Sulfenic Acid Modification Research Articles

Cysteinyl and methionyl redox switches: Structural prerequisites and consequences

Redox modifications of specific cysteinyl and methionyl residues regulate key enzymes and signal-transducing proteins in various pathways. Here, we analyzed the effect of redox modifications on protein structure screening the RCSB protein data bank for oxidative modifications of proteins, i.e. protein disulfides, mixed disulfides with glutathione, cysteinyl sulfenic acids, cysteinyl S-nitrosylation, and methionyl sulfoxide residues. When available, these structures were compared to the structures of the same proteins in the reduced state with respect to both pre-requirements for the oxidative modifications as well as the structural consequences of the modifications. In general, the conformational changes induced by the redox modification are small, i.e. within the range of normal fluctuations. Some redox modifications, disulfides in particular, induces alterations in the electrostatic properties of the proteins. Solvent accessibility does not seem to be a strict pre-requirement for the redox modification of a particular residue. We identified an enrichment of certain other amino acid residues in the vicinity of the susceptible residues, for disulfide and sulfenic acid modifications, for instance, histidyl and tyrosyl residues. These motifs, as well as the specific features of the susceptible sulfur-containing amino acids, may become helpful for the prediction of redox modifications.

Chemical proteomics reveals new targets of cysteine sulfinic acid reductase.

Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function, but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid (S-sulfenylation) and S-sulfinylation dynamic response to oxidative stress, indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore unknown protein substrates of the cysteine sulfinic acid reductase, sulfiredoxin (SRX), extending its function well beyond 2-Cys peroxiredoxins (2-Cys PRDX1–4), and offering new insight into the role of this unique oxidoreductase as a central mediator of ROS-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.

P-22 - Identification of cysteine residues responsible for redox regulation of phospholipase iPLA2γ

The mitochondrial Ca2+-independent phospholipase 2, isoform γ, (iPLA2γ) is directly activated by H2O2. Activation could be achieved by H2O2 reaction with reactive thiol groups. Our computational predictions revealed Cys559 and Cys714, to be the potential targets of human iPLA2γ to oxidative modifications. Purified recombinant iPLA2γ wild type and C559S, C714S, and C559S/C714S mutants were quantified for sulfenic acid modifications. Two methods utilizing BIAM addiction or Click-PEGylation reactions with subsequent Western analysis were used to test the reactivity and accessibility of the protein to redox modifications. Fully oxidized and reduced iPLA2γ forms exhibited different electrophoretic mobilities. Activity assays of recombinant iPLA2γ showed that all three mutants had lower catalytic activity induced by H2O2. Data are consistent with the upregulation of iPLA2γ by redox modification of reactive thiols and suggest S-sulfenylation as the post-translational modification of iPLA2γ.

Pathological hydrogen peroxide triggers the fibrillization of wild-type SOD1 via sulfenic acid modification of Cys-111

Amyotrophic lateral sclerosis (ALS) involves the abnormal posttranslational modifications and fibrillization of copper, zinc superoxide dismutase (SOD1) and TDP-43. However, how SOD1-catalyzed reaction product hydrogen peroxide affects amyloid formation of SOD1 and TDP-43 remains elusory. 90% of ALS cases are sporadic and the remaining cases are familial ALS. In this paper, we demonstrate that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 both in vitro and in SH-SY5Y cells. Using an anti-dimedone antibody that detects sulfenic acid modification of proteins, we found that Cys-111 in wild-type SOD1 is oxidized to C-SOH by pathological concentration of H2O2, followed by the formation of sulfenic acid modified SOD1 oligomers. Furthermore, we show that such SOD1 oligomers propagate in a prion-like manner, and not only drive wild-type SOD1 to form fibrils in the cytoplasm but also induce cytoplasm mislocalization and the subsequent fibrillization of wild-type TDP-43, thereby inducing apoptosis of living cells. Thus, we propose that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 and subsequently induces SOD1 toxicity and TDP-43 toxicity in neuronal cells via sulfenic acid modification of Cys-111 in SOD1. Our Western blot and ELISA data demonstrate that sulfenic acid modified wild-type SOD1 level in cerebrospinal fluid of 15 sporadic ALS patients is significantly increased compared with 6 age-matched control patients. These findings can explain how H2O2 at pathologic concentrations regulates the misfolding and toxicity of SOD1 and TDP-43 associated with ALS, and suggest that sulfenic acid modification of wild-type SOD1 should play pivotal roles in the pathogenesis of sporadic ALS.

Aldosterone does not alter endothelin B receptor signaling in the inner medullary collecting duct.

Recent studies suggest that aldosterone‐mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB‐stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin‐6c, an ETB‐selective agonist, and extracellular signal‐related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total‐ERK ratio similarly in control and aldosterone‐treated cells (aldosterone alone increased phospho/total‐ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP‐stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na+ diet, or low Na+ diet + desoxycorticosterone acetate. S6c inhibited AVP‐stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c‐stimulated cGMP accumulation in cultured IMCD, or S6c‐stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP‐dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD. Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD.

Sulfenic Acid Modification of Endothelin B Receptor is Responsible for the Benefit of a Nonsteroidal Mineralocorticoid Receptor Antagonist in Renal Ischemia.

AKI is associated with high mortality rates and the development of CKD. Ischemia/reperfusion (IR) is an important cause of AKI. Unfortunately, there is no available pharmacologic approach to prevent or limit renal IR injury in common clinical practice. Renal IR is characterized by diminished nitric oxide bioavailability and reduced renal blood flow; however, the mechanisms leading to these alterations are poorly understood. In a rat model of renal IR, we investigated whether the administration of the novel nonsteroidal mineralocorticoid receptor (MR) antagonist BR-4628 can prevent or treat the renal dysfunction and tubular injury induced by IR. Renal injury induced by ischemia was associated with increased oxidant damage, which led to a cysteine sulfenic acid modification in endothelin B receptor and consequently decreased endothelial nitric oxide synthase activation. These modifications were efficiently prevented by nonsteroidal MR antagonism. Furthermore, we demonstrated that the protective effect of BR-4628 against IR was lost when a selective endothelin B receptor antagonist was coadministered. These data describe a new mechanism for reduced endothelial nitric oxide synthase activation during renal IR that can be blocked by MR antagonism with BR-4628.

Proteomic analysis of peptides tagged with dimedone and related probes

Owing to its labile nature, a new role for cysteine sulfenic acid (-SOH) modification has emerged. This oxidative modification modulates protein function by acting as a redox switch during cellular signaling. The identification of proteins that undergo this modification represents a methodological challenge, and its resolution remains a matter of current interest. The development of strategies to chemically modify cysteinyl-containing peptides for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has increased significantly within the past decade. The method of choice to selectively label sulfenic acid is based on the use of dimedone or its derivatives. For these chemical probes to be effective on a proteome-wide level, their reactivity toward -SOH must be high to ensure reaction completion. In addition, the presence of an adduct should not interfere with electrospray ionization, the efficiency of induced dissociation in MS/MS experiments or with the identification of Cys-modified peptides by automated database searching algorithms. Herein, we employ a targeted proteomics approach to study the electrospray ionization and fragmentation effects of different -SOH specific probes and compared them to commonly used alkylating agents. We then extend our study to a whole proteome extract using shotgun proteomic approaches. These experiments enable us to demonstrate that dimedone adducts do not interfere with electrospray by suppressing the ionization nor impede product ion assignment by automated search engines, which detect a + 138 Da increase from unmodified peptides. Collectively, these results suggest that dimedone can be a powerful tool to identify sulfenic acid modifications by high-throughput shotgun proteomics of a whole proteome.

RegB Kinase Activity Is Repressed by Oxidative Formation of Cysteine Sulfenic Acid

RegB/RegA comprise a global redox-sensing signal transduction system utilized by a wide range of proteobacteria to sense environmental changes in oxygen tension. The conserved cysteine 265 in the sensor kinase RegB was previously reported to form an intermolecular disulfide bond under oxidizing conditions that converts RegB from an active dimer into an inactive tetramer. In this study, we demonstrate that a stable sulfenic acid (-SOH) derivative also forms at Cys-265 in vitro and in vivo when RegB is exposed to oxygen. This sulfenic acid modification is reversible and stable in the air. Autophosphorylation assay shows that reduction of the SOH at Cys-265 to a free thiol (SH) can increase RegB kinase activity in vitro. Our results suggest that a sulfenic acid modification at Cys-265 performs a regulatory role in vivo and that it may be the major oxidation state of Cys-265 under aerobic conditions. Cys-265 thus functions as a complex redox switch that can form multiple thiol modifications in response to different redox signals to control the kinase activity of RegB.

Endoplasmic Reticulum Thiol Oxidase Deficiency Leads to Ascorbic Acid Depletion and Noncanonical Scurvy in Mice

SummaryEndoplasmic reticulum (ER) thiol oxidases initiate a disulfide relay to oxidatively fold secreted proteins. We found that combined loss-of-function mutations in genes encoding the ER thiol oxidases ERO1α, ERO1β, and PRDX4 compromised the extracellular matrix in mice and interfered with the intracellular maturation of procollagen. These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins. Tissue ascorbic acid content was lower in mutant mice, and ascorbic acid supplementation improved procollagen maturation and lowered sulfenic acid content in vivo. In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H2O2-generating system. Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

Redox-Sensitive Sulfenic Acid Modification Regulates Surface Expression of the Cardiovascular Voltage-Gated Potassium Channel Kv1.5

Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

Activity of the tetrapyrrole regulator CrtJ is controlled by oxidation of a redox active cysteine located in the DNA binding domain

CrtJ from Rhodobacter capsulatus is a regulator of genes involved in the biosynthesis of haem, bacteriochlorophyll, carotenoids as well as structural proteins of the light harvesting-II complex. Fluorescence anisotropy-based DNA-binding analysis demonstrates that oxidized CrtJ exhibits ~20-fold increase in binding affinity over that of reduced CrtJ. Liquid chromatography electrospray tandem ionization mass spectrometric analysis using DAz-2, a sulfenic acid (-SOH)-specific probe, demonstrates that exposure of CrtJ to oxygen or to hydrogen peroxide leads to significant accumulation of a sulfenic acid derivative of Cys420 which is located in the helix-turn-helix (HTH) motif. In vivo labelling with 4-(3-azidopropyl)cyclohexane-1,3-dione (DAz-2) shows that Cys420 also forms a sulfenic acid modification in vivo when cells are exposed to oxygen. Moreover, a Cys420 to Ala mutation leads to a ~60-fold reduction of DNA binding activity while a Cys to Ser substitution at position 420 that mimics a cysteine sulfenic acid results in a ~4-fold increase in DNA binding activity. These results provide the first example where sulfenic acid oxidation of a cysteine in a HTH-motif leads to differential effects on gene expression.

Quantification of Protein Sulfenic Acid Modifications Using Isotope‐Coded Dimedone and Iododimedone

Quantitative proteomics: The new technique mentioned in the title—in short, ICDID—enables quantification of sulfenic acid modifications in proteins (see picture). The approach permits S-hydroxylation site occupancy to be monitored at individual cysteines within a single protein and is compatible with peptide-based proteomic strategies.

Quantification of Protein Sulfenic Acid Modifications Using Isotope‐Coded Dimedone and Iododimedone

Quantitative Proteomik: Die im Titel genannte Technik – kurz ICDID – ermöglicht die Quantifizierung und Verfolgung von Sulfensäuremodifikationen in Proteinen (siehe Bild), insbesondere die S-Hydroxylierung individueller Cysteine in einem einzelnen Protein. Die Methode ist mit peptidbasierten Proteomikstrategien kompatibel.

Sulfenic Acid Modification of Kv1.5: A Redox‐Sensitive Fate Switch for Channel Surface Expression

Atrial fibrillation, a life‐threatening arrhythmia, is strongly associated with oxidative stress. The voltage‐dependent potassium (Kv) channel, Kv1.5 is a prominent cardiovascular K+ channel that is vital for atrial repolarization. Numerous studies suggest that Kv channels are redox‐sensitive; however, a molecular link between oxidative stress and altered Kv1.5 expression/activity has not been established. We have discovered that Kv1.5 is modified with sulfenic acid, a novel, redox‐sensitive posttranslational modification to this channel. In HL‐1 cells, stabilizing sulfenic acid during acute oxidative stress decreases Kv1.5 cell surface expression and channel current. Sulfenic acid modification inhibits recycling of internalized Kv1.5 back to the cell surface and promotes channel degradation after chronic oxidant exposure. Thus, sulfenic acid modification of Kv1.5 represents a fate switch, in which further oxidation diverts the channel from a recycling to a degradation pathway, providing a molecular link between oxidative stress and altered channel expression in atrial fibrillation. Support: NIHGrants HL0270973 and T32ES07062 and AHA Predoctoral Fellowship 0910001G.

Possibilities and pitfalls in quantifying the extent of cysteine sulfenic acid modification of specific proteins within complex biofluids

BackgroundCysteine sulfenic acid (Cys-SOH) plays important roles in the redox regulation of numerous proteins. As a relatively unstable posttranslational protein modification it is difficult to quantify the degree to which any particular protein is modified by Cys-SOH within a complex biological environment. The goal of these studies was to move a step beyond detection and into the relative quantification of Cys-SOH within specific proteins found in a complex biological setting--namely, human plasma.ResultsThis report describes the possibilities and limitations of performing such analyses based on the use of thionitrobenzoic acid and dimedone-based probes which are commonly employed to trap Cys-SOH. Results obtained by electrospray ionization-based mass spectrometric immunoassay reveal the optimal type of probe for such analyses as well as the reproducible relative quantification of Cys-SOH within albumin and transthyretin extracted from human plasma--the latter as a protein previously unknown to be modified by Cys-SOH.ConclusionsThe relative quantification of Cys-SOH within specific proteins in a complex biological setting can be accomplished, but several analytical precautions related to trapping, detecting, and quantifying Cys-SOH must be taken into account prior to pursuing its study in such matrices.

Structural Basis for Catalytic Activation of Thiocyanate Hydrolase Involving Metal-Ligated Cysteine Modification

Thiocyanate hydrolase (SCNase) is a member of a family of nitrile hydratase proteins, each of which contains a unique noncorrin cobalt center with two post-translationally modified cysteine ligands, cysteine-sulfenic acid or -sulfenate (Cys-SO(H)), and cysteine-sulfininate (Cys-SO(2)(-)), respectively. We have found that a partially matured recombinant SCNase was activated during storage. The crystal structures of SCNase before and after storage demonstrated that Cys-SO(2)(-) modification of gammaCys131 proceeded to completion prior to storage, while Cys-SO(H) modification of gammaCys133 occurred during storage. SCNase activity was suppressed when gammaCys133 was further oxidized to Cys-SO(2)(-). The correlation between the catalytic activity and the extent of the gammaCys133 modification indicates that the cysteine sulfenic acid modification of gammaCys133 is of primary importance in determining the activity of SCNase.

Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies

Hydrogen peroxide (H2O2) functions as a second messenger that can activate cell proliferation through chemoselective oxidation of cysteine residues in signaling proteins. The connection between H2O2 signaling, thiol oxidation, and activation of growth pathways has emerged as fertile ground for the development of strategies for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the molecular events associated with tumorigenesis and evaluation of patient response to therapy. Here we report on the development of an immunochemical method for detecting sulfenic acid, the initial oxidation product that results when a thiolate reacts with H2O2. For this approach, the sulfenic acid is derivatized with a chemical tag to generate a unique epitope for recognition. The elicited antibody is exquisitely specific, context-independent, and capable of visualizing sulfenic acid formation in cells. Applying this approach to several systems, including cancer cell lines, shows it can be used to monitor differences in thiol redox status and reveals a diverse pattern of sulfenic acid modifications across different subtypes of breast tumors. These studies demonstrate a general strategy for producing antibodies against a specific oxidation state of cysteine and show the utility of these reagents for profiling thiol oxidation associated with pathological conditions such as breast cancer.

Mining the Thiol Proteome for Sulfenic Acid Modifications Reveals New Targets for Oxidation in Cells

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. We recently reported DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid modified proteins directly in living cells. Here we describe DAz-2, an analogue of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid modified proteins: 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Cross-comparison of these results with those from disulfide, S-glutathionylation, and S-nitrosylation proteomes reveals moderate overlap, suggesting fundamental differences in the chemical and biological basis for target specificity. The combination of selective chemical enrichment and live-cell compatibility makes DAz-2 a powerful new tool with the potential to reveal new regulatory mechanisms in signaling pathways and identify new therapeutic targets.

Crystal Structures of the Reduced, Sulfenic Acid, and Mixed Disulfide Forms of SarZ, a Redox Active Global Regulator in Staphylococcus aureus

SarZ is a global transcriptional regulator that uses a single cysteine residue, Cys(13), to sense peroxide stress and control metabolic switching and virulence in Staphylococcus aureus. SarZ belongs to the single-cysteine class of OhrR-MgrA proteins that play key roles in oxidative resistance and virulence regulation in various bacteria. We present the crystal structures of the reduced form, sulfenic acid form, and mixed disulfide form of SarZ. Both the sulfenic acid and mixed disulfide forms are structurally characterized for the first time for this class of proteins. The Cys(13) sulfenic acid modification is stabilized through two hydrogen bonds with surrounding residues, and the overall DNA-binding conformation is retained. A further reaction of the Cys(13) sulfenic acid with an external thiol leads to formation of a mixed disulfide bond, which results in an allosteric change in the DNA-binding domains, disrupting DNA binding. Thus, the crystal structures of SarZ in three different states provide molecular level pictures delineating the mechanism by which this class of redox active regulators undergoes activation. These structures help to understand redox-mediated virulence regulation in S. aureus and activation of the MarR family proteins in general.

Sulfenic Acid Modification: a Novel Link Between the Cardiovascular K+ Channel, Kv1.5, and Oxidative Stress

Atrial fibrillation, a life‐threatening arrhythmia, is strongly associated with oxidative stress. The voltage‐dependent potassium (Kv) channel, Kv1.5 is a prominent cardiovascular K+ channel that is vital for atrial repolarization. Numerous studies suggest that Kv channels are redox‐sensitive; however, a molecular link between oxidative stress and altered Kv1.5 expression/activity has not been established. Kv1.5 possesses intracellular cysteine residues that may be redox‐sensitive. Reversible formation of cysteine sulfenic acid in response to oxidative stress is a potentially important means of posttranslational protein regulation. Using a novel chemical probe, we find that Kv1.5 is modified with sulfenic acid in a redox‐sensitive manner. Mutagenesis to identify the redox‐sensitive cysteine residues reveals that sulfenic acid formation occurs on the COOH terminus. Using immunohistochemistry, we find that acute oxidant treatment decreases cell surface expression of Kv1.5, with no significant effect on Kv1.5 protein expression. Together, these data suggest that cellular redox state can induce formation of sulfenic acid and alter Kv1.5 cell surface density. Redox‐mediated alterations in cell surface density of ion channels may affect cellular excitability, leading to pro‐arrhythmic changes in cardiac action potentials. Research support: NIH Grants HL0270973 and T32ES07062.