Cysteine Residues In Function Research Articles

Xanthohumol ameliorates drug-induced hepatic ferroptosis via activating Nrf2/xCT/GPX4 signaling pathway

BackgroundAs a canonical iron-dependent form of regulated cell death (RCD), ferroptosis plays a crucial role in chemical-induced liver injuries. Previous studies have demonstrated that xanthohumol (Xh), a natural prenylflavonoid isolated from hops, exhibits anti-inflammatory, anti-antioxidative and hepatoprotective properties. However, the regulatory effects of Xh on hepatic ferroptosis and the underlying mechanism have not yet been fully elucidated. PurposeTo investigate the hepatoprotective effects of Xh against drug-induced liver injury (DILI) and the regulatory effects of Xh on hepatic ferroptosis, as well as to reveal the underlying molecular mechanisms. Methods/study designThe hepatoprotective benefits of Xh were investigated in APAP-induced liver injury (AILI) mice and HepaRG cells. Xh was administered intraperitoneally to assess its in vivo effects. Histological and biochemical studies were carried out to evaluate liver damage. A series of ferroptosis-related markers, including intracellular Fe2+ levels, ROS and GSH levels, the levels of MDA, LPO and 4-HNE, as well as the expression levels of ferroptosis-related proteins and modulators were quantified both in vivo and in vitro. The modified peptides of Keap1 by Xh were characterized utilizing nano LC-MS/MS. ResultsXh remarkably suppresses hepatic ferroptosis and ameliorates AILI both in vitro and in vivo, via suppressing Fe2+ accumulation, ROS formation, MDA generation and GSH depletion, these observations could be considerably mitigated by the ferroptosis inhibitor ferrostatin-1 (Fer-1). Mechanistically, Xh could significantly activate the Nrf2/xCT/GPX4 signaling pathway to counteract AILI-induced hepatocyte ferroptosis. Further investigations showed that Xh could covalently modify three functional cysteine residues (cys151, 273, 288) of Keap1, which in turn, reduced the ubiquitination rates of Nrf2 and prolonged its degradation half-life. ConclusionsXh evidently suppresses hepatic ferroptosis and ameliorates AILI via covalent modifying three key cysteines of Keap1 and activating Nrf2/xCT/GPX4 signaling pathway.

Improving Catalytic Activity, Acid-Tolerance, and Thermal Stability of Glutathione Peroxidase by Systematic Site-Directed Selenocysteine Incorporation.

Glutathione peroxidase (GPx) is an important antioxidant enzyme. Selenocysteine (Sec)-containing GPxs (Sec-GPxs) are usually superior to their conventional cysteine-containing counterparts (Cys-GPxs), which make up the majority of the natural GPxs but display unsuitable activity and stability for industrial applications. This study first heterologously expressed and characterized a Cys-GPx from Lactococcus lactis (LlGPx), systematically exchanged all the three Cys to Sec and introduced an extra Sec. The results showed that the insertion of Sec at the active site could effectively increase the enzyme activity and confer a lower optimal pH value on the mutants. The double mutant C36U/L157U increased by 2.65 times (5.12 U/mg). The thermal stability of the C81U mutant was significantly improved. These results suggest that site-directed Sec incorporation can effectively improve the enzymatic properties of LlGPx, which may be also used for the protein engineering of other industrial enzymes containing catalytic or other functional cysteine residues.

Inflammation and Nitro-oxidative Stress as Drivers of Endocannabinoid System Aberrations in Mood Disorders and Schizophrenia.

The endocannabinoid system (ECS) is composed of the endocannabinoid ligands anandamide (AEA) and 2-arachidonoylgycerol (2-AG), their target cannabinoid receptors (CB1 and CB2) and the enzymes involved in their synthesis and metabolism (N-acyltransferase and fatty acid amide hydrolase (FAAH) in the case of AEA and diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL) in the case of 2-AG). The origins of ECS dysfunction in major neuropsychiatric disorders remain to be determined, and this paper explores the possibility that they may be associated with chronically increased nitro-oxidative stress and activated immune-inflammatory pathways, and it examines the mechanisms which might be involved. Inflammation and nitro-oxidative stress are associated with both increased CB1 expression, via increased activity of the NADPH oxidases NOX4 and NOX1, and increased CNR1 expression and DNA methylation; and CB2 upregulation via increased pro-inflammatory cytokine levels, binding of the transcription factor Nrf2 to an antioxidant response element in the CNR2 promoter region and the action of miR-139. CB1 and CB2 have antagonistic effects on redox signalling, which may result from a miRNA-enabled negative feedback loop. The effects of inflammation and oxidative stress are detailed in respect of AEA and 2-AG levels, via effects on calcium homeostasis and phospholipase A2 activity; on FAAH activity, via nitrosylation/nitration of functional cysteine and/or tyrosine residues; and on 2-AG activity via effects on MGLL expression and MAGL. Finally, based on these detailed molecular neurobiological mechanisms, it is suggested that cannabidiol and dimethyl fumarate may have therapeutic potential for major depressive disorder, bipolar disorder and schizophrenia.

SP3-FAIMS Chemoproteomics for High-Coverage Profiling of the Human Cysteinome*.

Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line high-field asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of >14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only ∼28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.

Budlein A methylacrylate demonstrates potent activity against triple-negative breast cancer by targeting IκBα kinase and exportin-1

Triple-negative breast cancer (TNBC) remains the most challenging breast cancer subtype to treat because there are no targeted therapies. Currently, chemotherapy is the only clinical option for TNBC despite development of resistance. New therapeutic agents with unique mechanisms of action are urgently needed; therefore, this study investigated the potential anti-TNBC effects of budlein A methylacrylate (BAM), a natural sesquiterpene lactone isolated from plants of the Helianthus genus. We discovered that BAM selectively suppressed and induced apoptosis TNBC cell growth versus other breast cancer or normal mammary epithelial cells. Mechanistically, BAM co-inhibited inhibitor of nuclear factor κBα (IκBα) kinase subunit β (IKKβ) and exportin-1 (XPO-1; chromosome region maintenance 1, CRM1), which are two dysregulated onco-related proteins in TNBC cells, by covalently modifying key functional cysteine residues (Cys179 of IKKβ, Cys528 of XPO-1). Dual inhibition led to the stabilization and nuclear retention of IκBα, impairment of NF-κB transcriptional activity, and consequent induction of TNBC cell apoptosis. In conclusion, this study provides evidence that co-inhibition of IKKβ and XPO-1 by BAM was effective against TNBC, demonstrating it as a representative new generation inhibitor with potential for TNBC treatment.

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect against the oxidative extracellular environment. These disulfide bonds are typically formed within the ER by a variety of oxidoreductases, including members of the protein disulfide isomerase (PDI) family. Here, we establish chemoproteomic platforms to identify oxidized and reduced cysteine residues within the ER. Subcellular fractionation methods were utilized to enrich for the ER and significantly enhance the coverage of ER-localized cysteine residues. Reactive-cysteine profiling ranked ∼900 secretory pathway cysteines by reactivity with an iodoacetamide-alkyne probe, revealing functional cysteines annotated to participate in disulfide bonds, or S-palmitoylation sites within proteins. Through application of a variation of the OxICAT protocol for quantifying cysteine oxidation, the percentages of oxidation for each of ∼700 ER-localized cysteines were calculated. Lastly, perturbation of ER function, through chemical induction of ER stress, was used to investigate the effect of initiation of the unfolded protein response (UPR) on ER-localized cysteine oxidation. Together, these studies establish a platform for identifying reactive and functional cysteine residues on proteins within the secretory pathway as well as for interrogating the effects of diverse cellular stresses on ER-localized cysteine oxidation.

Na,K-ATPase α-subunit conformation determines glutathionylation efficiency

The functioning of the N, K-ATPase depends on the redox status of cells and its activity is inhibited by oxidative stress and hypoxia. We previously found that redox sensitivity of the Na,K-ATPase is mediated by glutathionylation of the α-subunit. An increase in the level of glutathionylation of cysteine residues in the Na,K-ATPase α-subunit under stressful conditions leads to a decrease in the activity of the enzyme and a change in its receptor function. The structure of the Na,K-ATPase undergoes significant conformational changes during functioning. The effects of enzyme conformation on its ability to undergo glutathionylation are not clear. Here we show that the highest level of glutathionylation in the α-subunit of Na,K-ATPase is achieved in the E1 (Na+-induced) conformation. The transition of the Na,K-ATPase to the E2 (K+-induced) conformation leads to a decrease in the efficiency of glutathionylation. The lowest efficiency of Na,K-ATPase glutathionylation was observed in the E2P and E2P ouabain states. According to molecular modelling data, the maximum number of cysteine residues available for glutathionylation are present in the E1P conformation. In the E2P conformation, the main functional cysteine residues (Cys204, Cys242, Cys452, and Cys456) are buried from the solvent, which makes them inaccessible for glutathionylation. Thus, the efficiency of α-subunit glutathionylation depends on enzyme conformation, which is altered by bound ligands and proteins. A shift in the E1/E2 equilibrium towards prevalence of E1 can lead to better access for the relevant ligands and proteins to the binding site located in the Na,K-ATPase α-subunit. Na,K-ATPase.

Interrogation of Functional Mitochondrial Cysteine Residues by Quantitative Mass Spectrometry.

Mitochondria are cellular sites of diverse redox biology, including ROS production, iron-sulfur biogenesis, and secondary metabolism, which all rely on proteogenic reactive cysteine residues. Mass spectrometry-based proteomic methods to monitor the reactivity and functionality of cysteine residues across complex proteomes have greatly expanded over the past decade. Here we describe a mitochondrial isolation procedure coupled with cysteine-reactive IA labeling that affords identification and characterization of functional mitochondrial cysteine residues that were heretofore inaccessible to whole-cell proteomic analysis.

Phototaxis in a wild isolate of the cyanobacterium Synechococcus elongatus

Many cyanobacteria, which use light as an energy source via photosynthesis, have evolved the ability to guide their movement toward or away from a light source. This process, termed "phototaxis," enables organisms to localize in optimal light environments for improved growth and fitness. Mechanisms of phototaxis have been studied in the coccoid cyanobacterium Synechocystis sp. strain PCC 6803, but the rod-shaped Synechococcus elongatus PCC 7942, studied for circadian rhythms and metabolic engineering, has no phototactic motility. In this study we report a recent environmental isolate of S. elongatus, the strain UTEX 3055, whose genome is 98.5% identical to that of PCC 7942 but which is motile and phototactic. A six-gene operon encoding chemotaxis-like proteins was confirmed to be involved in phototaxis. Environmental light signals are perceived by a cyanobacteriochrome, PixJSe (Synpcc7942_0858), which carries five GAF domains that are responsive to blue/green light and resemble those of PixJ from Synechocystis Plate-based phototaxis assays indicate that UTEX 3055 uses PixJSe to sense blue and green light. Mutation of conserved functional cysteine residues in different GAF domains indicates that PixJSe controls both positive and negative phototaxis, in contrast to the multiple proteins that are employed for implementing bidirectional phototaxis in Synechocystis.

Chemical‐proteomic strategies to investigate reactive cysteines

Cysteine residues regulate the activity of diverse proteins via post translational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. To identify regulatory cysteines in human proteomes, we have developed chemical‐proteomic tools to monitor cysteine reactivity both in vitro as well as directly in living cells. These global profiling methods have been applied to monitor the susceptibility of cysteines to S‐nitrosation and to identify redox‐active disulfides induced by epidermal‐growth factor (EGF) stimulation. In order to identify cysteine residues hypersensitive to S‐nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of transnitrosating agents. Our proteomic studies identified several previously unannotated cysteines, which we proceeded to demonstrate were important regulators of protein activity. To identify cysteine oxidation events triggered by EGF stimulation, we developed a caged cysteine‐reactive probe to enable profiling of cysteine reactivity directly in living cells. We identified several redox‐active disulfides that were formed upon EGF stimulation for future functional characterization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isotopically-Labeled Iodoacetamide-Alkyne Probes for Quantitative Cysteine-Reactivity Profiling

Cysteine residues on proteins serve a variety of catalytic and regulatory functions due to the high nucleophilicity and redox activity of the thiol group. Quantitative proteomic platforms for profiling cysteine reactivity can provide valuable information related to the post-translational modification state and inhibitor occupancy of functional cysteine residues within a complex proteome. Cysteine-reactivity profiling typically monitors changes in the extent of cysteine labeling by cysteine-reactive chemical probes, such as iodoacetamide (IA)-alkyne. To enable accurate measurements of cysteine reactivity changes, isotopic labels are introduced into the two proteomes of interest using either isotopically tagged proteomes (SILAC) or cleavable linkers (isoTOP-ABPP) that are installed using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here we provide an alternative strategy for isotopic tagging of two proteomes for cysteine-reactivity profiling by developing IA-light and IA-heavy, a pair of isotopically labeled iodoacetamide-alkyne probes. These probes can be utilized for proteome samples that are not amenable to SILAC labeling and are facile to synthesize, especially when compared to the isotopically tagged cleavable linkers. We confirm the quantitative accuracy of IA-light and IA-heavy by assessing cysteine reactivity in a purified thioredoxin protein, as well as globally within a complex proteome where IA-light treatment generates mass-spectrometry identification of 992 cysteine residues. Importantly, these isotopically tagged probes can also be utilized for quantifying the percentage of cysteine modification within a single sample. Preliminary data supports the use of these tags to quantify the stoichiometry of TCEP-susceptible cysteine oxidation events in cell lysates.

Quantitative Chemical Proteomic Profiling of the in Vivo Targets of Reactive Drug Metabolites.

Idiosyncratic liver toxicity represents an important problem in drug research and pharmacotherapy. Reactive drug metabolites that modify proteins are thought to be a principal factor in drug-induced liver injury. Here, we describe a quantitative chemical proteomic method to identify the targets of reactive drug metabolites in vivo. Treating mice with clickable analogues of four representative hepatotoxic drugs, we demonstrate extensive covalent binding that is confined primarily to the liver. Each drug exhibited a distinct target profile that, in certain cases, showed strong enrichment for specific metabolic pathways (e.g., lipid/sterol pathways for troglitazone). Site-specific proteomics revealed that acetaminophen reacts with high stoichiometry with several conserved, functional (seleno)cysteine residues throughout the liver proteome. Our findings thus provide an advanced experimental framework to characterize the proteomic reactivity of drug metabolites in vivo, revealing target profiles that may help to explain mechanisms and identify risk factors for drug-induced liver injury.

Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species. Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that react selectively with cysteine sulfenic acid formed in proteins via oxidation or hydrolysis reactions. Here, we report analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were identified for >1280 S-sulfenylated cysteines present in "druggable" proteins and orphan targets, revealing disparate reactivity profiles and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile that reacted preferentially with protein tyrosine phosphatases. Fragment-based covalent ligand discovery with C-nucleophiles affords an expansive snapshot of the ligandable "redoxome" with significant implications for covalent inhibitor pharmacology and also affords new chemical tools to investigate redox-regulation of protein function.

The Role of Cysteine Residues in Catalysis of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S–S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S–S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

A Chemical‐Proteomic Platform to Investigate Cysteine S‐Nitrosation

Cysteine residues on proteins play numerous functional roles as catalytic nucleophiles, redox‐active disulfides, or metal‐binding residues. Outside of their direct role in catalysis, cysteines can also regulate protein activity via posttranslational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. We hypothesize that cysteines that are hypersensitive to different oxidants or nitrosating agents are likely to be enriched in functional residues that serve to modulate protein activity. To explore this hypothesis, we focused our initial efforts on investigating cysteine susceptibility to nitrosation. S‐nitrosation is a unique posttranslational modification that serves to temporally and spatially control protein activity and localization. In order to identify cysteine residues hypersensitive to nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of nitric oxide donors. Our proteomic studies identified several known sites of nitrosation, as well as previously unannotated cysteines. We then proceeded to functionally characterize several of the unannotated cysteines using a variety of cell and molecular biology approaches. We show that many of these cysteines are located distal from the active site, and yet mediate essential protein functions such as substrate binding and localization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.

The succinated proteome

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pKa thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨm) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches.

Large-Scale Gene Disruption in Magnaporthe oryzae Identifies MC69, a Secreted Protein Required for Infection by Monocot and Dicot Fungal Pathogens

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

Role of Cysteine Residues in Cell Surface Expression and Function of the Human Riboflavin Transporter-2 (hRFT2) in Intestinal Epithelial Cells

Role of Cysteine Residues in Cell Surface Expression and Function of the Human Riboflavin Transporter-2 (hRFT2) in Intestinal Epithelial Cells

Redox biology: computational approaches to the investigation of functional cysteine residues.

Cysteine (Cys) residues serve many functions, such as catalysis, stabilization of protein structure through disulfides, metal binding, and regulation of protein function. Cys residues are also subject to numerous post-translational modifications. In recent years, various computational tools aiming at classifying and predicting different functional categories of Cys have been developed, particularly for structural and catalytic Cys. On the other hand, given complexity of the subject, bioinformatics approaches have been less successful for the investigation of regulatory Cys sites. In this review, we introduce different functional categories of Cys residues. For each category, an overview of state-of-the-art bioinformatics methods and tools is provided, along with examples of successful applications and potential limitations associated with each approach. Finally, we discuss Cys-based redox switches, which modify the view of distinct functional categories of Cys in proteins.