Redox-active Cysteine Research Articles

Redox Regulation of K+ Channel: Role of Thioredoxin.

Significance: Potassium channels regulate the influx and efflux of K+ ions in various cell types that generate and propagate action potential associated with excitation, contraction, and relaxation of various cell types. Although redox active cysteines are critically important for channel activity, the redox regulation of K+ channels by thioredoxin (Trx) has not been systematically reviewed. Recent Advances: Redox regulation of K+ channel is now increasingly recognized as drug targets in the pathological condition of several cardiovascular disease processes. The role of Trx in regulation of these channels and its implication in pathological conditions have not been adequately reviewed. This review specifically focuses on the redox-regulatory role of Trx on K+ channel structure and function in physiological and pathophysiological conditions. Critical Issues: Ion channels, including K+ channel, have been implicated in the functioning of cardiomyocyte excitation-contraction coupling, vascular hyperpolarization, cellular proliferation, and neuronal stimulation in physiological and pathophysiological conditions. Although oxidation-reduction of ion channels is critically important in their function, the role of Trx, redox regulatory protein in regulation of these channels, and its implication in pathological conditions need to be studied to gain further insight into channel function. Future Directions: Future studies need to map all redox regulatory pathways in channel structure and function using novel mouse models and redox proteomic and signal transduction studies, which modulate various currents and altered excitability of relevant cells implicated in a pathological condition. We are yet at infancy of studies related to redox control of various K+ channels and structured and focused studies with novel animal models. Antioxid. Redox Signal. 00, 00-00.

Benzophenanthridine Alkaloid Chelerythrine Elicits Necroptosis of Gastric Cancer Cells via Selective Conjugation at the Redox Hyperreactive C-Terminal Sec498 Residue of Cytosolic Selenoprotein Thioredoxin Reductase.

Targeting thioredoxin reductase (TXNRD) with low-weight molecules is emerging as a high-efficacy anti-cancer strategy in chemotherapy. Sanguinarine has been reported to inhibit the activity of TXNRD1, indicating that benzophenanthridine alkaloid is a fascinating chemical entity in the field of TXNRD1 inhibitors. In this study, the inhibition of three benzophenanthridine alkaloids, including chelerythrine, sanguinarine, and nitidine, on recombinant TXNRD1 was investigated, and their anti-cancer mechanisms were revealed using three gastric cancer cell lines. Chelerythrine and sanguinarine are more potent inhibitors of TXNRD1 than nitidine, and the inhibitory effects take place in a dose- and time-dependent manner. Site-directed mutagenesis of TXNRD1 and in vitro inhibition analysis proved that chelerythrine or sanguinarine is primarily bound to the Sec498 residue of the enzyme, but the neighboring Cys497 and remaining N-terminal redox-active cysteines could also be modified after the conjugation of Sec498. With high similarity to sanguinarine, chelerythrine exhibited cytotoxic effects on multiple gastric cancer cell lines and suppressed the proliferation of tumor spheroids derived from NCI-N87 cells. Chelerythrine elevated cellular levels of reactive oxygen species (ROS) and induced endoplasmic reticulum (ER) stress. Moreover, the ROS induced by chelerythrine could be completely suppressed by the addition of N-acetyl-L-cysteine (NAC), and the same is true for sanguinarine. Notably, Nec-1, an RIPK1 inhibitor, rescued the chelerythrine-induced rapid cell death, indicating that chelerythrine triggers necroptosis in gastric cancer cells. Taken together, this study demonstrates that chelerythrine is a novel inhibitor of TXNRD1 by targeting Sec498 and possessing high anti-tumor properties on multiple gastric cancer cell lines by eliciting necroptosis.

Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

Thioredoxin regulates the redox state and the activity of the human tRNA ligase complex.

The mammalian tRNA ligase complex (tRNA-LC) catalyzes the splicing of intron-containing pre-tRNAs in the nucleus and the splicing of XBP1 mRNA during the unfolded protein response (UPR) in the cytoplasm. We recently reported that the tRNA-LC coevolved with PYROXD1, an essential oxidoreductase that protects the catalytic cysteine of RTCB, the catalytic subunit of the tRNA-LC, against aerobic oxidation. In this study, we show that the oxidoreductase Thioredoxin (TRX) preserves the enzymatic activity of RTCB under otherwise inhibiting concentrations of oxidants. TRX physically interacts with oxidized RTCB, and reduces and reactivates RTCB through the action of its redox-active cysteine pair. We further show that TRX interacts with RTCB at late stages of UPR. Since the interaction requires oxidative conditions, our findings suggest that prolonged UPR generates reactive oxygen species. Thus, our results support a functional role for TRX in securing and repairing the active site of the tRNA-LC, thereby allowing pre-tRNA splicing and UPR to occur when cells encounter mild, but still inhibitory levels of reactive oxygen species.

DNA repair and oxidative stress defense systems in radiation-resistant Deinococcus murrayi.

Deinococcus murrayi is a bacterium isolated from hot springs in Portugal, and named after Dr. Robert G.E. Murray in recognition of his research on the genus Deinococcus. Like other Deinococcus species, D. murrayi is extremely resistant to ionizing radiation. Repair of massive DNA damage and limitation of oxidative protein damage are two important factors contributing to the robustness of Deinococcus bacteria. Here, we identify, among others, the DNA repair and oxidative stress defense proteins in D. murrayi, and highlight special features of D. murrayi. For DNA repair, D. murrayi does not contain a standalone uracil-DNA glycosylase (Ung), but it encodes a protein in which Ung is fused to a DNA photolyase domain (PhrB). UvrB and UvrD contain large insertions corresponding to inteins. One of its endonuclease III enzymes lacks a [4Fe-4S] cluster. Deinococcus murrayi possesses a homolog of the error-prone DNA polymerase IV. Concerning oxidative stress defense, D. murrayi encodes a manganese catalase in addition to a heme catalase. Its organic hydroperoxide resistance protein Ohr is atypical because the redox active cysteines are present in a CXXC motif. These and other characteristics of D. murrayi show further diversity among Deinococcus bacteria with respect to resistance-associated mechanisms.

New Dielis species and structural dichotomy of the mitochondrial cox2 gene in Scoliidae wasps

Some mitochondrial protein-coding genes of protists and land plants have split over the course of evolution into complementary genes whose products can form heteromeric complexes that likely substitute for the undivided proteins. One of these genes, cox2, has also been found to have split in animals, specifically in Scoliidae wasps (Hymenoptera: Apocrita) of the genus Dielis (Campsomerini), while maintaining the conventional structure in related Scolia (Scoliini). Here, a hitherto unrecognized Nearctic species of Dielis, D. tejensis, is described based on its phenotype and mtDNA. The mitogenome of D. tejensis sp. nov. differs from that of the sympatric sibling species Dielis plumipes fossulana by the reduced size of the cox2-dividing insert, which, however, still constitutes the fifth part of the mtDNA; an enlarged nad2-trnW intergenic region; the presence of two trnKttt paralogues; and other features. Both species of Dielis have a unique insertion of a threonine in COXIIA, predicted to be involved in COXIIA-COXIIB docking, and substitutions of two hydrophobic residues with redox-active cysteines around the CuA centre in COXIIB. Importantly, the analysis of mtDNA from another Campsomerini genus, Megacampsomeris, shows that its cox2 gene is also split. The presented data highlight evolutionary processes taking place in hymenopteran mitogenomes that do not fall within the mainstream of animal mitochondrion evolution.

Insights into the Thermodynamics and Kinetics of Amino-Acid Radicals in Proteins.

Some oxidoreductase enzymes use redox-active tyrosine, tryptophan, cysteine, and/or glycine residues as one-electron, high-potential redox (radical) cofactors. Amino-acid radical cofactors typically perform one of four tasks-they work in concert with a metallocofactor to carry out a multielectron redox process, serve as storage sites for oxidizing equivalents, activate the substrate molecules, or move oxidizing equivalents over long distances. It is challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single-amino-acid residue. The inherently reactive and highly oxidizing properties of amino-acid radicals increase the experimental barriers further still. This review describes a family of stable and well-structured model proteins that was made specifically to study tyrosine and tryptophan oxidation-reduction. The so-called α3X model protein system was combined with very-high-potential protein film voltammetry, transient absorption spectroscopy, and theoretical methods to gain a comprehensive description of the thermodynamic and kinetic properties of protein tyrosine and tryptophan radicals.

Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the Cterminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites.

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase.

Low G+C Gram-positive Firmicutes, such as the clinically important pathogens Staphylococcus aureus and Bacillus cereus, use the low-molecular weight thiol bacillithiol (BSH) as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. The protein YpdA has recently been shown to function as an essential NADPH-dependent reductase of oxidized bacillithiol disulfide (BSSB) resulting from stress responses and is crucial for maintaining the reduced pool of BSH and cellular redox balance. In this work, we present the first crystallographic structures of YpdAs, namely, those from S. aureus and B. cereus. Our analyses reveal a uniquely organized biological tetramer; however, the structure of the monomeric subunit is highly similar to those of other flavoprotein disulfide reductases. The absence of a redox active cysteine in the vicinity of the FAD isoalloxazine ring implies a new direct disulfide reduction mechanism, which is backed by the presence of a potentially gated channel, serving as a putative binding site for BSSB in the proximity of the FAD cofactor. We also report enzymatic activities for both YpdAs, which along with the structures presented in this work provide important structural and functional insight into a new class of FAD-containing NADPH-dependent oxidoreductases, related to the emerging fight against pathogenic bacteria.

Structure and Electron-Transfer Pathway of the Human Methionine Sulfoxide Reductase MsrB3.

Aims: The post-translational oxidation of methionine to methionine sulfoxide (MetSO) is a reversible process, enabling the repair of oxidative damage to proteins and the use of sulfoxidation as a regulatory switch. MetSO reductases catalyze the stereospecific reduction of MetSO. One of the mammalian MetSO reductases, MsrB3, has a signal sequence for entry into the endoplasmic reticulum (ER). In the ER, MsrB3 is expected to encounter a distinct redox environment compared with its paralogs in the cytosol, nucleus, and mitochondria. We sought to determine the location and arrangement of MsrB3 redox-active cysteines, which may couple MsrB3 activity to other redox events in the ER.Results: We determined the human MsrB3 structure by using X-ray crystallography. The structure revealed that a disulfide bond near the protein amino terminus is distant in space from the active site. Nevertheless, biochemical assays showed that these amino-terminal cysteines are oxidized by the MsrB3 active site after its reaction with MetSO.Innovation: This study reveals a mechanism to shuttle oxidizing equivalents from the primary MsrB3 active site toward the enzyme surface, where they would be available for further dithiol-disulfide exchange reactions.Conclusion: Conformational changes must occur during the MsrB3 catalytic cycle to transfer oxidizing equivalents from the active site to the amino-terminal redox-active disulfide. The accessibility of this exposed disulfide may help couple MsrB3 activity to other dithiol-disulfide redox events in the secretory pathway.

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF1-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF1-γ. Previous studies showed that the light-dependent reduction of CF1-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF1-γ, especially under physiological conditions. We therefore performed direct assessment of the CF1-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF1-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF1-γ. The results showed that CF1-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.

Methionine sulfoxide reductase B from Corynebacterium diphtheriae catalyzes sulfoxide reduction via an intramolecular disulfide cascade

Corynebacterium diphtheriae is a human pathogen that causes diphtheria. In response to immune system-induced oxidative stress, C. diphtheriae expresses antioxidant enzymes, among which are methionine sulfoxide reductase (Msr) enzymes, which are critical for bacterial survival in the face of oxidative stress. Although some aspects of the catalytic mechanism of the Msr enzymes have been reported, several details still await full elucidation. Here, we solved the solution structure of C. diphtheriae MsrB (Cd-MsrB) and unraveled its catalytic and oxidation-protection mechanisms. Cd-MsrB catalyzes methionine sulfoxide reduction involving three redox-active cysteines. Using NMR heteronuclear single-quantum coherence spectra, kinetics, biochemical assays, and MS analyses, we show that the conserved nucleophilic residue Cys-122 is S-sulfenylated after substrate reduction, which is then resolved by a conserved cysteine, Cys-66, or by the nonconserved residue Cys-127. We noted that the overall structural changes during the disulfide cascade expose the Cys-122-Cys-66 disulfide to recycling through thioredoxin. In the presence of hydrogen peroxide, Cd-MsrB formed reversible intra- and intermolecular disulfides without losing its Cys-coordinated Zn2+, and only the nonconserved Cys-127 reacted with the low-molecular-weight (LMW) thiol mycothiol, protecting it from overoxidation. In summary, our structure-function analyses reveal critical details of the Cd-MsrB catalytic mechanism, including a major structural rearrangement that primes the Cys-122-Cys-66 disulfide for thioredoxin reduction and a reversible protection against excessive oxidation of the catalytic cysteines in Cd-MsrB through intra- and intermolecular disulfide formation and S-mycothiolation.

Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other's virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicansIMPORTANCEEnterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.

On the role of the plant mitochondrial thioredoxin system during abiotic stress

ABSTRACTThiol-disulfide redox exchanges are widely distributed modifications of great importance for metabolic regulation in living cells. In general, the formation of disulfide bonds is controlled by thioredoxins (TRXs), ubiquitous proteins with two redox-active cysteine residues separated by a pair of amino acids. While the function of plastidial TRXs has been extensively studied, the role of the mitochondrial TRX system is much less well understood. Recent studies have demonstrated that the mitochondrial TRXs are required for the proper functioning of the major metabolic pathways, including stomatal function and antioxidant metabolism under sub-optimal conditions including drought and salinity. Furthermore, inactivation of mitochondrial TRX system leads to metabolite adjustments of both primary and secondary metabolism following drought episodes in arabidopsis, and makes the plants more resistant to salt stress. Here we discuss the implications of these findings, which clearly open up several research avenues to achieve a full understanding of the redox control of metabolism under environmental constraining conditions.

New red-shifted fluorescent biosensor for monitoring intracellular redox changes

Maintenance of intracellular redox homeostasis is critical for cell survival, proliferation, differentiation, and signaling. In this regard, major changes in the intracellular redox milieu may lead to cell death whereas subtle increases in the level of certain oxidizing species may act as signals that regulate a plethora of cellular processes. Redox-sensitive variants of green fluorescent proteins (roGFP2 and rxYFP) were developed and proved useful to monitor intracellular redox changes in a non-invasive and online manner. With the aim to extend the spectral range of the fluorescent redox biosensors, we here describe the generation, biochemical characterization and biological validation of a new redox reporter based on the red-shifted mRuby2 protein (rxmRuby2). Spectrofluorimetric analysis performed with the recombinant biosensor shows a reversible redox response produced by two redox-active cysteine residues predicted by molecular modeling. rxmRuby2 is highly selective for the couple glutathione/glutathione disulfide in the presence of the oxidoreductase glutaredoxin. The estimated redox potential of rxmRuby2 (E° −265 ± 22 mV) makes it suitable for its use in reducing subcellular compartments. Titration assays demonstrated the capacity of rxmRuby2 to monitor redox changes within a physiological pH range. rxmRuby2 responded sensitively and reversibly to different redox stimuli applied to HeLa and HEK293 cells expressing transiently and/or stable the biosensor. Fusing rxmRuby2 to the Clover fluorescent protein allowed normalization of the redox signal to the expression level of the reporter protein and/or to other factors that may affect fluorescence. The new red-shifted redox biosensor show promises for deep-tissue and in vivo imaging applications.

Redox Activation of Nox1 (NADPH Oxidase 1) Involves an Intermolecular Disulfide Bond Between Protein Disulfide Isomerase and p47phox in Vascular Smooth Muscle Cells.

Objective- PDI (protein disulfide isomerase A1) was reported to support Nox1 (NADPH oxidase) activation mediated by growth factors in vascular smooth muscle cells. Our aim was to investigate the molecular mechanism by which PDI activates Nox1 and the functional implications of PDI in Nox1 activation in vascular disease. Approach and Results- Using recombinant proteins, we identified a redox interaction between PDI and the cytosolic subunit p47phox in vitro. Mass spectrometry of crosslinked peptides confirmed redox-dependent disulfide bonds between cysteines of p47phox and PDI and an intramolecular bond between Cys 196 and 378 in p47phox. PDI catalytic Cys 400 and p47phox Cys 196 were essential for the activation of Nox1 by PDI in vascular smooth muscle cells. Transfection of PDI resulted in the rapid oxidation of a redox-sensitive protein linked to p47phox, whereas PDI mutant did not promote this effect. Mutation of p47phox Cys 196, or the redox active cysteines of PDI, prevented Nox1 complex assembly and vascular smooth muscle cell migration. Proximity ligation assay confirmed the interaction of PDI and p47phox in murine carotid arteries after wire injury. Moreover, in human atheroma plaques, a positive correlation between the expression of PDI and p47phox occurred only in PDI family members with the a' redox active site. Conclusions- PDI redox cysteines facilitate Nox1 complex assembly, thus identifying a new mechanism through which PDI regulates Nox activity in vascular disease.

Thiol stress-dependent aggregation of the glycolytic enzyme triose phosphate isomerase in yeast and human cells.

The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants, and xenobiotics. Cysteine-containing proteins are especially at risk, as the thiol side chain is subject to oxidation, adduction, and chelation by thiol-reactive compounds. The thiol-chelating heavy metal cadmium is a highly toxic environmental pollutant demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of cadmium toxicity responsible for these outcomes are largely unknown. Using fluorescent protein fusion to cytosolic proteins with known redox-active cysteines, we identified the yeast glycolytic enzyme triose phosphate isomerase as being aggregation-prone in response to cadmium and to glucose depletion in chronologically aging cultures. Cadmium-induced aggregation was limited to newly synthesized Tpi1 that was recruited to foci containing the disaggregase Hsp104 and the peroxiredoxin chaperone Tsa1. Misfolding of nascent Tpi1 in response to both cadmium and glucose-depletion stress required both cysteines, implying that thiol status in this protein directly influences folding. We also demonstrate that cadmium proteotoxicity is conserved between yeast and human cells, as HEK293 and HCT116 cell lines exhibit recruitment of the protein chaperone Hsp70 to visible foci. Moreover, human TPI, mutations in which cause a glycolytic deficiency syndrome, also forms aggregates in response to cadmium treatment, suggesting that this conserved enzyme is folding-labile and may be a useful endogenous model for investigating thiol-specific proteotoxicity.

Using Flow Cytometry to Detect and Measure Intracellular Thiol Redox Status in Viable T Cells from Heterogeneous Populations.

Increased production of reactive oxygen species (ROS) and deficiencies in cellular antioxidant defenses are the principal causes of cellular oxidative stress. ROS can react with a variety intracellular molecules, including redox active cysteine thiols (-SH) within proteins. Cysteine thiols can occupy several redox states and conversion between them is highly dynamic during, for example, cell growth, resulting in modification and subsequent loss of the "reduced thiol" form (-SH or -S-). The challenge lies with detecting and measuring thiol redox status inside viable heterogeneous cell populations (e.g., peripheral blood mononuclear cells (PBMCs)). Here we describe a flow cytometric approach for the evaluation of intracellular thiol redox status in human CD3+ T cells within a viable PBMC preparation. Using the thiol reactive probe, fluorescein-5 maleimide (F5M), we demonstrate that loss of reduced intracellular thiol correlates with a decrease in F5M fluorescence. We also detected a loss of F5M fluorescence in Jurkat cell cultures exposed to exogenous H2O2 generated by glucose oxidase. Since F5M binds irreversibly to reduced cysteine thiols, cells may be sorted based on F5M fluorescence intensity and redox active proteins can subsequently be extracted and separated using SDS-PAGE. This final step facilitates identification of redox active proteins from individual cell populations in live heterogeneous cell mixes using proteomic analysis.