Thiol-Disulfide Exchange Reactions in the Mammalian Extracellular Environment.

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Annu Rev Chem Biomol Eng. Author manuscript; available in PMC 2017 June 07. Published in final edited form as: Annu Rev Chem Biomol Eng. 2016 June 7; 7: 197–222. doi:10.1146/annurevchembioeng-080615-033553.

Thiol--Disulfide Exchange Reactions in the Mammalian Extracellular Environment Michael C. Yi1 and Chaitan Khosla1,2 Michael C. Yi: [email protected]; Chaitan Khosla: [email protected]

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1Department

of Chemical Engineering, Stanford University, Stanford, California 94305

2Department

of Chemistry, Stanford University, Stanford, California 94305

Abstract

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Disulfide bonds represent versatile posttranslational modifications whose roles encompass the structure, catalysis, and regulation of protein function. Due to the oxidizing nature of the extracellular environment, disulfide bonds found in secreted proteins were once believed to be inert. This notion has been challenged by the discovery of redox-sensitive disulfides that, once cleaved, can lead to changes in protein activity. These functional disulfides are twisted into unique configurations, leading to high strain and potential energy. In some cases, cleavage of these disulfides can lead to a gain of function in protein activity. Thus, these motifs can be referred to as switches. We describe the couples that control redox in the extracellular environment, examine several examples of proteins with switchable disulfides, and discuss the potential applications of disulfides in molecular biology.

Keywords redox switch; redox couple; functional disulfides; oxidoreductase

INTRODUCTION

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Mammalian life is driven by the demand for oxygen and the maintenance of aerobic processes. Reactive oxygen species are harmful by-products of aerobic metabolism that can have devastating effects on living organisms (1). As a consequence, nature has evolved intricate antioxidant systems to combat oxidative stress and maintain homeostasis. Inside living cells, the redox potential is tightly controlled in each compartment and organelle by a variety of mechanisms. In this review, we focus on the redox-active species and the redox reactions in the extracellular environment, which we define to be the space beyond the boundaries of the cell cytoskeleton but inclusive of the outer leaflet of the plasma membrane.

Correspondence to: Chaitan Khosla, [email protected]. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

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Redox potential (the electromotive force, Eh) is a measure of the tendency of a chemical species to accept or donate electrons, and it is mathematically described by the Nernst equation. For example, the glutathione/glutathione disulfide (GSH/GSSG) redox couple (2GSH ↔ GSSG + 2e−) can be written as Eh = E0 + (RT/nF) ln(Q), where R is the gas constant, T is absolute temperature, F is Faraday’s constant, n is the number of electrons transferred, and Q is the reaction quotient [(GSSG)/(GSH)2]. E0 represents the propensity of the chemical species to transfer electrons relative to the standard hydrogen electrode reaction (H2/2H+ + 2e−) at equilibrium (i.e., E0 = −(RT/nF) lnKeq = ΔG0/nF). Table 1 summarizes the reported redox potentials of two common redox couples in different extracellular environments. The lack of accurate methods to detect both oxidized and reduced forms of specific thiol-containing molecules has limited the availability of reliable data of this type and represents an important challenge for the molecular engineer.

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Regulation of the redox potential is critical to numerous biological processes, including cell growth, metabolism, signaling, gene transcription, and enzyme catalysis. Remarkably, the redox potentials of the intracellular and extracellular environments are quite different, with the former being considerably more reducing than the latter. The redox couples regulating each environment operate far from equilibrium (2) (Table 1) but work in concert (3). Within the cell, additional redox couples operate in a location-specific manner (4), thereby enabling independent control of the subcellular redox potential. For example, mitochondria have the most reducing environment and account for more than 90% of the electron transfer to O2, whereas the endoplasmic reticulum (ER) and Golgi complexes are more oxidizing to enable the proper folding of nascent proteins harboring disulfide bonds. To control the redox potential of the cytoplasm, the cell utilizes NADPH-dependent enzymes such as glutathione reductase (GR) and thioredoxin reductase (TrxR); these enzymes, in turn, influence the relative ratios of the dominant intracellular thiol-based redox couples [i.e., GSH/GSSG and the thioredoxin-1 (Trx)-based system Trxred/Trxox]. Because NADPH is absent from many subcellular compartments, other cofactors, such as flavins, directly transfer electrons to O2 or H2O2 in those environments.

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The intracellular concentration of low--molecular weight thiols is in the millimolar range, whereas the pool of free small-molecule thiols is considerably lower in the extracellular environment (12–20 μM). In this oxidizing milieu, the cysteine/cystine (Cys/CySS) couple, the most abundant low--molecular weight redox-active species present, is thus considered to dictate the redox state. Although Cys is a nonessential amino acid, it is principally obtained from the diet and from methionine (Met). Remarkably, a diet free of sulfur-containing amino acids results in only a modest shift in Cys/CySS ratios; replenishment of Met and Cys reverts these changes within 24 h (5). This suggests that the regulation of extracellular redox is governed by a multitude of biological processes, as discussed in sections that follow. The extracellular Eh of Cys/CySS influences cell behavior, such as proliferation, differentiation, and adhesion (6–8). As a corollary, plasma Cys/CySS and GSH/GSSG ratios have garnered interest as potential biomarkers for various malignancies. Human studies have demonstrated that these ratios shift toward more oxidizing potentials with age (9, 10), cardiovascular disease (11, 12), Parkinson’s disease (13), type 2 diabetes (14), atherosclerosis (6), cirrhosis (15), and in early experimental graft-versus-host disease (16).

Annu Rev Chem Biomol Eng. Author manuscript; available in PMC 2017 June 07.

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Besides peptide bonds, disulfide bonds are the most common covalent link between amino acids in proteins. These linkages generally form in the oxidizing environment of the ER. Accordingly, most disulfide bonds exist in proteins that are exported to the extracellular environment. Traditionally, it was believed that disulfide bonds participated solely in stabilizing the structures of extracellular proteins, and they were inert for the life of the protein. Only 10% of mammalian proteins are estimated to contain disulfide bonds (17), and these have been acquired through evolution to stabilize tertiary or quaternary structures (18). Redox biology is being rewritten to acknowledge the ability of these motifs to engage in redox reactions and even act as switches in response to environmental cues. As such, when redox-active disulfide bonds in proteins are cleaved, these events can lead to a gain of function in the oxidizing environment outside of the cell.

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Disulfide bonds are made up of six atoms linking two Cα of two Cys molecules: Cα--Cβ-Sγ--Sγ′--Cβ′--Cα′ (Figure 1). There are five χ angles (χ1, χ2, χ3, χ2′, and χ1′) rotating about the bonds that contribute to the 20 different possible configurations of disulfide bonds, as well as to the overall dihedral strain energy (DSE). These configurations can be group into three basic types based on the sign (positive or negative) of the χ2, χ3 and χ2′ torsional angles: spirals, hooks, and staples (Table 2). These are further characterized into left-handed (LH) or right-handed (RH) based on the sign of χ3 (17).

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Schmidt and coworkers (17) found that the −LHSpiral configuration is the most prevalent and has the lowest DSE. Thus, it is not surprising that −LHSpiral disulfides are associated primarily with structure and stability (Figure 2a). Catalytic disulfides have the highest mean DSE at 20.8 kJ mol−1 (compared with 14.8 kJ mol−1 for all disulfides) and prefer the +/− RHHook configuration. For example, active-site Cys molecules in Trx and protein disulfide isomerase (PDI) share this motif when bonded (Figure 2b). This is an important factor in the redox activity of these proteins, as high-energy disulfide bonds are more easily cleaved than bonds with lower stored energy. Negative RHStaple disulfides have relatively high DSE (18.1 kJ mol−1) and are associated with known allosteric disulfides. This phrase was coined by Schmidt et al. (17) to describe disulfide bridges that regulate protein function by nonenzymatically altering intermolecular or intramolecular structures. Negative RHStaple disulfides also have the shortest Cα--Cα bond distance (4.32 Å compared with 5.63 Å for all disulfides), which likely contributes to the high strain from increased deformation and puckering. This is evidenced by the fact that the group is populated by many disulfides that link adjacent strands in the same antiparallel β-sheet, which is unusual because they occur in regions that are already bound together by noncovalent interactions.

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Disulfide-bond cleavage can be catalyzed by dithiol--disulfide exchange, a process that is typically facilitated by oxidoreductases of the PDI superfamily. These groups of enzymes have a characteristic Cys--X--X--Cys active site. During disulfide exchange, one of the thiol groups deprotonates and performs a nucleophilic attack on the substrate disulfide bond, forming a mixed disulfide intermediate. The other thiol group is responsible for freeing up the reduced substrate, leaving an oxidized enzyme. As is discussed below, a growing body of evidence suggests that these reactions can occur in the oxidizing extracellular milieu. Although the formation and cleavage of a disulfide bond is ultimately driven by thermodynamics, this process is kinetically sluggish. Furthermore, the major redox couples,


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Cys/CySS and GSH/GSSG, are present in concentrations that are orders of magnitude lower than their intracellular amounts. Interestingly, several oxidoreductases are localized in extracellular compartments, enabling rapid catalytic cleavage of disulfide linkages. As mentioned above, the reduction of allosteric disulfide bonds on extracellular proteins can have a major impact on their biological activity. In this review, prototypical examples of these gain-of-function disulfides are introduced. We primarily focus on proteins whose activity is dormant until disulfide cleavage has occurred. In addition, we explore the major redox couples in the extracellular environment capable of inducing these shifts in functionality. Last, we discuss engineering strategies used to identify and modulate extracellular thiols for various applications in biology and chemistry.

EXTRACELLULAR REDOX COUPLES INVOLVING THIOL--DISULFIDE Author Manuscript

EXCHANGE REACTIONS Cysteine Cys and its oxidized dimer CySS are abundant substances in mammalian extracellular fluids, and they represent the major small molecule thiol/disulfide couple in plasma (19). CySS is present at 40–50 μM, whereas Cys is estimated to be between 8 and 10 μM. In humans, the physiological Cys/CySS redox potential (Eh) in the plasma of healthy individuals is approximately −80 mV (Table 1). Under disease conditions characterized by oxidative stress, this potential increases to between −62 mV and −20 mV (10). As mentioned in the Introduction, higher than average Eh values have been observed in participants who abuse alcohol, have diabetes, and smoke cigarettes, and higher values have also been correlated with aging (9, 10).


A substantial amount of evidence supports the notion that the Cys/CySS redox couple has a major role in redox communication between cells and organs. This is in contrast to the GSH/ GSSG couple (discussed in the next section), which functions principally in the detoxification of reactive substances. For example, Ramirez and coworkers (8) showed that in lung fibroblasts the more oxidizing Eh values of Cys/CySS resulted in induction of intracellular signals that stimulate cell proliferation and extracellular matrix production. In particular, oxidizing Eh values enhanced extracellular fibronectin production in both NIH/3T3 fibroblasts and primary lung fibroblasts via the protein kinase C pathway and induction of transforming growth factor-β1. In contrast, a more reduced Cys/CySS Eh favored proliferation of the Caco-2 intestinal cell line via activation of the mitogenic p44/p42 mitogen-activated protein kinase pathway (20, 21). Reducing Cys/CySS Eh also favored growth in normal retinal epithelial cells (7), suggesting that the effect is tissuespecific. In the case of endothelial cells (which most commonly line the walls of blood vessels), a more oxidizing Cys/CySS redox state stimulated H2O2 production, activated the master proinflammatory regulatory molecule NF-κB, and consequently increased expression of cell--cell adhesion molecules, including intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, and P-selectin (6). Thus, an increase in Eh Cys/CySS values appears to sensitize cells to oxidative stress responses.


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In contrast to other extracellular redox couples discussed in the section titled Extracellular Protein Redox Switches, which are synthesized inside cells and then exported, Cys is primarily derived from the diet. Human studies have demonstrated that sulfur-containing amino acids from meals can significantly alter the extracellular Cys/CySS ratio (5, 22). Individuals between 18 and 36 years of age were given chemically defined diets without any Met or Cys, and plasma was collected over the course of 4 days (5). Plasma Cys levels decreased during this period, whereas CySS levels were unchanged, leading to a more oxidized Eh of −67 ± 2 mV. After sulfur-containing amino acids were reintroduced, the redox potential reverted to the steady-state value of −80 mV. However, the GSH/GSSG couple was unaffected by the change in diet. These findings underscore the tight regulation of the extracellular redox potential.

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Unlike GSSG and Trxox, there is no known dedicated enzyme for CySS reduction. Reduction of extracellular CySS to Cys may be achieved by GSH. However, this process appears be sluggish (19) and may be limited by GSH availability. Additionally, γglutamylcysteine synthetase knockout mouse embryonic fibroblasts with a cellular GSH deficiency were still able to reduce intracellular CySS (23). Other schools of thought point to the extracellular Cys/CySS couple being regulated via transport systems such as the xc− antiporter system for CySS uptake and the neutral amino acid LAT2 transporter for Cys export (Figure 3). However, expression of this transporter is fairly low in many cell types and is absent in T cells. Thus, T cells are dependent on Cys supplied by antigen-presenting cells, such as dendritic cells (24). It has been shown that this transport system protects against oxidative stress-induced cell death by lipid peroxidation by driving a highly efficient Cys/CySS redox couple and not by boosting intracellular GSH levels (25). Because of the existence of multiple Cys and CySS transporters (26) and their varied distribution and expression in different tissues, extracellular Cys/CySS is likely to be intricately regulated in vivo. Alternative theories imply that CySS could be reduced by extracellular oxidoreductases, such as Trx, directly without the need for intracellular transport (23). For example, it has been shown that Trx could reduce CySS to Cys and then be reabsorbed by lymphoid cells (27).

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Glutathione

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GSH and its oxidized dimer GSSG also comprise a biologically important small-molecule redox couple. In both intracellular and extracellular environments, GSH is primarily used to protect mammals from oxidative damage by reducing H2O2 and organic hydroperoxides. It also functions as a xenobiotic detoxifier and regulator of cell signaling via the reversible glutathionylation of proteins. GSH is abundant inside cells, with steady-state concentrations of 1–10 mM, and consequently, it is a major intracellular thiol antioxidant. The ratio of GSH to GSSG in the cytosol is typically 50:1, but it increases to 2:1 in the ER, thus facilitating disulfide-bond formation, mainly via PDI. In contrast, its levels are typically very low in extracellular fluids. In plasma, the GSH concentration is 2–4 μM, whereas the GSSG concentration is at least an order of magnitude lower (~0.1 μM), giving an Eh value of −137 ± 9 mV in healthy adults (28).


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GSH is synthesized primarily by hepatic cells, and it is exported to the extracellular environment by members of the multidrug-resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette proteins, as well as some members of the organic anion-transporting polypeptide (OATP) family (29, 30). Mrp2 mediates not only the efflux of GSH but also of oxidized GSH derivatives, such as GSSG and S-nitrosoglutathione, into bile. Export is driven by concentration. However, Mrp1 and Oatp1 are responsible for exporting GSH into blood by a poorly understood mechanism. Oxidoreductive turnover of GSH is relatively rapid, with half-lives of between 2 and 6 h (31). Also, inhibitors of GSH synthesis have been demonstrated to substantially decrease plasma GSH levels. Similar to Cys and CySS, this suggests that there is an intimate relationship between intracellular GSH and extracellular GSH regulation and that the transport of GSH is a key controller.

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In the extracellular milieu, GSH is cleaved by the ectoenzyme γ-glutamyl transpeptidase, resulting in the release of glutamate (Glu) and cysteinylglycine (Figure 3). The cysteinylglycine is ultimately converted into its free amino acid components by dipeptidase, thus keeping plasma GSH levels low and allowing cellular utilization of these products through uptake. Individuals with γ-glutamyl transpeptidase deficiency have increased circulating levels of GSH that are associated with severe behavioral problems in the disorders glutathionuria and glutathionemia (32). Remarkably, the half-life of blood GSH is on the order of seconds to minutes (33), indicating rapid degradation. Regardless, the liver is considered the major site of GSH synthesis and release, and this mechanism provides a systemic source of Cys for extrahepatic tissues (34).

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Inside cells, GSSG is regenerated to GSH via the NADPH-dependent enzyme, GR. In the extracellular environment, the plasma selenoenzyme glutathione peroxidase (GPx-3) can reduce H2O2 and lipid hydroperoxides using GSH as a reducing agent. Whether this is a physiological process is debatable, as GPx-3 has a tenfold higher KM for GSH compared with the other GPx isoforms, and plasma GSH concentrations are three orders of magnitude lower than intracellular GSH concentrations. Another potential mechanism for extracellular GSH oxidation involves glutaredoxins (Grx), small, 11-kDa proteins that engage in dithiol and monothiol reactions with a variety of protein substrates. Grx proteins are considered to be key regulators of redox signaling, and although they are secreted proteins (35), their extracellular role has not been determined. Last but not least, the oxidation of extracellular GSH can also occur nonenzymatically: through transhydrogenation between GSH and cystinyl-bis-glycine that is most likely mediated by metal ions, as evidenced by inhibition from the metal ion chelator ethylenediaminetetraacetic acid.

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Cys is a precursor for GSH biosynthesis. Like Cys/CySS, oxidizing GSH/GSSG ratios in plasma are also correlated with aging, smoking, and cardiovascular disease. Despite this correlation, the Cys/CySS redox couple functions independently of the GSH/GSSG couple (19). For example, plasma concentrations of Cys and GSH were analyzed in 122 participants between the ages of 19 and 85 years. Remarkably, there was a linear oxidation rate of Cys/ CySS over the entire age span (0.16 mV/year), whereas there was no appreciable change in the oxidation of GSH/GSSG prior to 45 years of age. After age 45, the rate of oxidation in the GSH/GSSG couple was 0.7 mV/year (36). Additionally, the plasma Cys concentration peaks around 3 h after a meal, whereas GSH concentration peaks around 9 h after feeding


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(22). The proliferation rate of cultured Caco-2 cells could be adjusted by varying Eh Cys/ CySS potentials with no change in GSH/GSSG (37). Thus, small-molecule thiol-redox couples do not exist in equilibrium, nor are they rapidly interacting or mutually redundant.

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GSH/GSSG ratios are useful indicators of oxidative stress. For example, rats were dosed with diquat to induce oxidative stress, resulting in a 17-fold increase in plasma GSSG concentration while GSH was not significantly altered (38). GSH efflux is also a hallmark of apoptosis and associated with early stages of cell-death progression (39). In the brain, GSH has been considered to be a neurological hormone due to its ability to bind specific receptors and stimulate signal cascades in astrocytes (40); however, the precise biological functions of extracellular GSH are unknown. Given the low abundance of GSH in plasma and its rapid oxidation and degradation, it is possible that plasma GSH is simply a consequence of the liver acting as a supplier for other organs, such as the lung and kidney (41). Alternatively, studies of the lung (42) and intestine (43) have suggested that extracellular GSH may have an antioxidant role analogous to intracellular GSH. Thioredoxin

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Thioredoxin (Trx)-1 is a ubiquitous protein that is the most important protein disulfide reductant inside cells. Its intracellular targets include ribonucleotide reductase (44), peroxiredoxins, and several important transcription factors, making it a key regulator in cell proliferation and survival (45). Trx also protects cytosolic proteins from aggregation and inactivation via the formation of intramolecular or intermolecular disulfide bonds. Trx is widely distributed in subcellular compartments: It is found in the cytosol, nucleus, and mitochondria (Trx-2). The necessity of this redox protein is highlighted by the fact that TXN knockout mice are embryonically lethal (46). Trx can also be found in extracellular environments in mammals (both membrane-bound and soluble), even though the protein lacks a canonical secretion-signal sequence (47). Trx has a conserved active site motif (--Cys--Gly--Pro--Cys--) that undergoes reversible oxidation to form an intramolecular disulfide bridge via the transfer of reducing equivalents from its active site Cys residues to a disulfide-bonded protein substrate. Reduced Trx is regenerated via the NADPH-dependent flavoprotein TrxR. Inside cells, reduced Trx predominates with (Eh) = −280 mV (48).

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Human Trx was originally observed as a secreted protein from human T cell leukemia virus type-1-transformed lymphocytes, and is, therefore, cited as an adult T cell leukemia--derived factor (49). Extracellular Trx is present in the low nanomolar range in healthy individuals, and in response to oxidative stress-related signals, Trx is released from cells (50). As much as 100 nM extracellular Trx has been reported in the synovial fluid of patients with rheumatoid arthritis (51). In fact, Trx is emerging as a recurring biomarker for several diseases, including cancer, diabetes, and AIDS. Outside the cell, Trx exerts both cytokine- and chemokine-like properties, and these have been shown to result in an overall cytoprotective effect (52, 53). For instance, exogenously added Trx suppresses tumor necrosis factor-α-mediated cytotoxicity in U937 monocytes (54). In addition, extracellular Trx supports the mitogen- or cytokine-induced proliferation


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of lymphocytes (55), as well as the proliferation of normal fibroblasts (56) and various tumor cell lines (57). These effects depend on the redox-active Cys residues. Trx has also been shown to act as a chemotactic protein, promoting the migration of neutrophils, monocytes, and T cells (53). Although a few examples of extracellular Trx substrates have been identified (discussed below), their relationships, if any, to these phenotypic consequences are unknown. Trx is rapidly oxidized in the circulation of patients undergoing cardiac surgery with cardiopulmonary bypass (35). Although TrxR is released by normal and neoplastic cells (58), the absence of cofactor (NADPH) and cofactor-regeneration mechanisms in the extracellular milieu leaves open the question of how Trx is regenerated in this environment. Protein Disulfide Isomerase

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PDI promotes the formation, reduction, or rearrangement of protein disulfide bonds. It is principally responsible for the folding of nascent proteins via disulfide-bond formation in the ER. PDI is organized into five domains; two are homologous to Trx. Two other domains adopt the Trx fold but lack an active Cys pair. Thus, PDI is a member of the Trx superfamily, and with an E0 of −190 mV, PDI is one of the better oxidants in this family of protein cofactors (59).

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PDI is also expressed on the surface of various cells---for example, fibrosarcoma cells (60), platelets (61), and lymphocytes (62). Its best understood role in the extracellular environment is in the context of thrombus formation (63, 64). Using a flow chamber assay under arteriolar stress conditions, blood from wild-type mice exhibited extensive thrombus formation on collagen surfaces, whereas blood from PDI-knockout mice showed less formation (65). When purified PDI was exogenously added to blood from PDI-knockout mice, coverage was similar to that of the wild-type blood sample. Additionally, extracellular PDI has been shown to modulate the density of free thiol on the surfaces of fibrosarcoma cells (60). A potentially important pathogenic role of extracellular PDI is in mediating the reduction of two disulfide bonds of the HIV glycoprotein, gp120, which is responsible for cell recognition and fusion by the virus (62). Quiescin Q6 Sulfhydryl Oxidases

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Quiescin Q6 sulfhydryl oxidases (QSOX) are flavin-dependent enzymes that are also involved in protein folding in the ER. These multidomain proteins are characterized by two Trx--PDI domains at their N termini and a C-terminal domain homologous to the augmenter of liver regeneration (ALR) protein [also known as the essential for respiration and vegetative growth (ERV) protein]. The C-terminal domain also houses a bound flavin adenine dinucleotide cofactor and a catalytically active CXXC motif. QSOX was first discovered in the male reproductive tract of rodents as a protein that can produce disulfide bonds as well as H2O2 at the expense of O2 (66). Since then, a number of sulfhydryl oxidases have been discovered and characterized, including those found in egg whites (67) and seminal vesicles (68). Besides being localized in the ER, QSOX is also found in the Golgi complex, secretory granules, and extracellular space. QSOX exists as two splice variants: QSOX1a and QSOX1b. The former is different from QSOX1b in that the C-


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terminal transmembrane helix is truncated and transits the ER, with a fraction being secreted from cells.

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The physiological role of QSOX is not well understood, and although its substrate specificity has been extensively studied (69), its physiological targets are unknown. Reports have suggested that QSOX is involved in the formation of the extracellular matrix (70). Specifically, QSOX incorporated laminin into the extracellular matrix of WI-38 fibroblasts. Strikingly, small interfering RNA mediated knockdown of QSOX resulted in increased cell detachment and higher levels of soluble laminin. Addition of recombinant QSOX reversed these effects. Furthermore, exposure to an inhibitory antibody against QSOX resulted in the presence of fewer fibroblasts in the monolayer. QSOX is present in the plasma at concentrations of 25 nM (71) and is upregulated in several maladies, such as pancreatic cancer and heart failure, suggesting that it could be used as a diagnostic marker for these disorders.

EXTRACELLULAR PROTEIN REDOX SWITCHES The activity of proteins with allosteric disulfide bonds can be modulated by the presence of reducing (GSH or Trxred) or oxidizing (CySS or GSSG) agents. In the extracellular environment, oxidizing conditions prevail, leading to the preexistence of such disulfide bonds; however, release of an appropriate reducing agent by a cell can result in local bond cleavage and switching on of protein activity. In this section, we highlight some well-studied examples of switchable disulfide-containing proteins and the implications of this in human health. CD4


CD4 is a 55-kDa membrane-bound glycoprotein consisting of C-terminal transmembrane and cytosolic segments and an N-terminal extracellular region containing four immunoglobulin-like domains (D1--D4). D1, D2, and D4 each contain a single Cys pair. CD4 exists in three different forms on the surface of cells, as determined by the state of D2 Cys: (a) an oxidized monomer, (b) a reduced monomer, and (c) a homodimer linked via inter-D2 Cys residues (72). The last of these configurations is the preferred immune coreceptor. During adaptive immunity, CD4 recognition is required for T cell activation and proliferation, in addition to formation of the ternary major histocompatibility complex classII antigen--T cell receptor complex. CD4 is also the primary receptor for HIV entry into a T cell due to its recognition by the viral surface glycoprotein gp120. It is well known that the intramolecular disulfide bond in D2 behaves as an allosteric disulfide due to its high torsional strain. Cleavage of this disulfide is critical for HIV entry, and it is promoted by Trx (73) but not PDI (74). Trx-mediated bond cleavage also promotes homodimer formation, likely through two intermolecular disulfides between Cys130 in one monomer and Cys159 in the other. Consistent with this model, mutagenesis of the Cys residues in D2 resulted in enhanced HIV entry but impaired coreceptor function (75). D2 has a truncated β-barrel and an unusual disulfide bridge between Cys130 and Cys159 that links adjacent strands in the same β-sheet, as opposed to linking across different βsheets. Remarkably, primates and rodents are the only animals that possess this cross-strand


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disulfide and appear to have acquired Cys130 independently (76). The geometry and strain of this disulfide are also special (Figure 4). It displays a −RHStaple configuration with a relatively short Cα--Cα bond (i.e., 3.92 Å compared with 6.6 Å for standard immunoglobulin disulfides) (77). Because of this, it has been speculated that puckering between the β-sheets results in high strain energy (4.74 kcal mol−1) compared with the D1 (2.28 kcal mol−1) and D4 (1.74 kcal mol−1) disulfide bonds, correlating with ease of reduction. The D2 bond also makes the least contribution to the overall stability of CD4, with an enthalpy contribution of 3.65 kcal mol−1 compared with the D1 (4.36 kcal mol−1) and D4 (3.97 kcal mol−1) disulfides (73). High-Mobility Group Box 1

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High-mobility group box 1 (HMGB1) is a nonhistone nuclear protein containing two positively charged, proximal, DNA-binding domains (A box and B box) and a negatively charged, C-terminal tail. In the nucleus, HMGB1 sustains chromosome structure and stability. During stress, HMGB1 translocates to the cytosol and is eventually released into the extracellular medium via a nonclassical secretory pathway or by lysis. In the extracellular medium, HMGB1 exhibits various cytokine and chemokine functions through the receptor for advanced glycation end products (RAGE) (78) or the Toll-like receptors (TLRs) (79), and the CXCL12--CXCR4 complex, each of which activates downstream signaling pathways, such as NF-κB and possibly phosphatidylinositol-3 kinase (PI3K) (80). Serum HMGB1 is an important pathological marker of inflammation, with increased levels correlating with poor prognosis in cancer, sepsis, and autoimmunity (81).

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There are three Cys residues in mammalian HMGB1: Cys106 in the B box, and Cys23 and Cys45 in the A box. All three are redox active, and the state of these thiols has major implications for protein function (82). The A box Cys residues are spaced 4.3 Å apart (83) and can form an intramolecular disulfide bond that helps to stabilize the folded state of the protein (84). Interestingly, this disulfide exists in both the +/−RHHook and −LHSpiral configurations (Figure 5). Thus, it serves to stabilize the protein but can also be high in energy, making it susceptible to reduction. Cys106 is typically found in the reduced, thiol state, although dimer formation is possible in vitro (85).

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HMGB1 migrates to the extracellular space by active secretion or by passive release via necrosis, pyroptosis, autophagy, or apoptosis. The released, cytokine-active form of HMGB1 contains a Cys23--Cys45 disulfide bond with a reduced Cys106 residue. In a mouse model of hepatic inflammation induced by acetaminophen, this was the predominant form of serum HMGB1 (85). As inflammation resolved, the Cys106 residue was irreversibly oxidized to −SO3H, which restricted the cytokine-stimulating activity. Thus, Cys106 appears to be critical for cytokine activity and may have been evolutionarily conserved to allow inactivation after cytokine-stimulating activity is no longer needed. This was supported with a C106A mutation; the resulting protein was no longer able to bind TLR4--MD2 and activate macrophages or monocytes (86). Conversely, HMGB1 functions as a chemokine when all Cys residues are reduced (87). In this conformation, HMGB1 forms a heterocomplex with the chemokine CXCL12, which then acts exclusively through CXCR4 (88). This interaction is critical for cell migration, as Annu Rev Chem Biomol Eng. Author manuscript; available in PMC 2017 June 07.

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antibodies against CXCL12 block the HMGB1-induced migration of leukocytes. The disulfide-bonded state of the protein (Cys23--Cys45) does not form this heterodimer and does not abrogate binding of the fully reduced form, suggesting that the cytokine and chemokine functions of HMGB1 are mutually exclusive. A summary of the chemokine and cytokine functions of HMGB1 is presented in Figure 6. Although the physiological mechanism of cleavage of the intramolecular disulfide is unknown, various redox-active proteins can promote this transformation in vitro. For example, thiol--disulfide exchange can be achieved by Trx, albeit not with high specificity (84), and also by glutaredoxin (89). β-Defensin 1

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β-Defensins are small (4–5 kDa), cationic, Cys-rich, antimicrobial peptides secreted by a wide array of cell types. In the intestinal lumen, they form an essential component of innate immunity by binding to negatively charged microbial membranes. Although the mechanisms are not definitively understood, it has been suggested that this interaction ultimately leads to membrane depolarization and eventual cell death (90). Defective expression or function of βdefensins, or both, is associated with several maladies, such as inflammatory bowel diseases and infectious colitis. β-Defensins are composed of 36–47 amino acids and contain a characteristic Cys1--Cys5, Cys2--Cys4, and Cys3--Cys6 disulfide-bridge connection (91). These three disulfides link three β-strands that are arranged in an antiparallel sheet and exist in multiple configurations (e.g., −LHHook, −LHSpiral, −RHHook, and −RHStaple).

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Human β-defensin 1 (hBD1) is one of the most prominent members of its class, with ubiquitous constitutive expression by all epithelia (92). Reduction of the three disulfide bonds enables hBD1 to become a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, gram-positive commensals of Bifidobacterium and Lactobacillus species (93). In the presence of 2 mM dithiothreitol, hBD1 became as effective as hBD3, which is one of the most potent antimicrobial peptides in oxygen-rich environments. This finding was significant because previous reports had found that hBD-1 had paradoxically low antimicrobial activity, despite predominant expression in the intestine. The reduced form of hBD1 exhibits an unstructured, highly flexible polypeptide chain and likely contributes to its microbe-killing attribute. However, preservation of the Cys residues was critical, as mutation of any Cys to alanine (Ala) resulted in loss of activity.

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Trx, and to a lesser extent Grx, can also reduce hBD1 (94). Genetic knockdown of Trx in Caco-2 cells, as well as inhibition with a TrxR inhibitor (95), resulted in diminished hBD1 reduction, suggesting that the Trx/TrxR system may be the major physiological mechanism for redox regulation of hBD1. In fact, in vivo staining of colonic mucosal tissue in healthy individuals displayed colocalization of Trx with reduced hBD1 (94). Transient Receptor Potential Channel 5 The transient receptor potential (TRP) is a large family of multimeric cation channels that are either Ca2+- or Na+-permeable. This family includes transient receptor potential canonical (TRPC) channels, of which there are seven members. TRPC channels are Annu Rev Chem Biomol Eng. Author manuscript; available in PMC 2017 June 07.

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ubiquitously expressed in various cell types, including smooth muscle (96, 97) and endothelial (98) cells, fibroblast-like synoviocytes (99), and brain cells (100). Their ability to transport cations is primarily activated in response to phospholipase C--coupled receptors (101) and Ca2+ store depletion (102), and it is further modulated by Ca2+ and calmodulin binding (103). Once activated, these channels have a role in diverse physiological processes, ranging from smooth muscle contraction to neurological behavior.

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This class of proteins is further subdivided into two groups based on structural and functional similarities: TRPC1/4/5 and TRPC3/6/7. Furthermore, these subgroups can associate with one another to form functional homo- or heterotetrameric macrostructures. TRPs exhibit broad parallels to voltage-gated potassium subunits such as Kv1.2 (104, 105). A distinguishing feature is an extracellular loop between the fifth membrane-spanning segment and the amino acids responsible for ion selectivity. Within this loop is Glu543, which is responsible for lanthanide-dependent activation in TRPC5 (106), and two disulfideforming Cys residues at positions 553 and 558. These thiol residues are conserved in the TRPC1/4/5 group and have recently been shown to be susceptible to Cys-modifying agents such as NO (107, 108). Xu and coworkers (99) found that dithiothreitol, tris(2carboxyethyl)phosphine, and Trx can lead to increased activity of TRPC5 in HEK-293 cells, whereas other TRP channels lacking the vicinal Cys pair were unresponsive. This observation suggests that the disulfide bridge may conformationally constrain the channel, and it hints at redox regulation of ion transport, a hypothesis that is reinforced by the finding that Cys→Ala mutants of TRPC5 lead to constitutive channel activation. Activation has also been observed for fibroblast-like synoviocytes, whereas oxidized Trx had no effect (99). β2-Glycoprotein I and Von Willebrand Factor

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β2-glycoprotein I (β2GPI), also known as apolipoprotein H, is an abundant, 50-kDa plasma protein that has been implicated in apoptotic cell clearance and coagulation through interactions with serine proteases, anionic phospholipids, and cell-surface receptors (109– 111). Interest in β2GPI was sparked when this protein was found to be a dominant antigen in the autoimmune disorder antiphospholipid syndrome (112).

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β2GPI belongs to the complement control protein (CCP) superfamily. It has four CCP domains and an unusual fifth domain. Each of the four classical CCP domains contains two conserved disulfide bonds, whereas the fifth domain contains three disulfide bridges. In the unorthodox C-terminal domain, the Cys288--Cys326 bridge is exposed to solvent and has been demonstrated to be recognized by both Trx and PDI (113, 114). This disulfide bond displays a −/+RHHook configuration in both available crystal structures (115, 116) that is not particularly strained, meaning that solvent exposure is likely the reason it can be cleaved. Ioannou et al. (113) have demonstrated that free, thiol-containing β2GPI can be found in blood from healthy donors and that reduced β2GPI had a key protective role against oxidative stress-induced injury of endothelial cells in tissue culture. Trx-catalyzed reduction of β2GPI increased the glycoprotein’s affinity to von Willebrand Factor (vWF), a plasma protein that chaperones blood coagulation factor VIII and tethers platelets to injured blood vessel walls. This interaction helps promote the adhesion of GPIbα and platelets to activated vWF, which is critical for hemostasis.


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The vWF protein circulates as a set of 500-kDa homodimers; it can form higher-order multimers through building interdimeric disulfide bridges between Cys379 in one dimer and one or more of the Cys residues at positions 459, 462, and 464 in another. Multimers of up to 20,000 kDa can be formed; these large complexes are responsible for its hemostatic activity (117–119). The presence of very large multimers is associated with thrombosis. Reductions in the size of vWF multimers have been correlated with the generation of new thiols, suggesting that vWF activity could be controlled through disulfide-bond cleavage and that this modification could be catalyzed by Trx or PDI in vitro (120). This result led to the proposal that a reduction of the dimeric disulfides, which are formed between Cys residues located in the C-terminal domains, can lead to the formation of vicinal disulfides. Vicinal disulfides on two vWF molecules could then interchange to form interdimeric multimers. In a follow-up study, the physiologically relevant regulator of vWF size was shown to be thrombospondin 1, a multimeric glycoprotein produced by mast cells and platelets (121).

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Transglutaminase 2

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Transglutaminase 2 (TG2) is the best-studied and most prevalent member of the mammalian transglutaminase family of enzymes, which has abundant expression in both intracellular and extracellular regions. Although the exact biological role of TG2 remains to be elucidated, the protein has been implicated in the pathogenesis of a wide array of maladies, including celiac disease (122, 123), cancer (124), and several neurological disorders (125, 126). TG2 catalyzes the calcium- and thiol-dependent transamidation or deamidation of glutamine residues in protein or peptide substrates. These modifications are intricately regulated by several mechanisms at the posttranslational level (127). To gain activity, calcium ions must bind to TG2, allowing the enzyme to adopt an open conformation and enabling access to the active site (128, 129). In the presence of guanine nucleotides, the domains of TG2 collapse into a more compact structure, limiting the availability of the active site. Furthermore, it has been found that a vicinal disulfide (Cys370--Cys371) requires cleavage for activity (131). As shown in Figure 7, this bond forms a −RHStaple configuration, which is characteristic of allosteric disulfides (17).

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Due to the oxidizing extracellular environment, it has been postulated that TG2 remains catalytically inactive as a result of the vicinal disulfide bridge, despite the abundance of Ca2+ and scarcity of guanine nucleotides (130). The formation of this bond is mediated by an additional thiol, Cys230, and the mutation of this residue to Ala has been shown to reduce the enzyme’s susceptibility to oxidation (131). The three Cys residues are in close proximity to one another, and it has been shown that Cys370 can form an intermediate covalent link with Cys230. Cys230 is also unique to TG2 and is thought to act as a redox sensor, as it controls the formation of the vicinal disulfide bridge. Although the mechanism of TG2 inactivation by oxidation is not well understood, it has been proposed that oxidation can obstruct Ca2+ binding through making structural changes in the protein’s architecture (132). Despite TG2’s susceptibility to oxidation in the extracellular environment, it has also been demonstrated that the enzyme can be activated upon injury or stress (130). Jin et al. (133) and DiRaimondo et al. (134) found that Trx activated TG2 and that this interaction was comparable to an established natural substrate of Trx, insulin. Additionally, the calculated


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redox potential of the Cys370--Cys371 disulfide bridge is −190 mV, which makes it a good target for oxidation by Trx, whose E0 is −230 mV (Table 1). Interestingly, Trx secretion can be triggered by interferon (IFN)-γ, which is the predominant cytokine in celiac disease, making it plausible that TG2 activity could be facilitated by the redox protein (133, 135). Figure 8 outlines a possible mechanism for Trx-mediated TG2 activation in the pathogenesis of celiac disease.

IDENTIFYING AND EXPLOITING EXTRACELLULAR REDOX-SENSITIVE THIOLS AND DISULFIDES

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Cys is one of the most remarkable amino acids in biology. The side chain thiol has been implicated in a variety of functions, including metal bonding, redox chemistry, and disulfidebond formation. It is no wonder that Cys is the second most conserved amino acid in evolution (76), and because of this, it is commonly accepted that disulfide bonds are also highly conserved in evolution. Once a protein has acquired a disulfide bond through evolution, it rarely loses it, highlighting its significance in physiology (76). Engineering of Cys residues, therefore, may offer a powerful tool for probing important unanswered biological questions and for treating disease.

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As discussed in the section titled Protein Disulfide Isomerase, redox-active exofacial thiols on cell surfaces can be modulated by PDI (60). By using cell-impermeable thiol-modifying agents, such as N-(biotinoyl)-N′-(iodoacetyl) ethylendiamine, in combination with mild reducing agents, extracellular redox-sensitive proteins can be extracted and identified using immunoprecipitation and mass spectrometry (136). This approach has enabled the discovery of integrin α-4 as a redox-sensitive protein that is involved in cell adhesion to fibronectin, an effect that is enhanced by GSH. It is well established that integrins promote adhesion through disulfide cleavage, highlighting the importance of these global approaches for identifying new, extracellular, redox-sensitive proteins. Although these techniques often use small-molecule reducing agents, the use of proteins capable of disulfide cleavage, such as PDI and Trx, may be more suitable and physiologically relevant.

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As mentioned above, the DSE of a disulfide bond can be used to predict its conformation. The advent of bioinformatics and high-resolution, three-dimensional methods of determining protein structure has enabled the identification of 12,682 disulfide chains in 8,318 crystal structures of human proteins (137). However, it has been shown that there is a discrepancy in the calculations of DSE between NMR structure disulfides and X-ray structure disulfides (138). Protein structures in the literature are most commonly derived from X-ray crystallography data (91%), as there were past technical limitations in using NMR for structure determination. Specifically, NMR spectroscopy could be applied only to proteins with a molecular weight of no more than 30 kDa. However, the development of methyl transverse relaxation-optimized spectroscopy (TROSY) and freezing rotational diffusion of protein solutions at low temperature and high viscosity (FROSTY) may enable analysis of high--molecular weight proteins and protein complexes (139). Of the current analytical techniques, NMR spectroscopy is the ideal method for studying protein dynamics because the data are collected in solution. Crystal structures are static in nature and,


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therefore, may not represent the solution structure of the disulfide bond. Thus, an in silico approach to identify allosteric disulfides will be predicated on the development of more sophisticated techniques that can produce high-quality structures that accurately reflect a protein’s conformation in vivo. Regardless, current approaches still hold merit, as disulfides in TG2 and vWF were speculated to be allosteric before validation in subsequent functional studies (140).

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More recently, synthetic biomolecules have been engineered to harbor a terminal thiol, resulting in their enhanced cellular uptake (141–144). These molecules range from oligonucleotides to peptides to nanoparticles. These studies have been critical in optimizing the delivery of cargo, such as small interfering RNA, for transfection reagents. The delivery of these particles is significantly enhanced by the addition of a Cys residue, and this property depends on the reactivity of the thiol: The addition of thiol alkylating agents, such as N(ethylmaleimide) and iodoacetamide, that sequester the active thiol revert the enhanced uptake to baseline levels. The precise mechanism of delivery is unknown, but one study has suggested that there are multiple mechanisms (145). In the context of cell-penetrating peptides, the mode of transport depends on sequence, concentration, and temperature. This dependence may be due to the fact that the mode of import depends on the interaction of the biomolecule with its cognate receptor. These receptors may be internalized through native pathways, and the biomolecules may be simply piggybacking into the cell. Regardless, the fate of these particles is an important unanswered question that is biomolecule-dependent but warrants elucidation, especially for applications that require compartment-specific targeting, such as to the nucleus for small interfering RNAs.


Engineering switchable disulfides onto proteins is likely to be challenging because changes to a protein’s conformation and stability will be effected, and these changes could have outstanding downstream impacts on cell biology. One can easily envision unwanted disulfide-bond formation occurring between an inserted Cys and a native Cys resulting in misfolding. To circumvent this issue, groups have developed tools to predict stereochemical shifts and overall tolerance to the insertion of disulfide bridges (146, 147). Matsumura & Matthews (148) were pioneers in this engineering strategy; they performed site-directed mutagenesis on the active site cleft of bacteriophage T4 lysozyme. Mutation of threonine (Thr) 21 and Thr142 to Cys led to spontaneous formation of a disulfide bond under oxidizing conditions, completely nullifying the enzyme’s catalytic activity. Activity could be rapidly restored by adding a reducing agent. Similar strategies have been employed to understand the substrate-recognition machinery of insulin-degrading enzyme (149) and the regulation of P-glycoprotein (150). Thus far, these engineered switches have involved the use of a chemical thiol-reducing agent to cleave the disulfide bond. These agents are generally nonspecific and could lead to off-target effects. Instead, these disulfides should be engineered to have high substrate recognition by oxidoreductases such as Trx and PDI. In addition to higher specificity and better control of the engineered system, the rate of activation of the switch could be further accelerated. Alternatively, rapid oxidation by PDI or QSOX could also improve the kinetics of the switch, as oxidation is sluggish in the extracellular environment. A vast majority of disulfide-bond engineering has involved improving the stability of proteins, but the utilization of disulfides in toggling protein activity warrants further comprehensive evaluation. Annu Rev Chem Biomol Eng. Author manuscript; available in PMC 2017 June 07.

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CONCLUDING REMARKS During the past few decades, the discovery of functional, extracellular disulfides has piqued the interest of redox biologists. The textbook definition of disulfide bonds emphasized the importance of these posttranslational modifications to the stability of proteins, but now, disulfide bonds are appreciated for their versatility not only in maintaining protein integrity but also for their ability to engage in redox catalysis and regulate protein activity. Disulfides that modulate protein function are commonly referred to as allosteric disulfides (17). Here, we narrowed our scope to allosteric disulfides that, when cleaved, lead to a gain of function. The ability of the disulfide to control the activity of a protein in a binary manner makes it natural to refer to them as switches. Interestingly, these switches are turned on in response to a host of biological signals that are involved in both immunity and malignancy.

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A majority of these bonds exist in the −RHStaple configuration. This observation has enabled allosteric bonds to be predicted using computational software packages in conjunction with experimental data about protein structure. Thus, the discovery of new, functional disulfides will depend on the expansion of the protein-structure database, but this database must be validated in a physiological context. Many studies on extracellular redoxsensitive thiols involve the use of chemical thiol-reducing agents. Because these are not found in vivo and the low--molecular weight thiol redox couples (i.e., GSH/GSSG and Cys/ CySS) lean toward oxidizing potentials, specific interactions with oxidoreductases found in the extracellular space, such as Trx and PDI, should be investigated.

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Our knowledge of the role and maintenance of oxidoreductases in the extracellular environment is limited. Many of these proteins are nonclassically secreted by mechanisms that remain to be elucidated, and their secretion appears to be triggered by a variety of stimuli. Nature has evolved robust NADPH-dependent regeneration systems for GSH and Trx, but it is unclear whether these exist beyond the cellular borders. Perhaps these systems are tightly regulated due to the potential effects of activating these redox switches. Regardless, this area of biology demands further study.

Acknowledgments The authors thank Sooyun Choi for her assistance with Adobe Illustrator. Research on this topic in the authors’ laboratory is supported by a grant from the National Institutes of Health (number R01 DK063158).

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Author Manuscript Figure 1.

Author Manuscript

Disulfide bond conformation. The six atoms involved in disulfide bonding form five torsional angles, χ, and determine the overall configuration of a disulfide bond. Each angle can be positive or negative, giving rise to twenty unique states.

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Figure 2.

Examples of common disulfide configurations. (a) Crystal structure of the glucocorticoidinduced tumor necrosis factor receptor ligand, GITRL (Protein Data Bank identification number: 2Q1M). Cysteine (Cys) 58 and Cys78 form a +/− left-handed spiral, a signature configuration of stabilizing disulfides. (b) Crystal structure of human thioredoxin-1. Like most catalytic disulfides, Cys32 and Cys35 form a disulfide bond that follows the –righthanded hook configuration (Protein Data Bank identification number: 3M9K). These disulfides exhibit the highest mean dihedral strain energy when compared with all other disulfides. Disulfide bonds are represented as stick models.


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Author Manuscript Figure 3.


Cellular regulation of extracellular redox. The three major redox couples are Cys/CySS, GSH/GSSG, and Trxred/Trxox. GSH and Trx are maintained in thiol form by the NADPHdependent enzymes GR and TrxR. Although there is no known intracellular CySS reductase, CySS reduction is assumed to be achieved by GSH or Trx. GSH efflux is controlled by members of the multidrug-resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette proteins, as well as some members of the OATP family. The liver is the principal organ for GSH export. Extracellular GSH can undergo many different fates including oxidation to GSSG, scavenging by peroxides, S-glutathionylation of proteins, hydrolysis to Cys by γ-glutamyl transpeptidase and dipeptidase, and oxidation with CySS to yield CySSG. Cys transport is mediated by several Na+-dependent and -independent transporters, including LAT2. Extracellular Cys can undergo oxidation to CySS, Scysteinylation of proteins, or be reabsorbed by cells. Some cells express an xc− antiporter that facilitates CySS uptake, from where it can be reduced back to Cys in the reducing intracellular environment. Trx is secreted into the extracellular space by a nonclassical secretory mechanism and exerts chemokine and cytokine functions outside the cell (47). Abbreviations: Cys/CySS, cysteine/cystine; GPx-3, glutathione peroxidase; GR, glutathione reductase; GSH/GSSG, glutathione/glutathione disulfide; OATP, organic anion-transporting polypeptide; Trx, thioredoxin; TrxR, thioredoxin reductase.

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Figure 4.

Crystal structure of human CD4. Cysteine (Cys) 130 and Cys159 form an intramolecular disulfide bond with a –right-handed staple configuration (Protein Data Bank identification number: 3CD4). The cleavage of this disulfide is mediated by thioredoxin and is critical for HIV entry. Disulfide bonds are represented as stick models.


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Author Manuscript Author Manuscript Figure 5.

Author Manuscript

NMR protein structure of high-mobility group box 1 (HMGB1) protein (residues 1–84). Cysteine (Cys) 23 and Cys45 in the A box domain form a −/+ right-handed hook and –lefthanded spiral (Protein Data Bank identification number: 2RTU). When this disulfide bond is formed, HMGB1 exerts cytokine-like function. Upon reduction by either thioredoxin or glutaredoxin, HMGB1 switches function and serves as a chemokine. Disulfide bonds are represented as stick models.


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Figure 6.

Author Manuscript

The biological function of extracellular HMGB1 is redox dependent. HMGB1 contains three cysteines: two in the A box domain (Cys23 and Cys45) and one in the B box domain (Cys106). Fully reduced extracellular HMGB1 forms a heterodimer with CXCL12, which engages with CXCR4 to activate downstream signaling pathways, such as the PI3K pathway, leading to the chemotaxis of leukocytes (88). A disulfide bond can form between Cys23 and Cys45. This variant of HMGB1 can bind to RAGE (78) and TLR4 (79) to activate the NFκB pathway and induce production of proinflammatory cytokines. Terminal oxidation of Cys106 to −SO3H restricts the cytokine-stimulating activity of HMGB1 and is found after inflammation has resolved (85). Abbreviations: Cys, cysteine; HMGB1, high-mobility group box 1; PI3K, phosphatidylinositol-3 kinase; RAGE, receptor for advanced glycation end products; TLR4, Toll-like receptor-4.


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Figure 7.

Crystal structure of human transglutaminase 2. Cysteine (Cys) 370 and Cys371 form a vicinal disulfide bond that adopts the –right-handed staple configuration (Protein Data Bank identification number: 2Q3Z). This bond forms with the aid of the Cys230 residue, known as the redox sensor. Disulfide bonds are represented as stick models.


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Figure 8.

Author Manuscript

Proposed model for the activation of extracellular transglutaminase 2 (TG2) by thioredoxin (Trx) and implications in celiac disease. Interferon (IFN)-γ is the predominant cytokine in celiac disease and has been shown to trigger the release of Trx in human monocytes (133, 135). Trx can catalyze disulfide cleavage of Cys370--Cys371 on TG2, leading to its activation and subsequent deamidation of gluten-derived peptides. These peptides are presented on disease-associated human leukocyte antigen (HLA)-DQ2/8 molecules on antigen-presenting cells (APCs) to T cells, which become activated and secrete more IFN-γ (122, 123), creating an autoamplification loop.


Author Manuscript

Author Manuscript −168 ± 3 mV

−138 ± 7 mV

Data from Brown et al. (42) and Iyer et al. (11), obtained by collecting fluid via bronchoalveolar lavage in rats.

Data from Dahm & Jones (43), obtained from the lumen of rat jejunum.

Abbreviations: Cys/CySS, cysteine/cystine; GSH/GSSH, glutathione/glutathione disulfide; ND, not determined.

Data from Toghi et al. (153), obtained from the cerebrospinal fluid of healthy individuals aged approximately 68 years.

f

−80 ± 9 mV

−137 ± 9 mV

Data from Jones et al. (28), obtained from the plasma of healthy individuals aged 25–35 years.

d

e

−226 mV

Cys/CySS

Intestinal lumen redox potentiald (Eh)

Data from Clark (152) at pH 7.0, with adjustment from pH 7.4 using −59 mV/pH unit.

b

c

−240 ± 10 mV

GSH/GSSG

Plasma redox potentialc (Eh)

Data from Rost & Rapoport (151), obtained by a spectrophotometric assay at pH 7.0.

a

Standard potentiala,b (E0)

Redox couple

−108 ± 5 mV

−205 ± 2 mV

Lung redox potentiale (Eh)

Author Manuscript

Redox potentials of the GSH/GSSG and Cys/CySS couples in selected extracellular milieus

ND

−66 mV

Cerebrospinal fluid redox potentialf (Eh)

Author Manuscript

Table 1 Yi and Khosla Page 33


Author Manuscript

Author Manuscript

−

−

+

−

+

−

+

−

+

+

−LHSpiral

−RHHook

+/−LHSpiral

−LHHook

−/+RHHook

−RHStaple

+/−RHSpiral

−RHSpiral

−/+LHHook

+/−LHHook

−

+

+

+

−

+

−

−

+

−

χ2

−

−

+

+

+

+

−

−

+

−

χ3

+

−

+

+

−

−

+

−

−

−

χ2′

−

−

−

−

−

−

−

−

−

−

χ1′

+LHStaple

+RHStaple

+RHHook

+/−RHStaple

+LHSpiral

+LHHook

−LHStaple

+/−LHStaple

+/−RHHook

+RHSpiral

Configuration

Abbreviations: LH, left-handed; RH, right-handed.

χ1

Configuration

+

+

+

+

+

+

−

+

+

+

χ1

+

−

+

−

−

−

+

+

−

+

χ2

−

+

+

+

−

−

−

−

+

+

χ3

+

−

−

−

−

+

+

+

+

+

χ2′

+

+

+

−

+

+

−

−

−

+

χ1′

Author Manuscript

Possible configurations of a disulfide bond based on the torsional angles, χ

Author Manuscript

Table 2 Yi and Khosla Page 34


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