Redox-sensitive Cysteine Residues Research Articles

The Small Matter of a Red Ox, a Particularly Sensitive Pink Cat, and the Quest for the Yellow Stone of Wisdom

The article describes how recent advances in chalcogen Redox Biology shape the future of nutrition, drug design, agriculture, and environmental management. Since the turn of the Millennium, the biological chemistry of redox active sulfur species has witnessed various significant developments, with cysteine side-chains in proteins and enzymes emerging as centers of redox signaling and control and inspiring new concepts, such as the sulfur redoxome, the sulfenome, and the cellular thiolstat. Since then, it has emerged that redox sensitive cysteine residues are preferred targets of Reactive Sulfur Species (RSS), certain metal ions, and the emerging class of Reactive Selenium Species (RSeS). In addition, the cellular redoxome provides the basis for targeted redox modulation, for instance via nutritional intervention in the sick and elderly; it paves the way for highly selective catalytic sensor/effector agents active against a spectrum of redox-related diseases and lightens up possible avenues leading towards green phyto-protectants, often in cahoots with modern nanotechnology. Whilst the activity of redox-active food ingredients and multifunctional redox-modulating compounds on and in cells is complicated, modern techniques such as redox proteomics and chemogenetic phenotype profiling in combination with fluorescent-based “intracellular diagnostics” can be employed to illuminate certain changes, pathways, and eventually, also mode(s) of action. Undoubtedly, chalcogen-based redox systems will shape future research and development in nutrition, drug design, cosmetics, green agriculture, and waste management.

Bardoxolone methyl induces neuritogenesis in Neuro2a cells.

Bardoxolone methyl (RTA 402, CDDOMe) has been long known for its anti-inflammatory and exceptional cytotoxic activity. The biological responses to CDDOMe are truly dose dependent. And owing to the structural modifications introduced in its parent molecule oleanolic acid, CDDOMe is able to form reversible adducts with cellular proteins containing redox sensitive cysteine residues. This nature of CDDOMe makes it a multifunctional molecule targeting multiple signaling pathways. This study was initiated to study the response of Neuro2a, a mouse neuroblastoma cell line to CDDOMe. Neuro2a cells were treated with CDDOMe and all trans retinoic acid (ATRA) for 4days and observed for neurite outgrowth. The neurite length was estimated using ImageJ software (Neuron growth plugin). Cell viability was investigated using MTT dye reduction and trypan blue dye exclusion method. Gene expression of differentiation markers was analyzed using quantitative PCR. Cellular localization of Tuj1 and synaptophysin in differentiated Neuro2a cells was observed using immunofluorescence. CDDOMe ceased proliferation and induced dramatic neurite outgrowth in Neuro2a cells. These morphological changes were accompanied by time dependent increase in the mRNA levels of tyrosine hydroxylase, neurofilament 200 and synaptophysin. Besides, cytoskeleton protein Tuj1 and the synaptic vesicle protein synaptophysin were also observed to be localized in the neurites induced by CDDOMe. These early shreds of evidence suggest that CDDOMe induces differentiation in Neuro2a cells at concentrations ranging from 0.2 to 0.4μM and indeed contributes the existing knowledge on CDDOMe induced activities in cells.

Redox responses are preserved across muscle fibres with differential susceptibility to aging

Age-related loss of muscle mass and function is associated with increased frailty and loss of independence. The mechanisms underlying the susceptibility of different muscle types to age-related atrophy are not fully understood. Reactive oxygen species (ROS) are recognised as important signalling molecules in healthy muscle and redox sensitive proteins can respond to intracellular changes in ROS concentrations modifying reactive thiol groups on Cysteine (Cys) residues. Conserved Cys residues tend to occur in functionally important locations and can have a direct impact on protein function through modifications at the active site or determining protein conformation. The aim of this work was to determine age-related changes in the redox proteome of two metabolically distinct murine skeletal muscles, the quadriceps a predominantly glycolytic muscle and the soleus which contains a higher proportion of mitochondria. To examine the effects of aging on the global proteome and the oxidation state of individual redox sensitive Cys residues, we employed a label free proteomics approach including a differential labelling of reduced and reversibly oxidised Cys residues. Our results indicate the proteomic response to aging is dependent on muscle type but redox changes that occur primarily in metabolic and cytoskeletal proteins are generally preserved between metabolically distinct tissues. Biological significanceSkeletal muscle containing fast twitch glycolytic fibres are more susceptible to age related atrophy compared to muscles with higher proportions of oxidative slow twitch fibres. Contracting skeletal muscle generates reactive oxygen species that are required for correct signalling and adaptation to exercise and it is also known that the intracellular redox environment changes with age. To identify potential mechanisms for the distinct response to age, this article combines a global proteomic approach and a differential labelling of reduced and reversibly oxidised Cysteine residues in two metabolically distinct skeletal muscles, quadriceps and soleus, from adult and old mice. Our results indicate that the global proteomic changes with age in skeletal muscles are dependent on fibre type. However, redox specific changes are preserved across muscle types and accompanied with a reduction in the number of redox sensitive Cysteine residues.

A new mitofusin topology places the redox-regulated C terminus in the mitochondrial intermembrane space.

Mitochondrial fusion occurs in many eukaryotes, including animals, plants, and fungi. It is essential for cellular homeostasis, and yet the underlying mechanisms remain elusive. Comparative analyses and phylogenetic reconstructions revealed that fungal Fzo1 and animal Mitofusin proteins are highly diverged from one another and lack strong sequence similarity. Bioinformatic analysis showed that fungal Fzo1 proteins exhibit two predicted transmembrane domains, whereas metazoan Mitofusins contain only a single transmembrane domain. This prediction contradicts the current models, suggesting that both animal and fungal proteins share one topology. This newly predicted topology of Mfn1 and Mfn2 was demonstrated biochemically, confirming that the C-terminal, redox-sensitive cysteine residues reside within the intermembrane space (IMS). Functional experiments established that redox-mediated disulfide modifications within the IMS domain are key modulators of reversible Mfn oligomerization that drives fusion. Together, these results lead to a revised understanding of Mfns as single-spanning outer membrane proteins with an Nout-Cin orientation, providing functional insight into the IMS contribution to redox-regulated fusion events.

Involvement of Transient Receptor Potential Cation Channel Member A1 activation in the irritation and pain response elicited by skin-lightening reagent hydroquinone

Hydroquinone (HQ) is one of the most frequently used and effective skin-lightening products to treat skin hyperpigmentation disorders, including postinflammatory hyperpigmentation, melasma and solar lentigines. HQ is also widely used in cosmetic products for skin whitening. However, HQ treatment can evoke substantial skin irritation, a side effect that remains poorly understood. Here we demonstrate that HQ is an activator of the peripheral irritant receptor transient receptor potential (TRP) cation channel member A1 (TRPA1). HQ failed to activate TRPV1, TRPV4 or TRPM8. HQ-induced TRPA1 activation was dependent on essential redox-sensitive cysteine and lysine residues within N-terminus of channel protein. HQ elicited Ca2+ influx in a subpopulation of mouse sensory neurons sensitive to the TRPA1 agonist, mustard oil. HQ-induced neuronal responses were significantly reduced by TRPA1 inhibitors, and reduced in neurons isolated from Trpa1-deficient mice. In mice, intraplantar injection of HQ at clinically relevant concentrations elicited both acute pain and persistent mechanical hyperalgesia which were almost completely abolished by TRPA1 inhibitors. These findings identify TRPA1 as a molecular target for HQ and provide insights into the mechanism of HQ-induced skin irritation. These findings also suggest that selective TRPA1 antagonists may be useful to counteract HQ-induced skin irritation.

Peroxisomes as Modulators of Cellular Protein Thiol Oxidation: A New Model System.

Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H2O2 release. Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H2O2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H2O2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined. This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H2O2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches. Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiol-based signaling networks.

The Multifaceted Roles of DJ-1 as an Antioxidant.

The DJ-1 protein was originally linked with Parkinson's disease and is now known to have antioxidant functions. The protein has three redox-sensitive cysteine residues, which are involved in its dimerisation and functional properties. A mildly oxidised form of DJ-1 is the most active form and protects cells from oxidative stress conditions. DJ-1 functions as an antioxidant through a variety of mechanisms, including a weak direct antioxidant activity by scavenging reactive oxygen species. DJ-1 also regulates a number of signalling pathways, including the inhibition of apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis under oxidative stress conditions. Other proteins regulated by DJ-1 include enzymes, chaperones, the 20S proteasome and transcription factors, including Nrf2. Once activated by oxidative stress, Nrf2 upregulates antioxidant gene expression including members of the thioredoxin and glutathione pathways, which in turn mediate an antioxidant protective function. Crosstalk between DJ-1 and both the thioredoxin and glutathione systems has also been identified. Thioredoxin reduces a cysteine residue on DJ-1 to modulate its activity, while glutaredoxin1 de-glutathionylates DJ-1, preventing degradation of DJ-1 and resulting in its accumulation. DJ-1 also regulates the activity of glutamate cysteine ligase, which is the rate-limiting step for glutathione synthesis. These antioxidant functions of DJ-1 are key to its role in protecting neurons from oxidative stress and are hypothesised to protect the brain from the development of neurodegenerative diseases such as Parkinson's disease (PD)and to protect cardiac tissues from ischaemic-reperfusion injury. However, DJ-1, as an antioxidant, also protects cancer cells from undergoing oxidative stress-induced apoptosis.

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice.

BackgroundReactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants.MethodsWe used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2).ResultsWe identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells.ConclusionsThe findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-017-0115-3) contains supplementary material, which is available to authorized users.

The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H2O2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H2O2-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.

Characterization of novel small-molecule NRF2 activators: Structural and biochemical validation of stereospecific KEAP1 binding

BackgroundSemi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. MethodsWe measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. ResultsWe found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. ConclusionsAlthough KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. General significanceThis work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.

Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein.

HMGB1 protein and linker histone H1 have overlapping binding sites in the nucleosome. HMGB1 has been implicated in many DNA-dependent processes in chromatin involving binding of specific proteins, including transcription factors, to DNA sites pre-bent by HMGB1. HMGB1 can also act as an extracellular signaling molecule by promoting inflammation, tumor growth a metastasis. Many of the intra- and extracellular functions of HMGB1 depend on redox-sensitive cysteine residues of the protein. Here we report that mild oxidization of HMGB1 (and much less mutation of cysteines involved in disulphide bond formation) can severely compromise the functioning of the protein as a DNA chaperone by inhibiting its ability to unwind or bend DNA. Histone H1 (via the highly basic C-terminal domain) significantly inhibits DNA bending by the full-length HMGB1, and the inhibition is further enhanced upon oxidization of HMGB1. Interestingly, DNA bending by HMGB1 lacking the acidic C-tail (HMGB1ΔC) is much less affected by histone H1, but oxidization rendered DNA bending by HMGB1ΔC and HMGB1 equally prone for inhibition by histone H1. Possible consequences of histone H1-mediated inhibition of DNA bending by HMGB1 of different redox state for the functioning of chromatin are discussed.

Hydrogen peroxide - production, fate and role in redox signaling of tumor cells.

Hydrogen peroxide (H2O2) is involved in various signal transduction pathways and cell fate decisions. The mechanism of the so called “redox signaling” includes the H2O2-mediated reversible oxidation of redox sensitive cysteine residues in enzymes and transcription factors thereby altering their activities. Depending on its intracellular concentration and localization, H2O2 exhibits either pro- or anti-apoptotic activities. In comparison to normal cells, cancer cells are characterized by an increased H2O2 production rate and an impaired redox balance thereby affecting the microenvironment as well as the anti-tumoral immune response. This article reviews the current knowledge about the intracellular production of H2O2 along with redox signaling pathways mediating either the growth or apoptosis of tumor cells. In addition it will be discussed how the targeting of H2O2-linked sources and/or signaling components involved in tumor progression and survival might lead to novel therapeutic targets.

Acrolein Induces Epithelial Injury and Remodeling Through Direct Activation of Src

Cigarette smoke (CS)-derived reactive electrophiles such as acrolein affect biological systems largely by targeting redox-sensitive cysteine residues. In the present studies we examined the effects of acrolein exposure on the airway epithelium and importance of the redox-sensitive tyrosine kinase Src in these responses. Exposure of mouse tracheal epithelial (MTE) cells in Transwell® inserts to acrolein resulted in loss of trans-epithelial resistance (TER) and increased permeability to FITC-dextran, accompanied with a loss of epithelial junction proteins E-cadherin and zonal occludin-1 (ZO-1). These responses were prevented by pretreatment with the Src inhibitor PP2. Similar studies with human epithelial H292 cells indicated that inhibition of Src using PP2 or siRNA targeting prevented acrolein-induced phosphorylation of p120 and loss of E-cadherin. Acute exposure of C57BL/6 mice to acrolein (5 ppm for 4 hrs) increased epithelial permeability, measured by enhanced leakage of i.v. injected FITC-dextran into...

Redox regulation of mammalian 3-mercaptopyruvate sulfurtransferase.

A cystine-catabolizing enzyme, 3-mercaptopyruvate sulfurtransferase catalyzes the trans-sulfuration reaction of mercaptopyruvate or thiosulfate to thiol-containing compounds or cyanide. During the catalytic process, persulfide is formed at the catalytic site cysteine residue and a sulfur-acceptor substrate donates the outer sulfur of the persulfide to form a new persulfide molecule. Subsequently, the molecule can be reduced by thioredoxin to form hydrogen sulfide. The enzyme is regulated by redox changes via two redox-sensing molecular switches consisting redox-sensitive cysteine residues. One switch is the catalytic cysteine in itself, which is oxidized to form a cysteine-sulfenate resulting in inhibition of catalytic activity. The sulfenate can be reduced by thioredoxin resulting in restoration of the activity. The redox potential of sulfenate is lower than that of glutathione and greater than that of thioredoxin. The other switch involves cysteine residues positioned on the surface of the enzyme. The oxidation the intermolecular disulfide linkage at these cysteine residues, leading to dimer formation, inhibits enzyme activity. On the other hand, reduction-associated monomer formation increases catalytic activity. Thioredoxin reduces the disulfide bond more effectively than dithiothreitol, although the specificity mechanism has not been identified. Congenital defects in this enzyme result in, mercaptolactate-cysteine disulfiduria associated with or without mental retardation. However, the pathogenesis has not been identified. Because 3-mercaptopyruvate sulfurtransferase serves as a cellular antioxidative protein, the other biological functions related to the inhabitant disease are being investigated.

Cysteine-mediated redox signalling in the mitochondria.

The mitochondria are critical mediators of cellular redox homeostasis due to their role in the generation and dissipation of reactive oxygen/nitrogen species (ROS/RNS). Modulations in ROS/RNS levels in the mitochondria are often reflected through oxidation/nitrosation of highly redox-sensitive cysteine residues within this organelle. Oxidation/nitrosation of functional cysteines on mitochondrial proteins serves to modulate protein activity, localization, and complexation in response to cellular stress, thereby controlling critical processes such as oxidative phosphorylation, apoptosis, and redox signalling. In this review, we describe mitochondrial sources of ROS/RNS, cysteine modifications that are triggered by increased mitochondrial ROS/RNS, and examples of key mitochondrial proteins that are regulated through cysteine-mediated redox signalling. We highlight recent advancements in proteomic methods to study cysteine posttranslational modifications. These tools will further aid in illuminating the important role of cysteine in maintaining and transducing redox signals in the mitochondria.

Sulfhydryl-specific probe for monitoring protein redox sensitivity.

Reactive oxygen species (ROS) regulate various biological processes by modifying reactive cysteine residues in the proteins participating in the relevant signaling pathways. Identification of ROS target proteins requires specific reagents that identify ROS-sensitive cysteine sulfhydryls that differ from the known alkylating agents, iodoacetamide and N-ethylmaleimide, which react nonspecifically with oxidized cysteines including sulfenic and sulfinic acid. We designed and synthesized a novel reagent, methyl-3-nitro-4-(piperidin-1-ylsulfonyl)benzoate (NPSB-1), that selectively and specifically reacts with the sulfhydryl of cysteines in model compounds. We validated the specificity of this reagent by allowing it to react with recombinant proteins followed by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS), and mutant studies employed it to identify cellular proteins containing redox-sensitive cysteine residues. We also obtained proteins from cells treated with various concentrations of hydrogen peroxide, labeled them with biotinylated NPSB-1 (NPSB-B), pulled them down with streptavidin beads, and identified them with MS/MS. We grouped these proteins into four families: (1) those having reactive cysteine residues easily oxidized by hydrogen peroxide, (2) those with cysteines reactive only under mild oxidative stress, (3) those with cysteines reactive only after exposure to oxidative stress, and (4) those with cysteines that are reactive regardless of oxidative stress. These results confirm that NPSBs can serve as novel chemical probes for specifically capturing reactive cysteine residues and as powerful tools for measuring their oxidative sensitivity and can help to understand the function of cysteine modifications in ROS-mediated signaling pathways.

The role of redox-sensitive cysteines in multi sub-unit transcription factors

In response to oxidative stress, cells activate numerous processes and initiate the expression of genes via redox-sensitive transcription factors including the multi sub-unit transcription factors nuclear factor-κB (NFκB), activator protein 1 (AP-1), Hypoxia-inducible factor 1 α (HIF1α), Polyoma virus enhancer-binding protein 2 (PEBP2), cAMP-responsive element-bindingprotein (CREB), Nuclear Factor I/CCAAT Transcription Factor (NFI/CTF), Nuclear transcription factor Y (NF-Y) and E4 Transcription Factor 1 - 60kDa (E4TF1-60). Thioredoxin plays a critical role in this by directly or indirectly (via redox factor 1) reducing redox-sensitive cysteine residues in these transcription factors to modify their tertiary structure, sub-cellular localisation,proteinprotein interactions or DNA-binding affinity. The identity of the redox-sensitive cysteine in E4TF1-60 has not been investigated so far. Determining which cysteines are redox-sensitive within the E4TF1-60 protein sequence together with elucidation of which property is modified will help to better understand the mechanism by which the E4TF1 complex is involved in regulating the cellular oxidative stress response.

A redox-sensitive cysteine residue regulates the kinase activities of OsMPK3 and OsMPK6 in vitro

Two subgroup A rice mitogen-activated protein kinases (MAPKs), OsMPK3 and OsMPK6, have been implicated in multiple stress responses. However, the redox-control of the kinase activity of these proteins remains unknown. Here, immunoprecipitated OsMPK3 and OsMPK6 were initially activated in 15min, and this activation transiently increased in rice seedlings under H2O2 stress. Among the six conserved cysteine residues, only the fourth cysteine residues in the kinase domain VII, Cys179 and Cys210, were required for the in vitro kinase activities of OsMPK3 and OsMPK6, respectively. Moreover, the substitution of these specific cysteine residues with serine abrogated in vitro kinase responses to redox conditions. These results suggest a novel redox-control mechanism for the kinase activities of these MAPKs in vivo.

Structural and functional characterization of DUF1471 domains of Salmonella proteins SrfN, YdgH/SssB, and YahO.

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.

Quantitative label-free redox proteomics of reversible cysteine oxidation in red blood cell membranes

Reversible oxidation of cysteine residues is a relevant posttranslational modification of proteins. However, the low activation energy and transitory nature of the redox switch and the intrinsic complexity of the analysis render quite challenging the aim of a rigorous high-throughput screening of the redox status of redox-sensitive cysteine residues. We describe here a quantitative workflow for redox proteomics, where the ratio between the oxidized forms of proteins in the control vs treated samples is determined by a robust label-free approach. We critically present the convenience of the procedure by specifically addressing the following aspects: (i) the accurate ratio, calculated from the whole set of identified peptides rather than just isotope-tagged fragments; (ii) the application of a robust analytical pipeline to frame the most consistent data averaged over the biological variability; (iii) the relevance of using stringent criteria of analysis, even at the cost of losing potentially interesting but statistically uncertain data. The pipeline has been assessed on red blood cell membrane challenged with diamide as a model of a mild oxidative condition. The cluster of identified proteins encompassed components of the cytoskeleton more oxidized. Indirectly, our analysis confirmed the previous observation that oxidized hemoglobin binds to membranes while oxidized peroxiredoxin 2 loses affinity.