Modification Of Cysteine Residues Research Articles

A triple cysteine motif as major determinant of the modulation of neuronal KV7 channels by the paracetamol metabolite N-acetyl-p-benzo quinone imine.

The analgesic action of paracetamol involves KV7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H2O2, or nitric oxide has been found to modulate currents through KV7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal KV7 channels might be responsible for the effects of NAPQI. To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant KV7.1 to KV7.5 channels. Currents through the cardiac subtype KV7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (KV7.3) or by such a shift combined with increased maximal current levels (KV7.2, KV7.4, KV7.5). A stretch of three cysteine residues in the S2-S3 linker region of KV7.2 was necessary and sufficient to mediate these effects. The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of KV7 subunits and reinforces channel gating in homomeric and heteromeric KV7.2 to KV7.5, but not in KV7.1 channels. In KV7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol.

NADPH oxidase-mediated sulfenylation of cysteine derivatives is key regulatory events for plant immune responses.

Reactive oxygen species (ROS) are rapidly generated during plant immune responses by RBOH, which is a plasma membrane-localizing NADPH oxidase. Although regulatory mechanisms of RBOH activity have been well documented, the ROS-mediated downstream signaling is unclear. We here demonstrated that ROS sensor proteins play a central role in the ROS signaling via oxidative post-translational modification of cysteine residues, sulfenylation. To detect protein sulfenylation, we used dimedone, which specifically and irreversibly binds to sulfenylated proteins. The sulfenylated proteins were labeled by dimedone in Nicotiana benthamiana leaves, and the conjugates were detected by immunoblot analyses. In addition, a reductant dissociated H2O2-induced conjugates, suggesting that cysteine persulfide and/or polysulfides are involved in sulfenylation. These sulfenylated proteins were continuously increased during both PTI and ETI in a RBOH-dependent manner. Pharmacological inhibition of ROS sensor proteins by dimedone perturbated cell death, ROS accumulation induced by INF1 and MEK2DD, and defense against fungal pathogens. On the other hand, Rpi-blb2-mediated ETI responses were rather enhanced by dimedone. These results suggest that the sulfenylation of cysteine and its derivatives in various ROS sensor proteins are important events in downstream of RBOH-dependent ROS burst to regulate plant immune responses.

Electrophilic Compounds in the Human Diet and Their Role in the Induction of the Transcription Factor NRF2.

The phrase "Let food be thy medicine…" means that food can be a form of medicine and medicine can be a form of food; in other words, that the diet we eat can have a significant impact on our health and well-being. Today, this phrase is gaining prominence as more and more scientific evidence suggests that one's diet can help prevent and treat disease. A diet rich in fruits, vegetables, whole grains, and lean protein can help reduce the risk of heart disease, cancer, diabetes, and other health problems and, on the other hand, a diet rich in processed foods, added sugars, and saturated fats can increase the risk of the same diseases. Electrophilic compounds in the diet can have a significant impact on our health, and they are molecules that covalently modify cysteine residues present in the thiol-rich Keap1 protein. These compounds bind to Keap1 and activate NRF2, which promotes its translocation to the nucleus and its binding to DNA in the ARE region, triggering the antioxidant response and protecting against oxidative stress. These compounds include polyphenols and flavonoids that are nucleophilic but are converted to electrophilic quinones by metabolic enzymes such as polyphenol oxidases (PPOs) and sulfur compounds present in foods such as the Brassica genus (broccoli, cauliflower, cabbage, Brussel sprouts, etc.) and garlic. This review summarizes our current knowledge on this subject.

Hydrogen sulfide improves endothelial barrier function by modulating the ubiquitination degradation of KLF4 through TRAF7 S-sulfhydration in diabetic aorta

Anomalous vascular endothelium significantly contributes to various cardiovascular diseases. VE-cadherin plays a vital role in governing the endothelial barrier. Krüppel-like factor 4(KLF4), as a transcription factor, which binds the VE-cadherin promoter and enhances its transcription. Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an E3 ubiquitin ligase that has been shown to modulate the degradation of KLF4. H2S can covalently modify cysteine residues on proteins through S-sulfhydration, thereby influencing the structure and functionality of the target protein. However, the role of S-sulfhydration on endothelial barrier integrity remains to be comprehensively elucidated. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates endothelial integrity and its underlying mechanism.In this study, we observed that protein S-sulfhydration was reduced in the endothelium during diabetes and TRAF7 was the main target. Overexpression of TRAF7-Cys327 mutant could mitigate the endothelial barrier damage by weakening TRAF7 interaction with KLF4 and reducing ubiquitination degradation of KLF4. In conclusion, our research demonstrates that H2S plays a pivotal role in regulating S-sulfhydration of TRAF7 at Cys327. This regulation effectively inhibits the ubiquitin-mediated degradation of KLF4, resulting in an upregulation of VE-cadherin levels. This molecular mechanism contributes to the prevention of endothelial barrier damage.

AChE activity self-breathing control mechanisms regulated by H2Sn and GSH: Persulfidation and glutathionylation on sulfhydryl after disulfide bonds cleavage

Hydrogen sulfide (H2S), or dihydrogen sulfane (H2Sn), acts as a signal molecule through the beneficial mechanism of persulfidation, known as the post-translational transformation of cysteine residues to persulfides. We previously reported that Glutathione (GSH) could regulate enzyme activity through S-desulfurization or glutathionylation of residues to generate protein-SG or protein-SSG, releasing H2S. However, little is known about the mechanisms by which H2Sn and GSH affect the disulfide bonds. In this study, we provide direct evidences that H2Sn and GSH modify the sulfhydryl group on Cys272, which forms disulfide bonds in acetylcholinesterase (AChE), to generate Cys-SSH and Cys-SSG, respectively. Glutathionylation of disulfide is a two-step reaction based on nucleophilic substitution, in which the first CS bond is broken, then the SS bond is broken to release H2S. H2Sn and GSH controlled self-breathing motion in enzyme catalysis by disconnecting specific disulfide bonds and modifying cysteine residues, thereby regulating AChE activity. Here, we elucidated H2Sn and GSH mechanisms on disulfide in the AChE system and proposed a self-breathing control theory induced by H2Sn and GSH. These theoretical findings shed light on the biological functions of H2Sn and GSH on sulfhydryl and disulfide bonds and enrich the theory of enzyme activity regulation.

Multiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.

4-Octyl Itaconate Alleviates Airway Eosinophilic Inflammation by Suppressing Chemokines and Eosinophil Development.

4-Octyl itaconate (4-OI) is a derivative of the Krebs cycle-derived metabolite itaconate and displays an array of antimicrobial and anti-inflammatory properties through modifying cysteine residues within protein targets. We have found that 4-OI significantly reduces the production of eosinophil-targeted chemokines in a variety of cell types, including M1 and M2 macrophages, Th2 cells, and A549 respiratory epithelial cells. Notably, the suppression of these chemokines in M1 macrophages was found to be NRF2-dependent. In addition, 4-OI can interfere with IL-5 signaling and directly affect eosinophil differentiation. In a model of eosinophilic airway inflammation in BALB/c mice, 4-OI alleviated airway resistance and reduced eosinophil recruitment to the lungs. Our findings suggest that itaconate derivatives could be promising therapeutic agents for the treatment of eosinophilic asthma.

Regulation of ERK2 activity by dynamic S-acylation

Extracellular signal-regulated kinases (ERK1/2) are key effector proteins of the mitogen-activated protein kinase pathway, choreographing essential processes of cellular physiology. Here, we discover that ERK1/2 are subject to S-acylation, a reversible lipid modification of cysteine residues, at C271/C254. The levels of ERK1/2 S-acylation are modulated by epidermal growth factor (EGF) signaling, mirroring its phosphorylation dynamics, and acylation-deficient ERK2 displays altered phosphorylation patterns. We show that ERK1/2 S-acylation is mediated by "writer" protein acyl transferases (PATs) and "eraser" acyl protein thioesterases (APTs) and that chemical inhibition of either lipid addition or removal alters ERK1/2's EGF-triggered transcriptional program. Finally, in a mouse model of metabolic syndrome, we find that ERK1/2 lipidation levels correlate with alterations in ERK1/2 lipidation writer/eraser expression, solidifying a link between ERK1/2 activity, ERK1/2 lipidation, and organismal health. This study describes how lipidation regulates ERK1/2 and offers insight into the role of dynamic S-acylation in cell signaling more broadly.

Querying the In Vitro Proteome Cysteine Reactivity of 8:2 Fluorotelomer Acrylate.

Despite the phase out of legacy per- and polyfluoroalkyl substances (PFAS), fluorotelomer-based polymers (FTP) have been used for many applications, notably textile surface coatings. FTPs are of a health concern due to their breakdown into legacy PFAS and the co-occurrence of fluorotelomer acrylate (FTAC) monomers, of which the latter may potentially react with cellular thiols. To evaluate this hypothesis, we employed fluorous-solid-phase extraction (FSPE), to enrich peptides covalently modified by 8:2 fluorotelomer acrylate (8:2 FTAC) and coupled it to a modified nano-liquid chromatography method for the identification of in vitro protein adducts using bottom-up data-dependent proteomics analysis. Using this method, over 100 unique peptides were detected with 8:2 FTAC modifications, although none of the modified cysteine residues were annotated active site nucleophiles. In parallel, a synthetic C6F13-iodoacetamide (F13-IAM) chemical probe was used to gauge the upper bound of PFAS-thiol reactivity. Over seven hundred peptides were detected with modifications but only 9 of 28 annotated active site cysteines in this dataset were modified by F13-IAM. Further exploration of the impacts of 8:2 FTAC adducts on protein function revealed that 8:2 FTAC modification promotes protein aggregation in vitro. These results suggest that 8:2 FTAC may exhibit significant proteome thiol reactivity and imply a more general mechanism of toxicity of PFAS-induced protein aggregation.

Second-Generation AUTACs for Targeted Autophagic Degradation.

Targeted protein degradation via the ubiquitin-proteasome system has emerged as one of the most promising drug discovery modalities. Autophagy, another intracellular degradation system, can target a wide range of nonproteinous substrates as well as proteins, but its application to targeted degradation is still in its infancy. Our previous work revealed a relationship between guanine modification of cysteine residues on intracellular proteins and selective autophagy, resulting in the first autophagy-based degraders, autophagy-targeted chimeras (AUTACs). Based on the research background, all the reported AUTACs compounds contain cysteine as a substructure. Here, we examine the importance of this substructure by conducting SAR studies and report significant improvements in the degrader's activity by replacing cysteine with other moieties. Several derivatives showed sub-μM range degrading activity, demonstrating the increased practical value of AUTACs.

Enzymatic Generation of Thioaldehyde Motifs by Flavin-Dependent Cysteine Decarboxylases for Peptide Bioconjugation and Macrocyclization.

Thioaldehyde is a highly electrophilic group under aqueous conditions and can be generated via oxidative enzymatic modifications of cysteine residues in peptides and proteins. Herein, we report the installation of thioaldehyde and aldehyde groups at the C-terminus of peptides by flavin-dependent cysteine decarboxylases from the biosynthesis of ribosomally synthesized and post-translationally modified peptides. The in situ generated (thio)aldehyde is utilized as a reactive handle for peptide bioconjugation and macrocyclization.

Hydrogen sulfide signalling in neurodegenerative diseases.

The gaseous neurotransmitter hydrogen sulfide (H2 S) exerts neuroprotective efficacy in the brain via post-translational modification of cysteine residues by sulfhydration, also known as persulfidation. This process is comparable in biological impact to phosphorylation and mediates a variety of signalling events. Unlike conventional neurotransmitters, H2 S cannot be stored in vesicles due to its gaseous nature. Instead, it is either locally synthesized or released from endogenous stores. Sulfhydration affords both specific and general neuroprotective effects and is critically diminished in several neurodegenerative disorders. Conversely, some forms of neurodegenerative disease are linked to excessive cellular H2 S. Here, we review the signalling roles of H2 S across the spectrum of neurodegenerative diseases, including Huntington's disease, Parkinson's disease, Alzheimer's disease, Down syndrome, traumatic brain injury, the ataxias, and amyotrophic lateral sclerosis, as well as neurodegeneration generally associated with ageing.

Discovery of Itaconate-Mediated Lysine Acylation.

Itaconate is an important antimicrobial and immunoregulatory metabolite involved in host-pathogen interactions. A key mechanistic action of itaconate is through the covalent modification of cysteine residues via Michael addition, resulting in "itaconation". However, it is unclear whether itaconate has other regulatory mechanisms. In this work, we discovered a novel type of post-translational modification by promiscuous antibody enrichment and data analysis with the open-search strategy and further confirmed it as the lysine "itaconylation". We showed that itaconylation and its precursor metabolite itaconyl-CoA undergo significant upregulation upon lipopolysaccharides (LPS) stimulation in RAW264.7 macrophages. Quantitative proteomics identified itaconylation sites in multiple functional proteins, including glycolytic enzymes and histones, some of which were confirmed by synthetic peptide standards. The discovery of lysine itaconylation opens up new areas for studying how itaconate participates in immunoregulation via protein post-translational modification.

Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis

Catechols have been reported to be potent covalent inhibitors of ureases, and they exhibit activity by modifying cysteine residues at the entrance to enzymatic active sites. Following these principles, we designed and synthesized novel catecholic derivatives that contained carboxylate and phosphonic/phosphinic functionalities and assumed expanded specific interactions. When studying the chemical stability of the molecules, we found that their intrinsic acidity catalyzes spontaneous esterification/hydrolysis reactions in methanol or water solutions, respectively. Regarding biological activity, the most promising compound, 2-(3,4-dihydroxyphenyl)-3-phosphonopropionic acid (15), exhibited significant anti-urease potential (Ki = 2.36 μM, Sporosarcinia pasteurii urease), which was reflected in the antiureolytic effect in live Helicobacter pylori cells at a submicromolar concentration (IC50 = 0.75 μM). As illustrated by molecular modeling, this compound was bound in the active site of urease through a set of concerted electrostatic and hydrogen bond interactions. The antiureolytic activity of catecholic phosphonic acids could be specific because these compounds were chemically inert and not cytotoxic to eukaryotic cells.

Methylmercury directly modifies the 105th cysteine residue in oncostatin M to promote binding to tumor necrosis factor receptor 3 and inhibit cell growth.

We previously found that methylmercury induces expression of oncostatin M (OSM), which is released extracellularly and binds to tumor necrosis factor receptor 3 (TNFR3), possibly enhancing its own toxicity. However, the mechanism by which methylmercury causes OSM to bind to TNFR3 rather than to its known receptors, OSM receptor and LIFR, is unknown. In this study, we aimed to elucidate the effect of methylmercury modification of cysteine residues in OSM on binding to TNFR3. Immunostaining of TNFR3-V5-expressing cells suggested that methylmercury promoted binding of OSM to TNFR3 on the cell membrane. In an in vitro binding assay, OSM directly bound to the extracellular domain of TNFR3, and this binding was promoted by methylmercury. Additionally, the formation of a disulfide bond in the OSM molecule was essential for the binding of both proteins, and LC/MS analysis revealed that methylmercury directly modified the 105th cysteine residue (Cys105) in OSM. Next, mutant OSM, in which Cys105 was replaced by serine or methionine, increased the binding to TNFR3, and a similar effect was observed in immunoprecipitation using cultured cells. Furthermore, cell proliferation was inhibited by treatment with Cys105 mutant OSMs compared with wildtype OSM, and this effect was cancelled by TNFR3 knockdown. In conclusion, we revealed a novel mechanism of methylmercury toxicity, in which methylmercury directly modifies Cys105 in OSM, thereby inhibiting cell proliferation via promoting binding to TNFR3. This indicates a chemical disruption in the interaction between the ligand and the receptor is a part of methylmercury toxicity.

Protein Persulfidation: Recent Progress and Future Directions.

Significance: Hydrogen sulfide (H2S) is considered to be a gasotransmitter along with carbon monoxide (CO) and nitric oxide (NO), and is known as a key regulator of physiological and pathological activities. S-sulfhydration (also known as persulfidation), a mechanism involving the formation of protein persulfides by modification of cysteine residues, is proposed here to explain the multiple biological functions of H2S. Investigating the properties of protein persulfides can provide a foundation for further understanding of the potential functions of H2S. Recent Advances: Multiple methods have been developed to determine the level of protein persulfides. It has been demonstrated that protein persulfidation is involved in many biological processes through various mechanisms including the regulation of ion channels, enzymes, and transcription factors, as well as influencing protein-protein interactions. Critical Issues: Some technical and theoretical questions remain to be solved. These include how to improve the specificity of the detection methods for protein persulfidation, why persulfidation typically occurs on one or a few thiols within a protein, how this modification alters protein functions, and whether protein persulfidation has organ-specific patterns. Future Directions: Optimizing the detection methods and elucidating the properties and molecular functions of protein persulfidation would be beneficial for current therapeutics. In this review, we introduce the detailed mechanism of the persulfidation process and discuss persulfidation detection methods. In addition, this review summarizes recent discoveries of the selectivity of protein persulfidation and the regulation of protein functions and cell signaling pathways by persulfidation. Antioxid. Redox Signal. 39, 829-852.

Proteome Dynamics of Persulfidation in Leaf Tissue under Light/Dark Conditions and Carbon Deprivation.

Hydrogen sulfide (H2S) acts as a signaling molecule in plants, bacteria, and mammals, regulating various physiological and pathological processes. The molecular mechanism by which hydrogen sulfide exerts its action involves the posttranslational modification of cysteine residues to form a persulfidated thiol motif. This research aimed to study the regulation of protein persulfidation. We used a label-free quantitative approach to measure the protein persulfidation profile in leaves under different growth conditions such as light regimen and carbon deprivation. The proteomic analysis identified a total of 4599 differentially persulfidated proteins, of which 1115 were differentially persulfidated between light and dark conditions. The 544 proteins that were more persulfidated in the dark were analyzed, and showed significant enrichment in functions and pathways related to protein folding and processing in the endoplasmic reticulum. Under light conditions, the persulfidation profile changed, and the number of differentially persulfidated proteins increased up to 913, with the proteasome and ubiquitin-dependent and ubiquitin-independent catabolic processes being the most-affected biological processes. Under carbon starvation conditions, a cluster of 1405 proteins was affected by a reduction in their persulfidation, being involved in metabolic processes that provide primary metabolites to essential energy pathways and including enzymes involved in sulfur assimilation and sulfide production.

Identification and Quantification of S-Sulfenylation Proteome of Mycobacterium tuberculosis under Oxidative Stress.

The ability to maintain redox homeostasis is critical for Mycobacterium tuberculosis (Mtb) to survive the redox stress of the host. There are many antioxidant systems in Mtb to ensure its normal replication and survival in the host, and cysteine thiols are one of them. S-sulfenylation is one of the reversible modifications of cysteine thiols to resist oxidative stress. In the study, we investigated the total cysteine thiols modification and S-sulfenylation modification of Mtb proteome under the oxidative stress provided by hydrogen peroxide. To determine and quantify the S-sulfenylation modified proteins, high specific IodoTMT6plex reagents and high resolution mass spectrometry were used to label and quantify the peptides and proteins modified. There are significant differences for the total cysteine modification levels of 279 proteins and S-sulfenylation modification levels of 297 proteins under hydrogen peroxide stress. Functional enrichment analysis indicated that these cysteine-modified proteins were involved in the oxidation-reduction process, fatty acid biosynthetic process, stress response, protein repair, cell wall, etc. In conclusion, our study provides a view of cysteine modifications of the Mtb proteome under oxidative stress, revealing a series of proteins that may play a role in maintaining redox homeostasis. IMPORTANCE With the continuous spread of drug-resistant tuberculosis, there is an urgent need for new antituberculosis drugs with new mechanisms. The ability of Mtb to resist oxidative stress is extremely important for maintaining redox homeostasis and survival in the host. The reversible modifications of cysteine residues have a dual role of protection from irreversible damage to protein functions and regulation, which plays an important role in the redox homeostasis system. Thus, to discover cysteine modification changes in the proteome level under oxidative stress is quintessential to elucidate its antioxidant mechanism. Our results provided a list of proteins involved in the antioxidant process that potentially could be considered targets for drug discovery and vaccine development. Furthermore, it is the first study to determine and quantify the S-sulfenylation-modified proteins in Mtb, which provided better insight into the Mtb response to the host oxidative defense and enable a deeper understanding of Mtb survival strategies.

Aurora Kinase A Regulation by Cysteine Oxidative Modification

Aurora kinase A (AURKA), which is a member of serine/threonine kinase family, plays a critical role in regulating mitosis. AURKA has drawn much attention as its dysregulation is critically associated with various cancers, leading to the development of AURKA inhibitors, a new class of anticancer drugs. As the spatiotemporal activity of AURKA critically depends on diverse intra- and inter-molecular factors, including its interaction with various protein cofactors and post-translational modifications, each of these pathways should be exploited for the development of a novel class of AURKA inhibitors other than ATP-competitive inhibitors. Several lines of evidence have recently shown that redox-active molecules can modify the cysteine residues located on the kinase domain of AURKA, thereby regulating its activity. In this review, we present the current understanding of how oxidative modifications of cysteine residues of AURKA, induced by redox-active molecules, structurally and functionally regulate AURKA and discuss their implications in the discovery of novel AURKA inhibitors.

Thioredoxin 1 promotes autophagy through transnitrosylation of Atg7 during myocardial ischemia.

Modification of cysteine residues by oxidative and nitrosative stress affects structure and function of proteins, thereby contributing to the pathogenesis of cardiovascular disease. Although the major function of thioredoxin 1 (Trx1) is to reduce disulfide bonds, it can also act as either a denitrosylase or transnitrosylase in a context-dependent manner. Here we show that Trx1 transnitrosylates Atg7, an E1-like enzyme, thereby stimulating autophagy. During ischemia, Trx1 was oxidized at Cys32-Cys35 of the oxidoreductase catalytic center and S-nitrosylated at Cys73. Unexpectedly, Atg7 Cys545-Cys548 reduced the disulfide bond in Trx1 at Cys32-Cys35 through thiol-disulfide exchange and this then allowed NO to be released from Cys73 in Trx1 and transferred to Atg7 at Cys402. Experiments conducted with Atg7 C402S-knockin mice showed that S-nitrosylation of Atg7 at Cys402 promotes autophagy by stimulating E1-like activity, thereby protecting the heart against ischemia. These results suggest that the thiol-disulfide exchange and the NO transfer are functionally coupled, allowing oxidized Trx1 to mediate a salutary effect during myocardial ischemia through transnitrosylation of Atg7 and stimulation of autophagy.