The role of thioredoxin in the regulation of cellular processes by S-nitrosylation

Background

S-nitrosylation (or S-nitrosation) by Nitric Oxide (NO), i.e., the covalent attachment of a NO group to a cysteine thiol and formation of S-nitrosothiols (R-S-N=O or RSNO), has emerged as an important feature of NO biology and pathobiology. Many NO-related biological functions have been directly associated with the S-nitrosothiols and a considerable number of S-nitrosylated proteins have been identified which can positively or negatively regulate various cellular processes including signaling and metabolic pathways.

Scope of the review

Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by S-nitrosylation and Trx-mediated cellular homeostasis of S-nitrosothiols.

Major conclusions

Thioredoxin (Trx) system in mammalian cells utilizes thiol and selenol groups to maintain a reducing intracellular environment to combat oxidative/nitrosative stress. Reduced glutathione (GSH) and Trx system perform the major role in denitrosylation of S-nitrosylated proteins. However, under certain conditions, oxidized form of mammalian Trx can be S-nitrosylated and then it can trans-S-nitrosylate target proteins, such as caspase 3.

General significance

Investigations on the role of thioredoxin system in relation to biologically relevant RSNOs, their functions, and the mechanisms of S-denitrosylation facilitate the development of drugs and therapies. This article is part of a Special Issue entitled Regulation of Cellular Processes.     

Enhancement of Insulin Responsiveness by Nitric Oxide-mediated Inactivation of Protein-tyrosine Phosphatases

NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.     

S-Nitrosylation Inhibits Pannexin 1 Channel Function

S-Nitrosylation is a post-translational modification on cysteine(s) that can regulate protein function, and pannexin 1 (Panx1) channels are present in the vasculature, a tissue rich in nitric oxide (NO) species. Therefore, we investigated whether Panx1 can be S-nitrosylated and whether this modification can affect channel activity. Using the biotin switch assay, we found that application of the NO donor S-nitrosoglutathione (GSNO) or diethylammonium (Z)-1–1(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA NONOate) to human embryonic kidney (HEK) 293T cells expressing wild type (WT) Panx1 and mouse aortic endothelial cells induced Panx1 S-nitrosylation. Functionally, GSNO and DEA NONOate attenuated Panx1 currents; consistent with a role for S-nitrosylation, current inhibition was reversed by the reducing agent dithiothreitol and unaffected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a blocker of guanylate cyclase activity. In addition, ATP release was significantly inhibited by treatment with both NO donors. To identify which cysteine residue(s) was S-nitrosylated, we made single cysteine-to-alanine substitutions in Panx1 (Panx1^C40A, Panx1^C346A, and Panx1^C426A). Mutation of these single cysteines did not prevent Panx1 S-nitrosylation; however, mutation of either Cys-40 or Cys-346 prevented Panx1 current inhibition and ATP release by GSNO. This observation suggested that multiple cysteines may be S-nitrosylated to regulate Panx1 channel function. Indeed, we found that mutation of both Cys-40 and Cys-346 (Panx1^C40A/C346A) prevented Panx1 S-nitrosylation by GSNO as well as the GSNO-mediated inhibition of Panx1 current and ATP release. Taken together, these results indicate that S-nitrosylation of Panx1 at Cys-40 and Cys-346 inhibits Panx1 channel currents and ATP release.     

Protein Microarray Characterization of the S-Nitrosoproteome

Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO''s actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.It is known that NO regulates the majority of its physiologic function through S-nitrosylation (1). Protein-assisted or small molecule, S-nitrosoglutathione (GSNO)¹ trans-nitrosylation, oxidative S-nitrosation, and metalloprotein-catalyzed S-nitrosylation are the prominent cellular mechanisms that are utilized to S-nitrosylate proteins (2). A number of proteins are known to be S-nitrosylated and this post-translational modification can either activate or inactivate a protein''s biologic activity (1, 3). A number of attempts at probing tissue-specific S-nitrosoproteomes have been made, but the results of these are limited to proteins that are S-nitrosylated to a great degree and which are present at high concentrations (2, 4–6). Recently, to investigate determinants of S-nitrosylation, yeast and human target protein microarrays have been studied. However, these assay were limited because of the small number of proteins present on the chip (7). In addition, many proteins that are known to be S-nitrosylated have been studied through a targeted and biased approach (8). To overcome these shortcomings, we report the use of a 16,368 human protein microarray chip to better define the human S-nitrosoproteome.Ubiquitin is a 76-amino-acid long polypeptide that can be covalently added to lysine residues on targeted proteins either as single monomers or in chains. Ubiquitination of proteins can dramatically alter their function or localization depending on the number of ubiquitin attached and the nature of their linkages. The most well characterized ubiquitin-mediated process is targeting of the protein for degradation by the 26S proteasome, which occurs via poly-ubiquitination linked together through lysine 48 on the ubiquitin monomers. Ubiquitination occurs in a three-step enzymatic process in which the third enzyme, the ubiquitin protein ligase (E3) determines protein target specificity (9). NO S-nitrosylates the RING finger E3 ligases, parkin and XIAP, modifying their function (10, 11). In the case of parkin, S-nitrosylation transiently activates its E3 ligase activity, but ultimately inhibits its activity (12). In contrast, XIAP''s E3 ligase activity is unaffected by S-nitrosylation, but its anti-apoptotic function is compromised (11). Using the 16,368 human protein microarray, we identify a number of NO-regulated E3 ligases, the majority of which are activated by NO-dependent S-nitrosylation.     

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca²⁺-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.     

Structural Analysis of Cysteine S-Nitrosylation: A Modified Acid-Based Motif and the Emerging Role of Trans-Nitrosylation

S-Nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches that are capable of identifying nitrosylated Cys residues have been developed. However, the features underlying the specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid-base motif or hydrophobic areas and show high reactivity, low pK_a, and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated data set of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin, and physiological responses to NO. Analysis of this generic NO-Cys data set revealed that proximal acid-base motif, Cys pK_a, sulfur atom exposure, and Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid-base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly used for direct activation of Cys, the modified acid-base motif is engaged in protein-protein interactions thereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent.     

Regulation of Protein Function and Signaling by Reversible Cysteine S-Nitrosylation

NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications.     

S-nitrosylation of surfactant protein D as a modulator of pulmonary inflammation

Background

Surfactant protein D (SP-D) is a member of the family of proteins termed collagen-like lectins or “collectins” that play a role in non-antibody-mediated innate immune responses [1]. The primary function of SP-D is the modulation of host defense and inflammation [2].

Scope of review

This review will discuss recent findings on the physiological importance of SP-D S-nitrosylation in biological systems and potential mechanisms that govern SP-D mediated signaling.

Major conclusions

SP-D appears to have both pro- and anti-inflammatory signaling functions.SP-D multimerization is a critical feature of its function and plays an important role in efficient innate host defense. Under baseline conditions, SP-D forms a multimer in which the N-termini are hidden in the center and the C-termini are on the surface. This multimeric form of SP-D is limited in its ability to activate inflammation. However, NO can modify key cysteine residues in the hydrophobic tail domain of SP-D resulting in a dissociation of SP-D multimers into trimers, exposing the S-nitrosylated N-termini. The exposed S-nitrosylated tail domain binds to the calreticulin/CD91 receptor complex and initiates a pro-inflammatory response through phosphorylation of p38 and NF-κB activation [3,4]. In addition, the disassembled SP-D loses its ability to block TLR4, which also results in activation of NF-κB.

General significance

Recent studies have highlighted the capability of NO to modify SP-D through S-nitrosylation, causing the activation of a pro-inflammatory role for SP-D [3]. This represents a novel mechanism both for the regulation of SP-D function and NO's role in innate immunity, but also demonstrates that the S-nitrosylation can control protein function by regulating quaternary structure. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.     

Nitric oxide is required for the auxin-induced activation of NADPH-dependent thioredoxin reductase and protein denitrosylation during root growth responses in arabidopsis

Background and Aims Auxin is the main phytohormone controlling root development in plants. This study uses pharmacological and genetic approaches to examine the role of auxin and nitric oxide (NO) in the activation of NADPH-dependent thioredoxin reductase (NTR), and the effect that this activity has on root growth responses in Arabidopsis thaliana.Methods Arabidopsis seedlings were treated with auxin with or without the NTR inhibitors auranofin (ANF) and 1-chloro-2, 4-dinitrobenzene (DNCB). NTR activity, lateral root (LR) formation and S-nitrosothiol content were measured in roots. Protein S-nitrosylation was analysed by the biotin switch method in wild-type arabidopsis and in the double mutant ntra ntrb.Key Results The auxin-mediated induction of NTR activity is inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), suggesting that NO is downstream of auxin in this regulatory pathway. The NTR inhibitors ANF and DNCB prevent auxin-mediated activation of NTR and LR formation. Moreover, ANF and DNCB also inhibit auxin-induced DR5 : : GUS and BA3 : : GUS gene expression, suggesting that the auxin signalling pathway is compromised without full NTR activity. Treatment of roots with ANF and DNCB increases total nitrosothiols (SNO) content and protein S-nitrosylation, suggesting a role of the NTR-thioredoxin (Trx)-redox system in protein denitrosylation. In agreement with these results, the level of S-nitrosylated proteins is increased in the arabidopsis double mutant ntra ntrb as compared with the wild-type.Conclusions The results support for the idea that NTR is involved in protein denitrosylation during auxin-mediated root development. The fact that a high NO concentration induces NTR activity suggests that a feedback mechanism to control massive and unregulated protein S-nitrosylation could be operating in plant cells.     

Factors Associated with Nitric Oxide-mediated β2 Integrin Inhibition of Neutrophils

This investigation explored the mechanism for inhibition of β₂ integrin adhesion molecules when neutrophils are exposed to nitric oxide (^•NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a ^•NO flux of ∼28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400W), or with cells from mice lacking NOS-2 or the gp91^phox component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high ^•NO flux (∼112 nmol/min) that inactivates NOX.     

Post-translational S-Nitrosylation Is an Endogenous Factor Fine Tuning the Properties of Human S100A1 Protein

S100A1 is a member of the Ca²⁺-binding S100 protein family. It is expressed in brain and heart tissue, where it plays a crucial role as a modulator of Ca²⁺ homeostasis, energy metabolism, neurotransmitter release, and contractile performance. Biological effects of S100A1 have been attributed to its direct interaction with a variety of target proteins. The (patho)physiological relevance of S100A1 makes it an important molecular target for future therapeutic intervention. S-Nitrosylation is a post-translational modification of proteins, which plays a role in cellular signal transduction under physiological and pathological conditions. In this study, we confirmed that S100A1 protein is endogenously modified by Cys⁸⁵ S-nitrosylation in PC12 cells, which are a well established model system for studying S100A1 function. We used isothermal calorimetry to show that S-nitrosylation facilitates the formation of Ca²⁺-loaded S100A1 at physiological ionic strength conditions. To establish the unique influence of the S-nitroso group, our study describes high resolution three-dimensional structures of human apo-S100A1 protein with the Cys⁸⁵ thiol group in reduced and S-nitrosylated states. Solution structures of the proteins are based on NMR data obtained at physiological ionic strength. Comparative analysis shows that S-nitrosylation fine tunes the overall architecture of S100A1 protein. Although the typical S100 protein intersubunit four-helix bundle is conserved upon S-nitrosylation, the conformation of S100A1 protein is reorganized at the sites most important for target recognition (i.e. the C-terminal helix and the linker connecting two EF-hand domains). In summary, this study discloses cysteine S-nitrosylation as a new factor responsible for increasing functional diversity of S100A1 and helps explain the role of S100A1 as a Ca²⁺ signal transmitter sensitive to NO/redox equilibrium within cells.     

Protein S-nitrosylation: Role for nitric oxide signaling in neuronal death

Background

One of the signaling mechanisms mediated by nitric oxide (NO) is through S-nitrosylation, the reversible redox-based modification of cysteine residues, on target proteins that regulate a myriad of physiological and pathophysiological processes. In particular, an increasing number of studies have identified important roles for S-nitrosylation in regulating cell death.

Scope of review

The present review focuses on different targets and functional consequences associated with nitric oxide and protein S-nitrosylation during neuronal cell death.

Major conclusions

S-Nitrosylation exhibits double-edged effects dependent on the levels, spatiotemporal distribution, and origins of NO in the brain: in general Snitrosylation resulting from the basal low level of NO in cells exerts anti-cell death effects, whereas S-nitrosylation elicited by induced NO upon stressed conditions is implicated in pro-cell death effects.

General Significance

Dysregulated protein S-nitrosylation is implicated in the pathogenesis of several diseases including degenerative diseases of the central nervous system (CNS). Elucidating specific targets of S-nitrosylation as well as their regulatory mechanisms may aid in the development of therapeutic intervention in a wide range of brain diseases.     

Biochemical analysis of a Chinese cabbage phytocystatin-1

The phytocystatins are inhibitors of papain-like cysteine proteinases that are implicated in defense mechanisms and the regulation of protein turnover. BCPI-1, a Brassica rapa (Chinese cabbage) phytocystatin isolated from flower buds, contains an extended C-terminal region that contains a single Cys residue at position 102. In an effort to investigate the role of the C-terminus and this Cys residue in BCPI-1 activity, purified recombinant proteins of BCPI-1, including wild-type BCPI-1 (wtBCPI-1), N-terminus BCPI-1 (BCPI-1??C), C-terminus BCPI-1 (BCPI-1??N), and BCPI-1 with a single Cys residue exchange to Ser (BCPI-1C102S), were generated and their inhibitory activities against papain were investigated. Kinetic analysis revealed that the monomeric forms of wtBCPI-1 (K _i = 6.84 ± 0.3 × 10^?8 M) inhibited papain more efficiently than the dimeric forms of wtBCPI-1 (K _i = 1.01 ± 0.5 × 10^?7 M). Experiments with recombinant BCPI-1C102S demonstrated that the dimerization of wtBCPI-1 caused by the formation of an intermolecular disulfide bond at the cysteine residue. The inhibitory activity of the recombinant proteins, except BCPI-1??N, was reduced in the pH range of 7.0?C11.5 and was highly stable over a wide range of temperatures. Thus, dimerization mediated by the cysteine residue in the extended C-terminal region and alkaline conditions reduced the inhibitory activity of BCPI-1.     

Structural and molecular genetic insight into a widespread sulfur oxidation pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α₂β₂γ₂-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.     

Crystal structure of the Cys-NO modified YopH tyrosine phosphatase

Protein tyrosine phosphatases (PTPs) are key virulence factors in pathogenic bacteria, consequently, they have become important targets for new approaches against these pathogens, especially in the fight against antibiotic resistance. Among these targets of interest YopH (Yersinia outer protein H) from virulent species of Yersinia is an example. PTPs can be reversibly inhibited by nitric oxide (NO) since the oxidative modification of cysteine residues may influence the protein structure and catalytic activity. We therefore investigated the effects of NO on the structure and enzymatic activity of Yersinia enterocolitica YopH in vitro. Through phosphatase activity assays, we observe that in the presence of NO YopH activity was inhibited by 50%, and that this oxidative modification is partially reversible in the presence of DTT. Furthermore, YopH S-nitrosylation was clearly confirmed by a biotin switch assay, high resolution mass spectrometry (MS) and X-ray crystallography approaches. The crystal structure confirmed the S-nitrosylation of the catalytic cysteine residue, Cys403, while the MS data provide evidence that Cys221 and Cys234 might also be modified by NO. Interestingly, circular dichroism spectroscopy shows that the S-nitrosylation affects secondary structure of wild type YopH, though to a lesser extent on the catalytic cysteine to serine YopH mutant. The data obtained demonstrate that S-nitrosylation inhibits the catalytic activity of YopH, with effects beyond the catalytic cysteine. These findings are helpful for designing effective YopH inhibitors and potential therapeutic strategies to fight this pathogen or others that use similar mechanisms to interfere in the signal transduction pathways of their hosts.     

Periplasmic disulfide isomerase DsbC is involved in the reduction of copper binding protein CueP from Salmonella enterica serovar Typhimurium

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen with the ability to survive and replicate in macrophages. Periplasmic copper binding protein CueP is known to confer copper resistance to S. Typhimurium, and has been implicated in ROS scavenge activity by transferring the copper ion to a periplasmic superoxide dismutase or by directly reducing the copper ion. Structural and biochemical studies on CueP showed that its copper binding site is surrounded by conserved cysteine residues. Here, we present evidence that periplasmic disulfide isomerase DsbC plays a key role in maintaining CueP protein in the reduced state. We observed purified DsbC protein efficiently reduced the oxidized form of CueP, and that it acted on two (Cys104 and Cys172) of the three conserved cysteine residues. Furthermore, we found that a surface-exposed conserved phenylalanine residue in CueP was important for this process, which suggests that DsbC specifically recognizes the residue of CueP. An experiment using an Escherichia coli system confirmed the critical role played by DsbC in the ROS scavenge activity of CueP. Taken together, we propose a molecular insight into how CueP collaborates with the periplasmic disulfide reduction system in the pathogenesis of the bacteria.     

Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1

The loop following helix α2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k_cat value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the α2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K_M^GSH 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the α2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the α2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k_cat can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.     

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION*

In mammalian skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca²⁺-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO₂) through O₂-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O₂) versus low pO₂ (1% O₂), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO₂-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO₂ only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO₂-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO₂-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca²⁺ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in “hot spot” regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO₂-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.     

Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide sensor

OhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented by Bacillus subtilis OhrR) and 2-Cys (represented by Xanthomonas campestris OhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. The B. subtilis 1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, the X. campestris 2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-Cys B. subtilis OhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like the X. campestris OhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.     

The asymmetric trimeric architecture of [2Fe-2S] IscU: implications for its scaffolding during iron-sulfur cluster biosynthesis

IscU is a key component of the ISC machinery and is involved in the biogenesis of iron-sulfur (Fe-S) proteins. IscU serves as a scaffold for assembly of a nascent Fe-S cluster prior to its delivery to an apo protein. Here, we report the first crystal structure of IscU with a bound [2Fe-2S] cluster from the hyperthermophilic bacterium Aquifex aeolicus, determined at a resolution of 2.3 Å, using multiwavelength anomalous diffraction of the cluster. The holo IscU formed a novel asymmetric trimer that harbored only one [2Fe-2S] cluster. One iron atom of the cluster was coordinated by the S^γ atom of Cys36 and the N^ε atom of His106, and the other was coordinated by the S^γ atoms of Cys63 and Cys107 on the surface of just one of the protomers. However, the cluster was buried inside the trimer between the neighboring protomers. The three protomers were conformationally distinct from one another and associated around a noncrystallographic pseudo-3-fold axis. The three flexible loop regions carrying the ligand-binding residues (Cys36, Cys63, His106 and Cys107) and the N-terminal α1 helices were positioned at the interfaces and underwent substantial conformational rearrangement, which stabilized the association of the asymmetric trimer. This unique trimeric A. aeolicus holo-IscU architecture was clearly distinct from other known monomeric apo-IscU/SufU structures, indicating that asymmetric trimer organization, as well as its association/dissociation, would be involved in the scaffolding function of IscU.