynamic processes of phosphorylation/dephosphorylation, acet- without oxidant. Related methods used by Le Moan et al. [20], 80 15 ylation/deacetylation, methylation/demethylation, reversible metal Leichert et al. [21] and Go et al. [22,23] allow determination of the 81 16 ion binding, etc., may have an impact on in vivo properties. Most percentage reduction of Cys under control as well as experimen- 82 17 of the time, data and analytical methods are insufficient to fully tally treated conditions. For instance, proteins can be rapidly 83 18 evaluate these possibilities. Furthermore, site-directed mutagenesis extracted, treated with a heavy stable isotopic ICAT reagent, 84 19 studies to test the function of a Cys can be misleading if experimental treated with reductant, and finally treated with a light stable 85 20 conditions do not replicate conditions under which the Cys influ- isotopic ICAT reagent. With liquid chromatography–mass spectro- 86 21 ences function. Such issues represent challenges for the future to metry/mass spectrometry (LC–MS/MS), this allows measurement 87 22 improve integrated redox biology models. of the percentage oxidation of specific residues in peptide 88 23 sequences. Importantly, Le Moan et al. [20] showed that in yeast, 89 24 Protein thiols exist in a dynamic steady state Cys in many proteins are partially oxidized under control condi- 90 25 tions. Leichert et al. [21] obtained similar results in studies of 91 26 Targeted redox analyses of thioredoxin-1 (Trx1), thioredoxin Escherichia coli, and Go et al. found steady-state oxidation of 92 27 reductase-1, peroxiredoxin-1, mitochondrial Trx2, thioredoxin proteins in mouse aortic endothelial cells [22], HT29 colon 93 28 reductase-2, peroxiredoxin-3, NF-κB (p50), redox factor-1, protein carcinoma cells [6], lung fibroblasts [8], mouse liver [24], and liver 94 29 disulfide isomerase, and other proteins (for summary, see [15,16]) mitochondria [24]. 95 30 show partial oxidation in cell culture under control conditions. More sensitive mass spectrometers and newer peptide tagging 96 31 Studies of glutathionylation also reveal modified residues under methods are becoming available to further enhance sensitivity and 97 32 98 33 99 34 100 35 101 36 102 37 103 38 104 39 105 40 106 41 107 42 108 43 109 44 110 45 111 46 112 47 113 48 114 49 115 50 116 51 117 52 118 53 119 54 120 55 121 56 122 57 123 58 124 59 125 60 126 61 127 62 128 63 Fig. 1. The Cys proteome exists as an adaptive interface between the exposome and the functional genome. Research on integrated omics has emphasized the role of the 129 metabolome and the proteome as intermediates between environmental exposures and the genome, epigenome, and transcriptome. The Cys proteome has a more direct role 64 130 to support sensing and signaling of nutritional and environmental conditions and also to provide an array of reactive nucleophiles to neutralize toxic electrophilic chemicals. 65 Thus, the Cys proteome is an important functional component of the translated proteome, directly interacting with the metabolome to utilize environmental resources and 131 66 defend against environmental threats. 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 4 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 coverage of the Cys proteome [6], and efforts to identify oxida- Cys proteome is functional [16]. If such function were restricted to 67 2 tively sensitive Cys, the collective “sulfenylome” of protein Cys that catalytic or other “active sites,” then one or two Cys within each of 68 3 forms RSO in response to multiple stimuli, have yielded increas- the 20,000 gene products would account for only 10–20% of the 69 4 ingly promising results [25,26]. Although there remains a need for 214,000 Cys in the proteome. Instead, the data suggest that up to 70 5 caution until more independent analyses and confirmations are 80% could be functional, although most without essential “life/ 71 6 available, the mass spectrometry-based methods are enhancing death” function. Alternative functions for Cys include stabilization 72 7 the ability to characterize both stable and transient, partially of long-lived proteins by formation of Zn clusters [28], presenta- 73 8 oxidized Cys residues within the mammalian Cys proteome. tion of decoy thiols to protect critical thiols from oxidants and 74 9 Knowledge of the functions of the redox-sensitive Cys residues reactive electrophiles [27], and function as redox-sensing ele- 75 10 is of critical importance. The peptides detected are from relatively ments for coordinate regulation of functional pathways [16]. These 76 11 abundant proteins and ionize well with available instrumentation. are linked together by the concept that the Cys proteome functions 77 12 This leaves open the possibility that the Cys residues measured by as an interface between the genome and the exposome (Fig. 1) [1]. 78 13 LC–MS/MS are nonfunctional or function in a nonspecific way to Specifically, a Cys proteome network provides a defensive barrier 79 14 protect critical sites from thiol-reactive chemicals [27]. Three types and sensing system to enhance fitness to tolerate exogenous 80 15 of evidence suggest that many have more specific functions. oxidants, reactive electrophiles, and toxic trace metal ions and, 81 16 Comparison among species of the measured protein Cys to other at the same time, is used to optimize food utilization. 82 17 amino acid residues within a sampling of mitochondrial proteins Early forms of life evolved under significantly more reducing 83 18 showed that the Cys are more highly conserved (Fig. 2A) [6]. conditions than in the current age of highly efficient photosynth- 84

19 Examination of the positions of the measured protein Cys within esis. Before the evolution of an O2-rich atmosphere between 600 85 20 the three-dimensional crystal structures for homologous proteins and 900 million years ago, living systems were already utilizing 86 21 shows that the Cys are often in positions expected to be function- inorganic sulfur and Cys. Iron–sulfur clusters, for instance, are 87 22 ally important (Fig. 2B). These include positions near catalytic ancient prosthetic groups that may have functioned to abiotically 88 23 sites, protein–protein interaction sites, and RNA-binding or DNA- catalyze formation of C–C bonds [29]. Along with the evolving Met 89 24 interaction sites. Finally, the measured Cys peptides map to proteome and associated redox reactions to regulate physiology 90 25 functional pathways according to percentage oxidation (Fig. 3) [30,31], Cys became increasingly utilized with organizational 91 26 [6]. Some specific examples are discussed in more detail below. complexity [28]. Notably, selective pressure against the use of 92 27 The results are sufficient to conclude that this is a general Cys owing to vulnerability to oxidation is a result of the same 93 28 phenomenon, although not ubiquitous. Functional networks iden- chemical features that confer selective advantage when used as a 94 29 tified include the cytoskeleton, translation, nuclear import, RNA redox sensor and switch [28]. The Cys proteome thus enabled the 95

30 processing, energy metabolism, and oxidative stress signaling evolution of systems to regulate O2 availability in tissues and 96 31 [6,8,24]. subcellular compartments as well as support developmental 97 32 systems to tolerate and adapt to complex oxidative environments. 98 33 99 34 Selective pressure against Cys in protein 100 35 Redox compartmentalization 101 36 Cys codons represent 3.28% of all codons, yet the percentage of 102 37 Cys in protein is typically only 2.2% or less [28], indicating that Oxidation of protein Cys occurs within the secretory pathway 103 38 there is evolutionary selection against utilization of Cys. At the as part of a vectorial processing system for proteins targeted to 104 39 same time, the percentage of Cys in the proteome is substantially specific membrane regions and for protein export [32]. Measure- 105 40 greater in complex eukaryotes (2.2%) than in archaebacteria and ments of steady-state oxidation of proteins show that during 106 41 eubacteria (0.5%) [28]. The Cys residues are relatively conserved in processing, protein disulfide isomerase (PDI) is partially oxidized 107 42 metazoan evolution, leading to the interpretation that much of the [33,34]. Recent studies show that a similar redox processing of 108 43 109 44 110 45 111 46 112 47 113 48 114 49 115 50 116 51 117 52 118 53 119 54 120 55 121 56 122 57 123 58 124 59 125 60 126 61 127 62 128 63 Fig. 2. Multiple lines of evidence support function of redox-sensitive Cys measured by mass spectrometry. (A) Targeted searches show that Cys detected in redox proteomic 129 analyses are highly conserved across metazoans compared to other amino acids in the same proteins. (B) Examination of X-ray crystal structures shows that redox-sensitive 64 130 Cys are commonly present in functional motifs of proteins. Examples shown include specific Cys adjacent to the GTP-binding site in RhoQ, Cys in the RNA binding site of 65 heteronuclear ribonucleoprotein (HNRNP) A/B, and the DNA interaction site of PCNA. Graphs at the bottom show oxidation in response to challenge with cadmium or 131 66 inhibition of thioredoxin with auranofin (Aur) or GSH depletion with buthionine sulfoximine (BSO). 132

1 67 2 68 3 69 4 70 5 71 6 72 7 73 8 74 9 75 10 76 11 77 12 78 13 79 14 80 15 81 16 82 17 83 18 84 19 85 20 86 21 87 22 88 23 89 24 90 25 91 26 92 27 93 28 94 29 95 30 96 31 97 32 98 33 99 34 100 35 101 36 102 37 103 38 104 39 105 40 106 41 107 42 108 43 109 44 110 45 111 46 112 47 113 48 114 49 115 50 116 51 Fig. 3. Protein Cys map to functional pathways based upon percentage oxidation. Ingenuity pathway analysis of the most reduced protein Cys in colon carcinoma HT29 cells 117 52 under steady-state nonchallenged conditions showed associations with mitochondrial pathways for energy metabolism, cell growth, and proliferation; cytoplasmic 118 cytoskeletal pathways; and nuclear Ran-import mechanisms and RNA processing. Most oxidized protein Cys were associated with mitochondrial intermediary metabolism, 53 cytoplasmic cell survival, Myc-mediated apoptosis and 14-3-3 signaling, and nuclear granzyme B signaling. Data from Refs. [6,8,24,49]. 119 54 120 55 121 56 proteins occurs with mitochondrial protein import [35,36]. peroxiredoxin-1 to nuclei [45]. Increased abundance of nuclear Trx1 122 57 Dynamic redox processes are also involved in nuclear protein in a transgenic mouse model showed increased NF-κB activity and a 123 58 import and export [37–39]. These processes depend upon different proinﬂammatory state potentiating cardiovascular disease [46] and 124 59 control systems, discussed in more detail below, with many increased sensitivity to inﬂuenza infection [46]. Thus, control of the 125 60 utilizing thioredoxins and thioredoxin-like proteins. Cys proteome within subcellular compartments has an impact on 126 61 Targeted studies of steady-state oxidation of Trx in subcellular Cys reactivity and biologic function. 127 62 compartments [40] show that oxidation of Trx differs among major 128 63 compartments of mammalian cells, with oxidation in cytoplasm 4 Functional networks within the Cys proteome 129 64 nuclei 4 mitochondria [15,41–44]. Dynamic control within orga- 130 65 nelles is illustrated by NF-κB reporter activity being decreased by Differences in steady-state oxidation of Trx and glutathione (GSH) 131

66 generation of H2O2 within nuclei [9] and increased by targeting systems among compartments suggest an organizational structure in 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 6 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 which the central thiol redox systems control subcellular functions. derivatives, with protein Cys varying in selectivity for these 67 2 Studies using mass spectrometry-based redox proteomics revealed, modifications versus formation of internal disulfides [53]. 68 3 however, that steady-state percentage oxidation of Cys in proteins The sulfur of Cys can be sequentially oxidized by strong 69 4 within cytosol, mitochondria, and nuclei mapped to functional net- oxidants, progressing from sulfenic to sulfinic and sulfonic acids 70 5 works rather than to subcellular compartments [6]. These results (present as RSO2 and sulfonate, RSO3 , respectively). These higher 71 6 support a role for the Cys proteome in the metabolic and structural oxidation states of Cys are significantly more difficult to reduce 72 7 organization of cells, not just as a series of isolated elements in and may be considered irreversible, although reduction of certain 73 8 insulated pathways within subcellular compartments but rather as a Cys–SO2 (e.g., peroxiredoxin (Prx)-2) is catalyzed by sulfiredoxin 74 9 redox network connected throughout the cell. [54]. Higher oxidation states are detected in mass spectral analysis 75 10 Emerging data indicate that redox networks coordinate large of proteins but the extent of natural occurrence and possible 76 11 numbers of redox elements in function and resistance to environ- functions remain unclear. 77 12 mental challenges. Barabasi and coworkers described metabolic Cysteinyl sulfur undergoes a variety of other reversible and 78 13 organization in scale-free hierarchical structures that are inher- irreversible covalent modifications including nitrosylation, persul- 79 14 ently stable [47,48]. A bilateral structure for the Cys proteome can fidation, alkylation, and arylation that can have unique effects on 80 15 accommodate different oxidant and reductant systems and has Cys redox regulation, function, and signaling (Fig. 4). At least 18 81 16 advantages over parallel branching pathways in which protein Cys biologically occurring nonradical modifications of Cys thiol have 82 17 residues are physically or kinetically isolated from one another been described, including the above reactions. These have widely 83 18 [6,7]. Furthermore, redox proteomics studies with inhibition of varying stability and multiple reviews of their biological impacts 84 19 thioredoxin reductase (TrxR) show that many protein Cys are are available [55–57]. A remarkable selectivity in reaction with 85 20 simultaneously oxidized [49]. Along with the above evidence for different targets highlights the versatility of protein Cys in use. For 86 21 compartmentalization, and recent data showing that redox path- instance, the redox sensor Keap1 has multiple Cys residues that 87 22 ways can switch owing to abundance of reactive targets [50], these react independently with alkenals, metals, and nitric oxide to 88 23 observations indicate that interactions within the Cys proteome provide a selective detection system [58]. This system also 89

24 are best described as a network with spatial determinants [6,7]. discriminates acrylamide and H2O2 by reaction with different Cys 90 25 Redox signaling pathways are present within this network, char- residues [59,60].RS can also be converted to thiyl radicals during 91 26 acterized by being localized [51] and having rapid flux compared ionizing radiation and other conditions. These radicals rapidly 92 27 to the relatively slow and distributed election flow of the overall react with RS and O2 in a series of reactions that convert more 93 28 Cys proteome. We discuss this in more detail below under “Protein reactive radicals to superoxide [57,61]. A Cys thiyl radical inter- 94 29 Cys redox regulation, Trx and GSH systems.” mediate is used in the catalytic mechanism of all classes of 95 30 ribonucleotide reductase, abstracting a hydrogen from the 30 96 31 carbon of ribonucleotide ribose to permit deoxygenation at the 97 0 32 Cys proteome chemistry and functions 2 position for deoxyribonucleotide synthesis [62,63]. 98 33 In addition to Cys, the genome includes special instructions for 99 34 Chemical properties of cysteinyl sulfur the translation of Sec, the so-called 21st amino acid [64]. Sec is 100 35 identical to Cys except for the replacement of sulfur with selenium 101 36 The six valence electrons of sulfur in Cys are distributed among (Se). Cys and Sec undergo many comparable reactions, but Se is 102 37 four sp3 orbitals, which consist of two lone pairs and two sigma much more acidic and nucleophilic than sulfur [65]. Sec is 103 38 bonds with C and H, respectively. Compared to the more abundant incorporated into important thiol regulatory and antioxidant 104 39 element oxygen, sulfur is less electronegative and has a valence proteins, including TrxR, glutathione peroxidase (GPx), and Met- 105 40 shell radius nearly twice as large. In the absence of local effects in R-sulfoxide reductase B1, formerly known as selenoprotein R 106 41 protein structure, the thiol of Cys is 5–6 orders of magnitude more [64,66,67]. These interactions warrant inclusion of the selenopro- 107 42 acidic than the hydroxyl group of serine. At physiologic pH, local teome along with the Cys proteome and Met proteome in broader 108 43 environments in proteins can result in predominantly the thiol considerations of redox systems biology. 109 44 (most common) or the thiolate (most reactive) form. Sulfur also 110 45 has greater nucleophilicity, with reactivity being substantially Factors affecting protein thiol reactivity 111 46 enhanced by ionization and local protein structure. These proper- 112 47 ties favor the use of cysteinyl sulfur in biological switching, as it Common chemical features for thiol reactivity exist for reac- 113 48 has increased tendency both to donate electrons and to accept tions involving metal chelation, substrate reduction, and/or con- 114 – fi fi 49 them [52]. jugation or thiol disul de exchange. Speci c Cys are, however, 115 50 often limited to reaction with a fraction of potential thiol-reactive 116 51 chemicals, e.g., individual thiols within the same monomeric 117 52 Reactions of protein Cys thiol protein are selective for thiol–disulfide exchange or S-nitrosylation 118 53 [68]. Cys reactivity is mediated in part by the overall availability of 119 54 Cys thiolate donates an electron pair to thermodynamically the thiolate form because Cys thiol shares its donor electron pair in 120

55 favorable targets resulting in the oxidation of the thiol group. a sigma bond with hydrogen. As a consequence, pH and pKa 121 56 Sulfenic acids (RSOH), present mostly as RSO, are a common interdependently regulate cysteinyl sulfur reactivity. For two 122 57 product of Cys oxidation and rapidly resolve by reaction with a hypothetical thiolates in solvent pH 7.4, sharing a reaction 123 58 thiol, eliminating water to form RSSR. The reaction of RSO with mechanism for H2O2 removal and having pKa 7.4 (50% RS ) and 124 59 RSH to form RSSR is 4105 M1 s1 [52]. RSO are central to the 8.4 (9.1% RS), respectively, in the absence of other inﬂuences, the 125 60 oxidative modiﬁcation of large numbers of Cys targets [25]. more acidic thiol reacts approximately 5-fold more rapidly with 126

61 Dimeric or internal disulfides often describe a biologically relevant the target species. Therefore, pKa exerts influence on the relation- 127 62 half-cell with the reduced Cys species to form a redox couple ship of a thiol to solvent pH, which differs between subcellular 128 63 maintained in nonequilibrium steady state based on interacting compartments in eukaryotes. For instance, the mitochondrial 129 64 redox couples, compartmentation, cell cycle progression, and matrix is alkaline at pH 7.8 compared to the intermembrane space, 130 65 other parameters [40]. In addition to internal disulfides, protein which is maintained approximately 1 unit lower [69]. This affects 131 66 Cys react with GSH or free Cys to form S-glutathionyl or S-cysteinyl thiol redox potential with specificity to each thiol. For a thiol– 132

1 67 2 68 3 69 4 70 5 71 6 72 7 73 8 74 9 75 10 76 11 77 12 78 13 79 14 80 15 81 16 82 17 83 18 84 19 85 20 86 21 87 22 88 23 89 Fig. 4. Functional modifications of the Cys proteome. Protein Cys (R–SH) undergoes a range of enzymatic and nonenzymatic modifications, often mediated by pH-dependent 24 ionization to Cys thiolate (R–S; large yellow sphere). Interaction of R–S with metal ions (blue) is common as a structural element, as a binding motif for nucleic acids, and 90 25 as a component of catalytic sites. R–S is subject to oxidation ([O]) to Cys sulfenate (R–SO , yellow) by H2O2 and other two-electron oxidants. Thiol (R–SH) also undergoes 91 fi – – – fi – – — fi 26 exchange with disul de (R1 SS R1) to generate a different thiol (R1 SH) and disul de (R1 SS R) in a process termed thiol disul de exchange. Nonequilibrium steady-state 92 oxidation of specificR–SH occurs owing to the presence of low nanomolar concentrations of H2O2 in cells. R–SO reacts with R–SH or GSH to produce the respective 27 93 disulfides (R–SS–R, R–SSG; dark yellow, top). Many R–SO can undergo hyperoxidation to sulfinate (R–SO2 ; orange, bottom) and sulfonate (R–SO3 ; red) in the presence of 28 excess oxidant. R–S also reacts with hypothiocyanous acid (HOSCN) to produce sulfenyl thiocyanate (R–SSCN; dark yellow, upper left), which rapidly hydrolyzes, and with 94 29 nitric oxide through multiple means of nitronium ion transfer (NO þ) to produce a corresponding nitrosothiol (R–SNO; orange, bottom left). R–SO can also react with 95 ) 30 hydrogen sulfide (HS ) to form a cysteinyl persulfide (R–SS , green), which can be oxidized to a cysteinyl thiosulfate (R–SSO3 . Alternatively, R–SO can react with primary 96 31 amines of neighboring amino acids or separate biomolecules to form sulfenamide (R–SNRH), which can be further oxidized to sulfinamide (R–S(O)NRH) and sulfonamide (R– 97 32 S(O)2NRH), frequently resulting in cyclic intramolecular structures. 98 33 99 34 disulfide half-cell, the effect on the standard redox potential is maintain metal ion homeostasis to prevent so-called “mis-metala- 100 35 5.9 mV/0.1 pH unit. In the case of vicinal dithiols, the effect is tion” of maturing enzymes [81]. Cys involved in metal ion 101 36 more limited (2.95 mV/0.1 pH) [70]. In vicinal dithiols that coordination also participate in thiol–disulfide exchange during 102 37 collaborate in a single catalytic function, one is typically more changes in the steady-state potential of cellular antioxidants [82]. 103 38 acidic and the overall behavior depends upon the presence and Subcellular compartmentalization of metal ions (particularly in the 104 39 characteristics of other reactants. Stable RSO offer another means endomembrane system) is dependent on the expression and 105 40 of differentiating pH regulation of redox couples, as they tend to oxidation of cysteinyl redox-active transporters such as sarcoplas- 106 2þ 41 be very strong acids [71]. However, the pKa- and pH-determined mic reticulum Ca -ATPase (SERCA) [81,83]. 107 42 fraction of Cys available as thiolate varies over only a 102-fold 108 43 range and thus cannot solely explain the 106-fold range of The role of Cys in catalytic sites of enzymes 109

44 reactivity of protein thiols with H2O2 and similar ranges for other 110 45 oxidants [72,73]. The flexible reactivity of Cys allows it to confer a range of 111 46 The nucleophilicity index (determined by highest occupied functions on enzyme catalytic mechanisms. A common role of Cys 112 47 molecular orbital and lowest unoccupied molecular orbital) of a in enzyme catalysis is as a soft nucleophile, acting as the electron 113 48 thiol also greatly contributes to its reactivity. Specifically, a donor for a wide range of substrates. In addition, many proteins 114 49 strongly acidic thiol with poor nucleophilicity may be similar in utilize Cys to facilitate protein–protein (e.g., intermolecular dis- 115 50 reactivity to a weakly acidic thiol with high nucleophilicity [74]. ulfide formation), protein–DNA (e.g., promoter binding), and 116 51 The reactive thiolate species is a “soft” (polarizable) nucleophile protein–lipid interactions (e.g., S-palmitoylation) [84–86]. Further- 117 52 and targets of Cys therefore tend to be “soft” (polarizable) more, a number of proteins use Cys to permit the inclusion or 118 53 electrophiles, e.g., 4-hydroxynonenal [74]. function of enzyme prosthetic groups such as heme, Fe, and Zn 119 54 Protein Cys are also highly effective in coordinating metal ion [78,87,88], discussed elsewhere in this review. Cys has direct 120 55 complexes in enzyme active sites and are widely used to regulate catalytic involvement in at least five of the six Enzyme Commis- 121 56 protein structure, maintain metal ion homeostasis, or sequester a sion classes of enzymes as briefly described here and summarized 122 57 metal toxicant [75,76]. Cys cooperates with inorganic sulfur in in Table 1. 123 58 4100 iron–sulfur (Fe–S) proteins, such as ferredoxins and Rieske Major subtypes of oxidoreductases involving Cys in enzyme 124 59 proteins [77]. Proximal Cys interaction with heme iron is a catalysis include disulfide exchange, peroxidation, thiyl radical 125 60 conserved feature of cytochromes P450 (CYP), which enables formation, thioesterification, and persulfidation (Fig. 4). Each of 126 61 monooxygenase activity [78]. Without the coordination of Cys234, these reactions begins with reduced Cys thiol(ate) and involves the 127 62 CYP 2B4 is converted to a form with NADPH oxidase activity [79]. formation of covalent bonds between Cys thiol and substrates 128 63 Cysteinyl sulfur (alone or in combination with histidine nitro- containing carbon, oxygen, sulfur, or phosphorus but not nitrogen, 129 64 gen) coordinates Zn2 þ in 3% of human gene products [80].Cuþ which bonds with catalytic Cys only as a means of enzyme 130 65 generally has higher affinity for Cys than other metals, and Cys- regulation. Many of these reactions transfer electrons to or from 131 66 rich metal ion chaperones (e.g., metallothionein and ferritin) NAD(P)H via Cys. Note that whereas the Cys thiol is oxidized in the 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 8 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Table 1 67 2 Cys in catalytic sites of enzymes. 68 3 69 Function Enzyme Substrate(s) Ref. 4 70 5 Oxidoreductase GR GSSG [251] 71 6 Grx GSS–Cys, Trx–SS [96,252] 72 7 GPx Lipid, inorganic peroxides [64] 73 GST Electrophiles, lipid peroxides [253] 8 74 TrxR Trx–SS [64] 9 Prx Peroxides [60] 75 10 GAPDH G3P [59] 76 11 ADH Aldehydes [93] 77 12 RNR (radical-mediated reduction) Ribonucleotides [63,254] 78 Transferase Thiosulfate sulfurtransferase, mercaptopyruvate sulfurtransferase H2S, S2O3, mercaptopyruvate [91] 13 Thymidylate synthase dUMP [255] 79 14 Hydrolase γ-Glu hydrolase γ-Glu peptides [256] 80 15 Caspases, cathepsins, calpains Polypeptides [94] 81 16 PTP, Cdc25 pTyr [176] 82 Isomerase PDIs RSSR-X-X-X-RSH [89] 17 83 Ligase Parkin E3 ubiquitin ligase Cys-His-Glu [257] 18 84 19 The sulfur of Cys functions in the active site of at least five of the six classes of enzymes classified by the Enzyme Commission. These include various types of oxidoreductases 85 20 linked to the NADP or NAD system and also radical-mediated ribonucleotide reduction. Cys functions in the transfer of sulfane in some transferases. Cys is present in the 86 fi fi 21 active sites of hydrolases, such as cysteine proteases and phosphatases. Protein disul de isomerases (PDIs) catalyze the rearrangement of thiol and disul de bonds without 87 any net oxidation or reduction. Some ligases have active-site Cys functioning in covalent bond formation. 22 88 23 89 24 initial step of all of these reactions, the substrates and catalytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ALDH, 90 25 cycle determine whether energy (often in the form of NADPH) is Cys thioacyl formation is preceded by a thiohemiacetal intermedi- 91 26 generated or consumed. ate that donates a hydride to NADP þ [93]. Similarly, Cys proteases 92 27 Protein Cys thiyl radicals are known to occur only in ribonucleo- attack the amide carbon peptide backbones at select residues (e.g., 93 28 tide reductases (RNRs), which are essential for DNA synthesis in all aspartate), causing cleavage of the amide bond [94]. Cys proteases 94 29 kingdoms of life [63]. All RNRs utilize a Cys thiyl radical to initiate are unlike Asp proteases in that they do not participate in classical 95 30 the catalytic cycle but differ in how it is generated: class I RNR acid–base hydrolysis. Rather, the Cys thioacyl ester provides a 96 31 utilizes a stable tyrosyl radical, class II utilizes adenosylcobalamin, good leaving group to facilitate hydrolysis and protease regenera- 97 32 and class III utilizes a glycyl radical generated by Fe–Sclustersand tion. Similarly, the Cys–SPO3 thiophosphate ester formed after 98 33 S-adenosylmethionine [63]. The Cys thiyl radical initiates reaction nucleophilic attack on phosphotyrosine in the protein tyrosine 99 34 by oxidizing C3 of ribonucleotide ribose. The carbon-centered phosphate (PTP) catalytic cycle is an excellent leaving group [95]. 100 35 radical shifts to C2, dehydrates, and undergoes one-electron reduc- However, as in acid–base catalysis, these reactions are supported 101 36 tion (again forming a radical) by a pair of additional Cys residues. by negatively charged side chains such as Asp and Glu, which 102 37 Finally, the radical oxidizes the initiating Cys, regenerating the coordinate the protonation of water, substrates, and Cys itself. 103 38 original thiyl radical and releasing deoxyribonucleotide [62]. 104 39 Disulfide- and peroxide-reducing functions of catalytic Cys 105 40 have been extensively reviewed. These reactions transfer electrons Protein Cys redox regulation 106 41 to substrate sulfur or oxygen in an oxidation state of 0 (RSOH 107 42 sulfur) or 1 (RSSR and peroxides). The result is formation of Trx and GSH systems 108 43 disulfide or RSOH on the nucleophilic Cys and the liberation of 109

44 RSH, H2O, or ROH from the substrate. The initiating Cys, typically Extensive research in the past decades shows that redox 110 45 oxidized to RSSR, must then be reduced to regenerate the enzyme. responses of the Cys proteome occurring at the extracellular and 111 46 These reactions make up the bulk of redox regulation by members cellular levels are critical in the initiation and progression of 112 47 of the TrxR and glutathione reductase (GR) antioxidant nodes. disease. Two major cellular redox regulation systems, dependent 113 48 These reactions are also found in important chaperone proteins, Trx and GSH, are central to the regulation of the Cys proteome. 114 49 such as protein-disulfide isomerase, which are classified by the These systems maintain a reduced redox environment in cells by 115 50 Enzyme Commission as isomerases because they rearrange protein catalyzing electron flux from NADPH through TrxR and GR to Trx 116 51 disulfides rather than cause net oxidation or reduction [89]. and GSH, respectively. In addition, mixed disulfides of GSH with 117 52 Sulfurtransferases are proteins that catalyze the formation, protein Cys (S-glutathionylation) can have a broad impact on 118 53 interconversion, and reactions of compounds containing sulfane protein function and disease [88]. S-glutathionyl groups are 119 54 sulfur atoms [90]. Although characterization of many is incomplete, removed by glutaredoxin (Grx) plus GSH. In some cases Grx may 120 55 Cys persulfide (Cys–SSH) is an important catalytic intermediate for transfer electrons from the GR–GSH node into the TrxR–Trx node 121 56 some [91]. The catalytic Cys is persulfidated by thiosulfate, hydro- by reducing Trx disulfide [96]. Numerous studies of Cys modifica- 122 57 gen sulfide, or mercaptopyruvate, creating a highly reactive sulfur tion and redox regulation provide evidence that redox-sensitive 123 58 donor, which produces low-molecular-weight sulfur metabolites regulatory Cys residues (see Fig. 5 legend) play critical roles in cell 124

59 such as thiocyanate (SCN) and methanethiol [92].H2Scanbe signaling and control as part of a redox network structure. These 125 60 liberated from the Cys persulfide as well as the original persulfide systems are involved in multiple signaling pathways through 126 61 donor [92].Aspersulfidation is also a putative peptidyl Cys direct or indirect interactions with a variety of target proteins 127 62 regulatory mechanism, it is covered elsewhere in this review. controlled by the GSH and Trx redox systems (Fig. 5). Proteins and 128 63 Catalytic Cys-formed thioesters fall into two classes: thioacyl associated protein networks are often selectively regulated by Trx 129 64 esters (S–CR bond) and thiophosphate esters (S–PO3 bond). or GSH systems but also commonly regulated by both GSH and Trx 130 65 Thioacyl esters are intermediates of aldehyde dehydrogenases systems, e.g., S-glutathionylation in C374 and S-nitrosylation in C285 131 66 (ALDHs) and cysteine protease catalytic cycles. In the case of of actin (Fig. 5). 132

1 67 2 68 3 69 4 70 5 71 6 72 7 73 8 74 9 75 10 76 11 77 12 78 13 79 14 80 15 81 16 82 17 83 18 84 19 85 20 86 21 87 22 88 23 89 24 90 25 91 26 92 27 93 28 94 29 95 30 96 31 97 32 Fig. 5. Cys proteome networks regulated by the Trx and GSH systems. Accumulating evidence shows that redox networks exist in different subcellular compartments 98 supported by NADPH as a reductant functioning in opposition to oxidation by H2O2 and other oxidants. Trx is the most common intermediary reductant, with a smaller 33 number of proteins supported by GSH. Cysteine and cystine constitute the most abundant extracellular low-molecular-weight thiol/disulfide couple, apparently functioning 99 34 in the regulation of protein Cys on the extracellular surface. References provide detailed information on the interactions of Cys proteins and functional Cys residues 100 35 illustrated here: TrxR–actin [110], Trx-dependent regulation of proteins [139], Ref1–AP1 [258], TrxR/AP1 [259], PDGFR [260], xCT [261], PTEN–Trx [262,263], PTEN–GSH 101 – – – – – – – – – 36 [264], ASK1 Trx2 [264], GSTp1 Prx6 [265], GAPDH GSH [266,267],ND GSH [268], p53 Trx [269], Trx2 Prx3 [270], Srx Prx1 and Srx Prx3 [271,272], Grx2 Prx3 [273], 102 Grx2–Prx3–Trx2 [274], PDI–UPR sensor [275], Era–PDI [276], PDI–TF [277], Ero-α-PDI and GSH [278], GPx7–ER [279], Nrf2 regulatory Cys residues [280], Srx functional Cys 37 residues [281]. 103 38 104 39 105 40 As indicated above, mass spectrometry-based redox proteomics susceptible to multiple posttranslational modifications in Cys resi- 106 41 together with bioinformatics enables identification of redox- dues including reversible thiol oxidation (Cys217, 257, 272, 285, 374) 107 42 sensitive Cys residues, proteins, and networks of the Cys proteome [102–104], glutathionylation (Cys10,217,257,374) [105–107],and 108 43 in association with cellular conditions [6,8,49]. Studies in yeast nitrosylation (Cys217, 257, 285, 374) [108–110]. Oxidation disrupts actin 109 44 showed that the redox proteome was preserved in cells deficient in dynamics, e.g., filamentous (assembled) and monomeric (disas- 110 45 the GSH system but not in cells with compromised Trx systems. sembled) actin formation [111,112]. Glutathionylation/deglutathio- 111 46 These results show that the Trx system is most central in regulation nylation of actin, regulated by the concentration of GSH and Grx 112 47 of the yeast Cys proteome [97]. In mammalian cells, responses of activity, is also associated with actin dynamics [113–115]. 113 48 the Cys proteome were tested by selectively disrupting the Trx- and In addition to actin itself, a cohort of actin-associated cytoske- 114 49 GSH-dependent systems with inhibitors, auranofin for thioredoxin letal proteins controls actin dynamics. Oxidation of Cys139 and 115 50 reductase and buthionine sulfoximine for γ-glutamylcysteine Cys147 in actin-binding protein cofilin stimulates apoptotic signal- 116 51 synthetase, the first enzyme in the GSH synthesis pathway. Results ing as a consequence of inhibiting interaction with actin as well as 117 52 showed that the Trx and GSH systems support distinct functions in increasing mitochondrial translocation [116], and cofilin oxidation 118 53 cellular redox control by controlling different subsets of protein Cys. impairs cytoskeletal function in T cells [117]. Furthermore, destrin, 119 54 Consistent with the yeast findings, the Trx system regulated a much acofilin family member, is an actin depolymerizing factor regu- 120 55 broader range of proteins than the GSH system [49]. However, GSH lated by Cys redox status. Destrin nuclear translocation was 121 56 and Grx can act as a backup to the TrxR–Trx system to promote the associated with redox disruption of the actin cytoskeleton pro- 122 57 reduction of Trx during aurothioglucose exposure [96];theextent teome [8]. Actin cytoskeleton proteins were also susceptible to 123 58 to which this alters redox proteomic effects of TrxR inhibition is oxidation during inflammatory signaling associated with mito- 124 59 unclear. Examples from studies with selective inhibition of TrxR and chondrial oxidation [6,22]. Redox-dependent control of actin 125 60 GSH systems are available [49,98]. dynamics was observed from the interaction of actin with the 126 61 Trx system, Trx1 and TrxR1 [110,118,119]. Release of Trx1 from 127 62 Regulation of actin cytoskeleton proteins actin resulted in degradation of Trx1, which then stimulated cell 128 63 death [119]. Denitrosylation of actin by TrxR via focal adhesion 129 64 Cytoskeleton remodeling through actin dynamics regulates vital kinase restored cellular function [110]. 130 65 biological processes, e.g., cell morphogenesis, migration and growth, Extensive knowledge of the redox modulation of the cytoske- 131 66 membrane trafficking, exocytosis, and endocytosis [99–101].Actinis leton by the GSH and Trx systems shows the central importance of 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 10 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 redox control in cell function. The data demonstrate that GSH and high-glucose exposure caused increased reactive oxygen species 67 2 Trx do not indiscriminately “equilibrate” the peptidyl Cys environ- generation and caspase-3 activation [140]. The pathological role of 68 3 ment to a consistent reduction potential but sensitively and Txnip was revealed with the release of Txnip from oxidized Trx1 to 69 4 selectively maintain discrete nonequilibrium dynamics for indivi- stimulate inflammation [141] and cell death [140]. Similarly, the 70 5 dual proteins and protein networks. The following examples reduced form of Trx1 binds to apoptosis signal-regulating kinase-1 71 6 further illustrate the use of kinetically controlled systems in (ASK-1) and inhibits its activity to prevent stress- and cytokine- 72 7 nuclear protein trafficking and in maintenance of the integrity of induced cell death [142]. Activation of ASK-1 is stimulated under 73 8 the genome. Recent reviews are also available on the redox stress signaling after dissociation with Trx1 [142] and plays a key 74 9 processing in the secretory pathway [33,34,120] and in mitochon- role in apoptosis with increased DNA damage [143,144]. Direct 75 10 drial import [35,36]. Relatively less information is available on interaction of Trx with Txnip (Cys247) and ASK-1 (Cys250) via 76 11 vectorial redox processing during endocytosis and proteolysis in reduced Cys32 (Cys30 of Trx2) supports a key role for the Trx 77 12 lysosomes, but evidence is available suggesting that analogous thiol–disulfide redox regulatory function against oxidative stress- 78 13 systems are present [121,122]. mediated DNA damage and cell death signaling [141]. 79 14 Trx1 also plays a key role in DNA repair mechanisms by 80 15 Cys proteome in nuclear protein trafficking regulating the base excision repair pathway via the p53 down- 81 16 stream gene, growth arrest, and DNA-damage-inducible protein 82 17 Redox-dependent transcription has been known for many 45α and its interaction with apurinic/apyrimidinic endonuclease 1 83 18 years, with divergent mechanisms involved for AP-1, Nrf2, NF- (also called redox factor (Ref)-1) [145]. This mechanism is regu- 84 19 κB, glucocorticoid receptor, and other systems [38,123–126]. Both lated by the interaction between Trx1, Ref-1, and redox-sensitive 85 20 Trx1 and its inhibitory binding partner Txnip are translocated into p53 transcription factor [146,147]. TrxR–Trx1 also interacts with 86 21 nuclei to regulate the Cys proteome. For instance, nuclear accu- Ref-1, and this interaction controls p53 activity supporting the 87 22 mulation of Txnip was detected when cells were treated with the Trx1 redox system in modulation of redox-sensitive transcription 88 23 anti-cancer histone deacetylase inhibitor suberoylanilide hydro- factors [148]. Moreover, Trx1 protection against cell death was 89 24 xamic acid [127]. Rch1 (importin-α) is required for Txnip nuclear shown to occur in growth regulation in B cell chronic lymphocytic 90 25 translocation and growth control, and this interaction is controlled leukemia by controlling the interaction of protein disulfide iso- 91 26 by a Trx-dependent redox system [128]. Interestingly, Trx1 is also merase with tumor necrosis factor receptor [149]. This finding 92 27 translocated into nuclei, and import requires karyopherin-α additionally supports the beneficial role of Trx1 in survival, 93 28 (importin-α) and nitric oxide (NO). Site-directed mutagenesis of perhaps via activation of NF-κB transcription factor [147]. 94 29 Lys81, 82 of Trx1 showed that these residues are important for the 95 30 translocation of Trx1 into nuclei [129], suggesting that the protein 96 Oxidation of the Cys proteome by H O and derivative species 31 translocation mechanism is complex, requiring localized signal 2 2 97 32 motifs in addition to redox-dependent regulation. 98 Measurements of bulk protein Cys in mouse liver and HT29 cell 33 In addition to enhanced Trx1 translocation to nuclei, transport 99 culture show that 92 and 88%, respectively, are in the thiol form 34 and translocation of a wide range of proteins are regulated in a 100 [6]. With procedures to minimize artifacts in mass spectrometry 35 redox-dependent manner. For instance, oxidative stress stimulates 101 measurements, similar mean (84.3%) and median (87.7%) values 36 nuclear translocation of SBP2 (selenocysteine insertions sequence 102 were obtained for HT29 cells in culture. Specific protein Cys had a 37 binding protein 2) via oxidation of Cys within a redox-sensitive Cys- 103 distribution of steady-state oxidation ranging from less than 8% to 38 rich domain. Reversal by Trx1 or Grx suggests that the redox state of 104 greater than 99% reduced. These observations raise key questions 39 SBP2 is critical to compartmental translocation and selenoprotein 105 about the oxidants involved and the underlying enzymatic and 40 synthesis [130]. In bronchopneumonia, protein S-glutathionylation 106 nonenzymatic mechanisms contributing to steady-state oxidation. 41 significantly colocalized with Fas ligand, and Grx1-knockout mice 107 The balance of oxidation and reduction of the Cys redox 42 had increased Fas S-glutathionylation and improved murine lung 108 proteome are governed by the thermodynamics, selectivity, and 43 infection survival [131]. Mass spectrometry-based redox proteomics 109 kinetics of various peptidyl Cys. As indicated above, details of the 44 also showed functional redox networks of nuclear import proteins 110 reduction systems are becoming clear. In contrast, the fine-tuned 45 (Fig. 3) [6]. Additionally, targeted expression of nuclear Trx1, stimula- 111 oxidative signaling has been ascribed extensively to H2O2,a 46 tion of nuclear H O production with nuclear-targeted D-amino acid 112 2 2 species that is relatively unreactive with most Cys thiols. At least 47 oxidase, and enhanced nuclear peroxidase activity by expression of 113 three factors influence the specificity of oxidation, i.e., localized 48 nuclear-targeted Prx1 further show distinct redox control in nuclei 114 high-activity generation of H O , catalytic mediators of oxidation, 49 [9,45]. Thus, multiple approaches show that the nuclear Cys pro- 2 2 115 and generation of alternative low-molecular-weight chemical 50 teome is controlled in a semiautonomous manner by transport and 116 oxidants. Enzymes with high reactivity with H O (e.g., members 51 compartment-specificregulation. 2 2 117 of the Prx and GSH S-transferase (GST) families) may act as special 52 118 carriers of the oxidative signal to interacting proteins [150]. 53 Nuclear functions of the Cys proteome 119 Alternatively, H O is used to generate other oxidants with altered 54 2 2 120 kinetics (e.g., hypohalous acids) [151], which in turn can form 55 The dynamic nature and functional importance of 121 further derivative oxidizing species (e.g., haloamines). These 56 compartment-specific control of the Cys proteome are further 122 processes support speciation of an H O oxidative signal into 57 illustrated by Trx-dependent protection against cellular stresses 2 2 123 various paths, which regulate not only the fate of H O but also 58 associated with growth inhibition and cell death induced by H O , 2 2 124 2 2 the effect of its oxidative signal. 59 TNFα, and chemotherapeutic agents [132–134]. Trx-interacting 125 60 protein (Txnip/Trx binding protein/vitamin D3-upregulated pro- 126

61 tein) directly binds to Trx1 by forming a disulﬁde bond between H2O2 127 247 32 62 Cys of Txnip and catalytically active Cys residues of Trx (Cys Although O2 is present at 5 orders of magnitude higher 128 35 63 and Cys ), inhibiting the redox activity of Trx [135,136]. The concentration than H2O2 in aerobic organisms, H2O2 is usually 129 64 complex of Txnip-bound Trx contributes to the pathogenesis of considered the most central oxidant in the maintenance of steady- 130

65 multiple diseases including diabetes, autoimmune disease, state oxidation of the Cys proteome. H2O2 is a product of at 131 66 and cancer [137–139]. Upregulation of Txnip under sustained least 31 human enzymes (Table 2) and has been measured in 132

1 Table 2 67 2 Hydrogen peroxide-generating human enzymes. 68 3 69 Name Protein Gene Accession No. Location Product 4 70

5 Aldehyde oxidase ADO AOX1 Q06278 C H2O2 71 ﬂ 6 Amine oxidase ( avin-containing) A AOFA MAOA P21397 M H2O2 72 ﬂ 7 Amine oxidase ( avin-containing) B AOFB MAOB P27338 M H2O2 73 D-Amino acid oxidase OXDA DAO P14920 Px H O 8 2 2 74 L-Amino acid oxidase OXLA IL4I1 Q96RQ9 L H2O2

9 D-Aspartate oxidase OXDD DDO Q99489 Px H2O2 75

10 Amiloride-sensitive amino oxidase (copper containing ABP1 ABP1 P19801 S H2O2 76 11 FAD-linked sulfhydryl oxidase ALR GFER P55789 C, M, S H2O2 77 12 Hydroxyacid oxidase 1 HAOX1 HAO1 Q9UJM8 Px H2O2 78 Hydroxyacid oxidase 2 HAOX2 HAO2 Q9NYQ3 Px H O 13 2 2 79 Membrane primary amine oxidase AOC3 AOC3 Q16853 PM H2O2 1 14 Peroxisomal N -acetylspermine/spermidine oxidase PAOX PAOX Q6QHF9 Px, C H2O2 80 15 Peroxisomal acyl-coenzyme A oxidase 1 ACOX1 ACOX1 Q15067 Px H2O2 81 16 Peroxisomal acyl-coenzyme A oxidase 3 ACOX3 ACOX3 Q15254 Px H2O2 82 Peroxisomal sarcosine oxidase SOX PIPOX Q9P0Z9 Px H O 17 2 2 83 Prenylcysteine oxidase 1 PCYOX PCYOX1 Q9UHG3 L H2O2 18 84 Protein-lysine 6-oxidase LYOX LOX P28300 S H2O2

19 Pyridoxine-5-phosphate oxidase PNPO PNPO Q9NVS9 C H2O2 85 ﬁ 20 Retina-speci c copper amine oxidase AOC2 AOC2 O75106 PM, C H2O2 86 21 Spermine oxidase SMOX SMOX Q9NWM0 C, N H2O2 87 Sulfhydryl oxidase 1 QSOX1 QSOX1 O00391 G H O 22 2 2 88 Sulfhydryl oxidase 2 QSOX2 QSOX2 Q6ZRP7 N, PM, S H2O2

23 Sulﬁte oxidase, mitochondrial SUOX SUOX P51687 M H2O2 89 24 Xanthine dehydrogenase/oxidase XDH XDH P47989 C, Px, S H2O2 90 25 NADPH oxidase 1 NOX1 NOX1 Q9Y5S8 PM O2 91 phox Cytochrome b245 heavy chain (gp91 ) CY24B (NOX2) CYBB P04839 PM O2 26 92 NADPH oxidase 3 NOX3 NOX3 Q9HBY0 PM O2 27 93 NADPH oxidase 4 NOX4 NOX4 Q9NPH5 E, PM, N H2O2 28 NADPH oxidase 5 NOX5 NOX5 Q96PH1 E O2 94 29 Dual oxidase 1 DUOX1 DUOX1 Q9NRD9 PM H2O2 95 30 Dual oxidase 2 DUOX2 DUOX2 Q9NRD8 PM H2O2 96 31 97 Enzymes producing superoxide anion (O2 ) are included because O2 is commonly dismutated to produce H2O2. Abbreviations used for subcellular localization: C, 32 cytoplasm; E, endoplasmic reticulum; G, Golgi; L, lysosome; M, mitochondria; N, nucleus; PM, plasma membrane; Px, peroxisome; S, secreted. Other proteins are likely to 98 33 produce O2 or H2O2 but not included owing to limited characterization. Enzymes generating lipid peroxides are not included. 99 34 100 35 mammalian tissues at concentrations in the range of 1 to 50 nM and GSH correctly reproduced experimentally measured oxidation 101

36 [152–155]. Measurements of H2O2 based upon steady-state proﬁles for the GSH and Trx redox couples [159]. The model 102 37 kinetics of endogenous catalase in perfused rat liver developed suggested that approximately 10% of intracellular protein thiols 103

38 by Sies and Chance [156] may be the most reliable tissue react with H2O2 at substantial rates and predicted an unequal 104 39 measurements available because there are no possible artifacts distribution of the intracellular antioxidative burden between Trx- 105 40 due to transfection or calibration of imaging reagents. Catalase dependent and GSH-dependent antioxidant pathways, with the 106 41 concentration is too low to support this type of measurement in former contributing the majority of the cellular antioxidant 107 42 most cells and tissues, however, and more sensitive fluorescent defense due to Prx and protein disulfides. Importantly, if enzymes 108 43 probes have been widely used. Some, such as dichlorofluorescein with peroxidase activity, such as the Prx and GST families, use 109 44 diacetate, are nonspecific and require caution when evaluating cell protein Cys as a direct reductant, supported by Trx or GSH systems 110

45 and tissue H2O2 production. Other probes can also be nonspeciﬁc, to reduce the protein Cys back to thiol form, then a complete 111 46 insensitive to rapid ﬂux changes, or misleading owing to redis- enzymatic system exists to maintain the dynamic Cys proteome 112 47 tribution of probes within cells [157]. In contrast to high reactivity [7]. The overall organizational structure is one in which the Cys 113 þ 48 of H2O2 with peroxiredoxins, GSH peroxidases, and catalase, H2O2 proteome is not collectively equilibrated with NADP /NADPH, 114 49 reacts quite slowly with the majority of thiols. For low-molecular- with O2/H2O, or at any single redox potential. Instead, a continuum 115 50 weight thiolates, the rate constant of reaction with H2O2 is about of kinetically limited nonequilibrium steady states exists for 116 51 20 M1 s1 but the effective rate constant for total thiol (thiol þ protein Cys, varying in abundance and spatial and temporal 117 52 thiolate) is o5M1 s1 [158]. Using 5 M1 s1, total protein Cys distributions [40]. 118

53 at 20 mM, and H2O2 at 10 nM, noncatalytic oxidation of protein The system is dynamic and responds to changes in H2O2 during 119 54 Cys by H2O2 in cells occurs at 0.0003% per minute. This rate is too inﬂammation and signaling for proliferation, cell migration, or 120 55 slow to maintain the steady-state oxidation as indicated above. host defense [151,160–162]. Research shows substantial circadian 121 56 Multiple alternatives exist that could account for higher steady- variation [163], changes during development [164], and spatio- 122 57 state oxidation, e.g., a fraction of protein Cys with higher rate temporal temporal changes during wound healing [165]. The 123

58 constants, more reactive oxidants derived from H2O2, or catalytic oxidative second messenger [166] with localized generation [51] 124 59 oxidation of protein Cys by peroxiredoxins, glutathione S-trans- is ampliﬁed by a “ﬂoodgate” mechanism [167] involving excessive 125

60 ferases, or other enzymes in the presence of H2O2. Studies of oxidation of Prx and Trx1 (see below) [168]. This mechanism could 126 61 various protein Cys show that rate constants for the reaction with alter redox signaling by diverting H2O2 flux to oxidize other 127 62 H2O2 vary over more than 6 orders of magnitude [57]. A network proteins or, perhaps more likely, support enzymatic oxidation or 128 63 model of H2O2 clearance with 24 elements and 28 kinetic para- other redox-activated signaling mechanism [166]. 129 64 meters including pseudo-enzymatic oxidative turnover of protein Earlier analytic methods detected only protein disulfides; 130 65 thiols; the enzymatic actions of catalase, glutathione peroxidase, development of reagents for trapping RSO, however, showed 131 66 peroxiredoxin, and glutaredoxin; and the redox reactions of Trx that RSO is the predominant modification of the Cys proteome 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 12 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 [25] and central to oxidative Cys regulation (Fig. 4). For instance, activates Nrf2 and MAPK signaling in airway epithelia [189].A 67 2 RSO in multiple PTPs is associated with inhibition of CD8 þ T cell potential limitation for specific Cys oxidation is that HOCl and 68 3 activation [169], and formation of RSO in IQGAP1 plays a direct HOBr have high and indiscriminate reactivity with most Cys [190] 69 4 role in endothelial cell migration and angiogenesis [170]. Recent and also react with non-Cys residues, e.g., Met and primary amines 70 5 bioinformatics analysis using a dimedone-tagged click chemistry [191,192]. On the other hand, conversion of hypohalous acids into 71 6 probe has revealed multiple peptidyl Cys with basal RSO mod- less reactive haloamines (e.g., RNHCl) may confer specificity. For 72 7 ification, including Cys18 of SIRT6 and multiple Cys of pyruvate instance, taurine chloramine oxidizes Cys282 of creatine kinase 73 8 kinase M2 [26]. RSO were detected in solvent-accessible peptidyl with threefold increased selectivity compared to HOCl [193]. 74 9 Cys from proteins associated with most subcellular compartments Hypothiocyanous acid (HOSCN) is a related oxidant that is 75 10 and included enzymes regulating phosphorylation, acetylation, constantly produced from endogenous SCN, lactoperoxidase (LPO), 76 11 and ubiquitylation [26]. Peptidyl Cys with detectible RSO mod- and H2O2 in human secretory fluids (e.g., saliva, airway lining fluid, 77 12 ification were often associated with Glu and Lys residues and less tears, and milk) [194–196]. HOSCN has been studied extensively in 78 13 often with other Cys residues [26]. saliva and is maintained at concentrations greater than fivefold 79 14 Oxidation of RSO to RSO2 (sulfinate) in peroxiredoxin has a more abundant than the precursor H2O2 [197]. In addition to 80 15 special role in signaling because this oxidation inhibits peroxidase enzymatic production by LPO, MPO, and other peroxidases 81 16 activity, redirects reducing equivalents from the Trx system away [186,282], HOSCN is produced by the direct reduction of HOCl or 82 17 from Prx2, and changes protein Cys redox regulation [171]. This HOBr by SCN, a reaction that competes effectively with physio- 83 18 process is termed “hyperoxidation,” and detailed studies with logic concentrations of GSH [52,198]. HOSCN reacts with protein 84 19 Cys50 of Prx2 [172], including thermodynamics and contributions Cys to form RSO and SCN [52,198]. 85 20 of proximal amino acids [60,73,173,174] are available to describe The most critical aspect of HOSCN is that it is significantly less 86

21 the underlying mechanism. Ultimately, hyperoxidation of Prx2 reactive than H2O2, HOCl, or HOBr [173,199–201], which suggests 87 22 diverts H2O2 to other antioxidant systems. Such diversion alters greater specificity for Cys proteomic regulation. Support for this 88 23 redox signaling by changing elements of the Cys proteome that possibility is limited, but HOSCN, and not HOCl or HOBr, induces 89 24 react with oxidant [166]. Furthermore, hyperoxidation of Prx2 is cell signaling by reaction with serum factors [202,203]. HOSCN 90 25 biochemically regulated, as sulfiredoxin reduces Prx2 Cys sulfinate selectively targets salivary GSTP, but not GSTA or GSTM [204]. 91 26 back to the sulfenate form, which may be reduced by Trx [54]. Altered redox signaling of HOSCN may be reflected in altered 92 27 The Cys proteome includes sulfinic and sulfonic acids, as well as cellular responses to this oxidant instead of others, e.g., removal of 93 28 series of sulfenamides, sulfinamides, and sulfonamides. With the H2O2 and HOCl by SCN protects against mammalian cell death 94 29 exception of sulfinic acid and possible precursor RSONR2 [175] in and decreases inflammation [179,205]. Furthermore, mammalian 95 30 peroxiredoxins, there is little evidence that these represent func- innate immunity exploits specificity of HOSCN reactivity to place 96 31 tional variations. Similarly, cyclic sulfonamide (isothiazolidinone) selective oxidative pressure on microbes [206]. The biochemical 97 32 structures are formed from the catalytic Cys of certain phospha- and physiological effects of SCN and HOSCN have been recently 98 33 tases, e.g., Cys215 of PTP1B [175,176], but the functional and reviewed [207]. 99 34 physiologic relevance of these is unclear. Cysteinyl sulfur and 100 35 amine interaction also provides a means to cross-link molecules S-nitrosylation 101 36 by strong oxidants, observed in model cysteinyl peptides and GSH 102 37 and in vivo [177–179]. Thiosulfinates and thiosulfonates are Nitric oxide is a precursor for S-nitrosylation of the Cys 103 38 documented in thiol chemistry [52], and thiosulfinate has been proteome, governed by multiple reaction pathways, e.g., metal 104 39 found as an intermediate of the sulfiredoxin (Srx) Cys99 reduction catalysis of NOþ transfer, with the ultimate action being formation 105 40 of Prx2 sulfinic phosphoryl ester [180]. Thus, a diverse range of of protein S-nitrosocysteine (RSNO). S-nitrosylation is regulated by 106 41 oxidized sulfur and sulfamine modifications of the Cys proteome the GSH and Trx systems, which reduce RSNO [208,209]. Trans-S- 107 42 can occur, but only a very small number have been characterized nitrosylation is important in NO signaling and as a negative 108 43 in terms of biologic functions. feedback mechanism for NO synthases and effector proteins 109 44 [209]. SERCA is nitrosylated at Cys344 and Cys349 after ONOO 110 45 exposure, which enhances subsequent protein glutathionylation 111

46 Oxidant species derived from H2O2 [210]. The oxidation of critical SERCA Cys residues, e.g., S- 112 47 The Cys proteome is subject to oxidation by peroxide-derived nitrosylation and S-glutathionylation, may be a guanylate 113 48 oxidants, some of which have similar thermodynamic potential cyclase-independent means to alter vascular tone [211]. Skeletal 114 2 þ 3635 49 but lesser kinetic limitations than H2O2. Hypohalous acids (hypo- muscle ryanodine receptor/Ca -release channel Cys is regu- 115 50 chlorous acid, HOCl; hypobromous acid, HOBr) are strong oxidants lated by S-nitrosylation, whereas other Cys mediate redox sensi- 116 51 generated by phagocytic myeloperoxidase (MPO) during inflam- tivity through SNO-independent means [209,212,213]. Several 117 52 mation and widely recognized as dual agents of host defense and mitochondrial metabolic enzymes are targets of S-nitrosylation 118 53 host injury [151,181]. HOCl and HOBr rapidly react with thiolates [214]. Thus, S-nitrosylation represents a series of modifications of 119 54 by transfer of Clþ or Brþ [52]. The resulting sulfenyl halide is the Cys proteome, which can be considered parallel to the 120 55 unstable and rapidly hydrolyzes to release Cl or Br and RSO oxidative systems in providing a network of regulatory elements 121 56 [182]. Similarly, eosinophil peroxidase is released in allergic events influencing diverse aspects of cell function. 122 57 and generates HOBr, but little or no HOCl at physiologic pH 123 58 [183,184]. Persulfidation 124 59 Low-level generation of HOCl and HOBr can also occur in 125 60 tissues without inflammation via the activity of peroxidasins (also Persulfidation, also termed S-sulfhydration [215,216], is another 126 61 known as vascular peroxidases), which participate in collagen mechanism for modification of the Cys proteome that can operate 127 62 cross-linkage and are essential for tissue development [185–188]. in parallel with the oxidative and S-nitrosylation systems. The 128 63 However, the impact of constitutive HOCl and HOBr generation on protein persulfide (RSS ) is derived from H2S generated from Cys 129 64 Cys proteomic regulation has not been extensively characterized. via intestinal microbes, mercaptopyruvate sulfotransferase, 130 65 In principle, these oxidants could oxidize selected subsets of the cystathionine β-synthase (CBS), and cystathionine γ-lyase (CGL) 131 66 Cys proteome as suggested by the finding that HOCl independently [217,218]. At physiologic pH, H2S is principally ionized (HS ) and 132

1 reacts with a disulﬁde to produce RSS and release a thiol. For As described above, the Cys proteome is part of an adaptive 67 2 instance, GSH persulﬁde (GSS) is formed from the reaction of interface of an organism with its environment, and covalent 68

3 GSSG with H2S [219]. The sulfur added to the thiol in the persulﬁde modiﬁcations of the Cys proteome are useful to characterize 69 4 moiety is present at the oxidation state of elemental sulfur environmental exposures to pollutants, dietary metabolites, drug 70

5 (sulfane), which commonly exists as octasulfane (S8). Cysteinyl metabolites, and endogenous toxicants [237]. In human serum 71 6 polysulfides have been documented in crystal structures of bacter- albumin, 66 adducts of Cys34 have been detected [238]. Similarly 72 7 ial sulfide quinone oxidoreductases [215], and free polysulfides in drug metabolism, quinone structures from metabolites of 73

8 (H2Sn) in brain tissue can persulfidate multiple protein Cys targets acetaminophen (APAP), diclofenac, and related drugs have distinct 74 9 [220]. Sulfane sulfur can be transferred between protein thiols and 1,4-Michael addition reaction with thiols to form a covalently 75 10 low-molecular-weight thiols (trans-persulfidation or trans-sulfa- modified Cys34 of albumin. The APAP metabolite N-acetyl-p- 76 11 nation). The extent of persulfide modification of proteins is benzoquinone imine (NAPQI) targets peptidyl Cys of essential 77 12 currently unknown [221,222]. metabolic and regulatory proteins, including TrxR1, which is 78 13 Persulfide formation generally interferes with normal protein covalently modified by NAPQI at residues Cys59 (GR-homologous 79 14 Cys reactivity in much the same way as S-nitrosylation or active residue) and Cys497 and Sec498 (vicinal thiol–selenol pair), 80 15 sulfenylation [223] but differs in that the highly nucleophilic and associates these modifications with loss of TrxR activity in 81 16 auxiliary sulfane is more reactive toward electrophiles [224]. APAP-overdose mouse liver [239]. Other modifications also occur, 82 149 17 Inhibition of H2S generation leads to mitochondrial dysfunction such as GAPDH Cys modification by NAPQI [240]. 83 18 and increased O2 flux, suggesting a role for H2S in regulation of 84 19 mitochondrial function [225]. Persulfidation of 39 proteins, includ- 85 20 ing GAPDH Cys150, was detected by mass spectrometry [226]. Integrated function of the Cys proteome 86 21 Persulfidation of GAPDH Cys150 by treatment with NaSH increased 87 The proteome is the embodiment of the genome, serving to 22 enzyme activity, with an EC50 of 15 mM; GAPDH is also an S- 88 translate the genetic and epigenetic systems into function and 23 nitrosylation target, highlighting a potential for H2S/NO cross talk 89 24 [226]. After TNFα stimulation of CGL, increased persulfidation of interaction with the environment. The series of posttranslational 90 fi 25 p65 Cys38 regulated its DNA binding and antiapoptotic function modi cations of the translated proteome, collectively termed the 91 26 [227]. The C59S mutant of P66Shc is resistant to antiphosphoryla- epiproteome [241], dramatically expands the structural variation 92 and range of reactivity beyond that found in the translated 27 tion effects of H2S or CBS overexpression, implicating regulation by 93 proteome. Perhaps most importantly, bi- and multiphasic switches 28 persulfidation [228].H2S also regulates endothelial NO synthase 94 29 (NOS) activity by persulfidation of Cys443, which promotes activity of the Cys proteome provide organizational hubs to broadly 95 30 of the NOS in opposition to NO binding [216]. control and integrate complex biologic functions. 96 31 Although not fully elucidated, recent studies have begun to 97 fi 32 de ne multiphasic switching by the Cys proteome through detec- 98 fi 33 Alkylation tion of proteins with S-nitrosylation, S-glutathionylation, persul - 99 34 dation, and sequential oxygenation [242]. Some Cys residues can 100 fi 35 Many modifications of the Cys proteome are irreversible under undergo multiple modi cations, potentially providing pathway 101 374 36 normal circumstances in vivo. Some of these, such as modification switches to change biologic function. For instance, Cys of actin 102 37 by endogenously derived reactive lipids, function to provide a can be nitrosylated as well as glutathionylated [107,109]. However, 103 38 response to stress [229]. Reactive aldehydes make up a highly comparison of sulfenylated Cys residues in 193 proteins with 104 fi fi 39 significant and well-recognized fraction of permanent Cys adduc- previously identi ed targets of glutathionylation, disul de forma- 105 40 tion [230]. Many α/β-unsaturated aldehydes such as 4- tion, and S-nitrosylation shows minimal overlap [25]. These results 106 fi 41 hydroxynonenal (4-HNE) are “soft” electrophiles that react readily suggest that different Cys modi cations are selectively targeted 107 42 with soft nucleophiles and are chiefly regulated in vivo by Cys toward different subsets of the Cys proteome rather than serving 108 43 thiolate [74,174]. 4-HNE reacts with Cys residues of many impor- as multidirectional switches. Expansion of this pioneering 109 44 tant proteins. The thioredoxin system, including TrxR (Cys496 research is needed to provide an understanding of the integrated 110 45 along with Sec497 [65]) and Trx (Cys32, Cys35), is very sensitive to function of the Cys proteome. 111 46 inhibition by 4-HNE [231]. Similarly, the catalytic and modifying At the same time, multiple omics platforms are being used to 112 47 subunits of Glu–Cys ligase (GCLC/M) are targets of 4-HNE (GCLC translate basic science into medical applications [243]. Compar- 113 48 Cys553, GCLM Cys35) [232]. 4-HNE also induces antioxidant genes ison to genomics and transcriptomics illustrates current limita- 114 49 (e.g., TrxR1, heme oxygenase-1) through the induction of the Nrf2 tions at the level of the Cys proteome. Unlike the genome and 115 50 promoter by modifying Cys288 of Keap1 [58,233]. Conversely, 4- epigenome, for which systematic methods provide nearly com- 116 51 HNE inhibits the release of NF-κB by modifying Cys179 of IKK-β prehensive coverage, only a few percent of the Cys in the 117 52 [234]. Together, these examples emphasize that irreversible mod- proteome are captured in the best mass spectrometry proteomics 118 – 53 ification of Cys in signaling proteins is functionally important to analyses. In studies of gene expression redox proteomic interac- 119 54 respond to electrophile exposures [229]. tions [244] common pathway responses were observed, but the 120 55 Selectivity of protein Cys to electrophilic modification has been data were too sparse to allow direct correlation. On the other 121 56 demonstrated by comparisons of proteins modified by iodoacetyl hand, more targeted analysis of redox proteomic associations with 122 57 and maleimido reagents [235]. For instance, of 1255 distinct Cys- metabolomics in isolated mitochondria provided direct correla- 123 58 containing peptides from isolated mitochondria, the electrophiles tions [24]. The data indicate that with improved coverage of the 124 59 overlapped only 35% in Cys adduction and had distinctly different proteome, integrated omics research will enable mechanistic 125 60 effects on cytotoxicity and apoptosis. Similarly in nuclear–cytosolic understanding at the level of systems biology, i.e., directly linking 126 61 fractions, there was only 14% peptidyl Cys overlap between the changes in the Cys proteome with altered biologic function. 127 62 electrophiles [236]. The targeted residues represented proteins 128 63 involved in a variety of central cellular pathways including Trx2, Perspective and future directions 129 64 heat shock proteins, permeability transition pore complex, 130 65 nuclear/cytosolic cytoskeleton, DNA replication/repair, protein A conceptual framework is emerging for elucidation of the Cys 131 66 synthesis, and RNA processing [236]. proteome as a central omics layer within the continuum of 132

Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 14 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 16 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i 18 Y.-M. Go et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Please cite this article as: Go, Y-M; et al. The cysteine proteome. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j. freeradbiomed.2015.03.022i