Inhibition of Glutaredoxin 5 predisposes Cisplatin-resistant Head and Neck Cancer Cells to Ferroptosis.

Rationale: Loss of iron-sulfur cluster function predisposes cancer cells to ferroptosis by upregulating iron-starvation response, but the role of glutaredoxin 5 (GLRX5) silencing in ferroptosis remains unknown. We examined the role of GLRX5 functional loss in promoting ferroptosis in cisplatin-resistant head and neck cancer (HNC) cells. Methods: The effects of sulfasalazine treatment and GLRX5 gene silencing were tested on HNC cell lines and mouse tumor xenograft models. These effects were analyzed concerning cell viability and death, lipid reactive oxygen species (ROS) and mitochondrial iron production, labile iron pool, mRNA/protein expression, and malondialdehyde assays. Results: Cyst(e)ine deprivation, erastin, or sulfasalazine induced ferroptosis in HNC cells, which was relatively less sensitive in cisplatin-resistant HNC cells. Sulfasalazine or cyst(e)ine deprivation-induced ferroptosis resulted from increased lipid peroxidation and intracellular free iron, which were significantly promoted by short-interfering RNA or short hairpin RNA (shRNA) targeting GLRX5 (P<0.05). GLRX5 silencing activated iron-starvation response and boosted up intracellular free iron through the iron-responsive element-binding activity of increased iron regulatory protein (increased transferrin receptor and decreased ferritin). These effects were rescued by resistant GLRX5 cDNA but not by catalytically inactive mutant GLRX5 K101Q. The same results were noted in an in vivo mouse model transplanted with vector or shGLRX5-transduced HNC cells and treated with sulfasalazine. Conclusion: Our data suggest that inhibition of GLRX5 predisposes therapy-resistant HNC cells to ferroptosis.

Shared Mutations in a Novel Glutaredoxin Repressor of Multicellular Trichome Fate Underlie Parallel Evolution of Antirrhinum Species.

Most angiosperms produce trichomes-epidermal hairs that have protective or more specialized roles. Trichomes are multicellular in almost all species and, in the majority, secretory. Despite the importance of multicellular trichomes for plant protection and as a source of high-value products, the mechanisms that control their development are only poorly understood. Here, we investigate the control of multicellular trichome patterns using natural variation within the genus Antirrhinum (snapdragons), which has evolved hairy alpine-adapted species or lowland species with a restricted trichome pattern multiple times in parallel. We find that a single gene, Hairy (H), which is needed to repress trichome fate, underlies variation in trichome patterns between all Antirrhinum species except one. We show that H encodes a novel epidermis-specific glutaredoxin and that the pattern of trichome distribution within individuals reflects the location of H expression. Phylogenetic and functional tests suggest that H gained its trichome-repressing role late in the history of eudicots and that the ancestral Antirrhinum had an active H gene and restricted trichome distribution. Loss of H function was involved in an early divergence of alpine and lowland Antirrhinum lineages, and the alleles underlying this split were later reused in parallel evolution of alpines from lowland ancestors, and vice versa. We also find evidence for an evolutionary reversal from a widespread to restricted trichome distribution involving a suppressor mutation and for a pleiotropic effect of H on plant growth that might constrain the evolution of trichome pattern.

Production of adeno-associated virus vectors for in vitro and in vivo applications.

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010-1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

GLRX inhibition enhances the effects of geftinib in EGFR-TKI-resistant NSCLC cells through FoxM1 signaling pathway.

PURPOSE: Non-small-cell lung cancer (NSCLC) is the most common form of lung cancer. Gefitinib is one of the most accepted therapies against NSCLC in those carrying EGFR mutations, but it is only effective in approximately 20% of patients with NSCLC. Thus, alternative therapeutic interventions are urgently needed to overcome gefitinib resistance. Glutaredoxin (GLRX) plays a key role in oxidative stress. However, whether GLRX inhibition could enhance gefitinib efficacy in the gefitinib-resistant NSCLC cells is unknown. In this study, we aimed to determine whether combined inhibition of GLRX could enhance growth-inhibitory effects of gefitinib in gefitinib-resistant NSCLC cells. METHODS: Real-time PCR and western blotting were used to examine the mRNA and protein levels of GLRX in gefitinib-sensitive PC9 and HCC827 and -resistant human lung adenocarcinoma PC9R, HCC827R, and H1975 cells. Cell Counting Kit-8, flow cytometry, JC-1 staining, and reactive oxygen species (ROS) assays were used to evaluate cell proliferation, cell cycle progression, mitochondrial membrane potential, and ROS generation, respectively. Mouse tumor xenografts were used to assess the effect of GLRX in vivo. RESULTS: We found that GLRX was upregulated in gefitinib-resistant PC9R, HCC827R, and H1975 cells. GLRX inhibition enhanced the effects of geftinib in gefitinib-resistant cell proliferation in vitro and in vivo and promoted apoptosis and cell cycle arrest via the EGFR/Forkhead Box M1 (FoxM1) signaling pathway, indicating that combined inhibition of GLRX could enhance growth-inhibitory effects of gefitinib in gefitinib-resistant NSCLC cells. CONCLUSIONS: Our results suggest that GLRX inhibition enhances the effects of geftinib in EGFR-TKI-resistant NSCLC cells. Thus, GLRX may represent a therapeutic target for increasing the efficiency of gefitinib treatment.

Frenolicin B Targets Peroxiredoxin 1 and Glutaredoxin 3 to Trigger ROS/4E-BP1-Mediated Antitumor Effects.

Peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3) are two major antioxidant proteins that play a critical role in maintaining redox homeostasis for tumor progression. Here, we identify the prototypical pyranonaphthoquinone natural product frenolicin B (FB) as a selective inhibitor of Prx1 and Grx3 through covalent modification of active-site cysteines. FB-targeted inhibition of Prx1 and Grx3 results in a decrease in cellular glutathione levels, an increase of reactive oxygen species (ROS), and concomitant inhibition of cancer cell growth, largely by activating the peroxisome-bound tuberous sclerosis complex to inhibit mTORC1/4E-BP1 signaling axis. FB structure-activity relationship studies reveal a positive correlation between inhibition of 4E-BP1 phosphorylation, ROS-mediated cancer cell cytotoxicity, and suppression of tumor growth in vivo. These findings establish FB as the most potent Prx1/Grx3 inhibitor reported to date and also notably highlight 4E-BP1 phosphorylation status as a potential predictive marker in response to ROS-based therapies in cancer.

Identifying Ortholog Selective Fragment Molecules for Bacterial Glutaredoxins by NMR and Affinity Enhancement by Modification with an Acrylamide Warhead.

Illustrated here is the development of a new class of antibiotic lead molecules targeted at Pseudomonas aeruginosa glutaredoxin (PaGRX). This lead was produced to (a) circumvent efflux-mediated resistance mechanisms via covalent inhibition while (b) taking advantage of species selectivity to target a fundamental metabolic pathway. This work involved four components: a novel workflow for generating protein specific fragment hits via independent nuclear magnetic resonance (NMR) measurements, NMR-based modeling of the target protein structure, NMR guided docking of hits, and synthetic modification of the fragment hit with a vinyl cysteine trap moiety, i.e., acrylamide warhead, to generate the chimeric lead. Reactivity of the top warhead-fragment lead suggests that the ortholog selectivity observed for a fragment hit can translate into a substantial kinetic advantage in the mature warhead lead, which bodes well for future work to identify potent, species specific drug molecules targeted against proteins heretofore deemed undruggable.

Glutaredoxin-1 Silencing Induces Cell Senescence via p53/p21/p16 Signaling Axis.

Glutaredoxin-1 (Grx1) catalyzes deglutathionylation with glutathione as a cofactor. Accumulating evidence indicates important roles for Grx1 and S-glutathionylation in the aging process; however, further exploration of Grx1-regulated cellular processes is important to understand the functions of Grx1 in aging. In the present study, we constructed stable Grx1 knockdown or overexpression human cell lines. Grx1 silencing significantly decreased the cellular ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) (GSH/GSSG ratio), resulting in excessive reactive oxygen species (ROS) accumulation, whereas Grx1 overexpression decreased cellular ROS levels. Grx1 silencing also increased glutathionylation of DJ-1 and HSP60, contributing to decreased mitochondrial spare respiration capacity and ATP production. We applied quantitative proteomics to identify differentially expressed proteins between Grx1 knockdown and control cells and showed that Grx1 silencing inactivated DNA replication and damage repair pathways. p53 signaling was activated by Grx1 silencing, which inhibited the CDK4-mediated G1-S transition, resulting in G1 phase cell-cycle arrest and cell senescence, a known hallmark of aging. Taken together, our results indicate that Grx1 regulates DNA replication and damage repair processes and is a potential therapeutic target for aging-related diseases.

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor.

Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC50 value of 32 µM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC50 value (37 µM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.

Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1µM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity.

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress.

Engineered nanoparticles (ENPs) are increasingly utilized for commercial and medical applications; thus, understanding their potential adverse effects is an important societal issue. Herein, we investigated protein S-glutathionylation (SSG) as an underlying regulatory mechanism by which ENPs may alter macrophage innate immune functions, using a quantitative redox proteomics approach for site-specific measurement of SSG modifications. Three high-volume production ENPs (SiO2, Fe3O4, and CoO) were selected as representatives which induce low, moderate, and high propensity, respectively, to stimulate cellular reactive oxygen species (ROS) and disrupt macrophage function. The SSG modifications identified highlighted a broad set of redox sensitive proteins and specific Cys residues which correlated well with the overall level of cellular redox stress and impairment of macrophage phagocytic function (CoO > Fe3O4 ≫ SiO2). Moreover, our data revealed pathway-specific differences in susceptibility to SSG between ENPs which induce moderate versus high levels of ROS. Pathways regulating protein translation and protein stability indicative of ER stress responses and proteins involved in phagocytosis were among the most sensitive to SSG in response to ENPs that induce subcytoxic levels of redox stress. At higher levels of redox stress, the pattern of SSG modifications displayed reduced specificity and a broader set pathways involving classical stress responses and mitochondrial energetics (e.g., glycolysis) associated with apoptotic mechanisms. An important role for SSG in regulation of macrophage innate immune function was also confirmed by RNA silencing of glutaredoxin, a major enzyme which reverses SSG modifications. Our results provide unique insights into the protein signatures and pathways that serve as ROS sensors and may facilitate cellular adaption to ENPs, versus intracellular targets of ENP-induced oxidative stress that are linked to irreversible cell outcomes.

Redox regulation of metabolic and signaling pathways by thioredoxin and glutaredoxin in NOS-3 overexpressing hepatoblastoma cells.

Nitric oxide (NO) plays relevant roles in signal transduction in physiopathology and its effects are dependent on several environmental factors. NO has both pro-apoptotic and anti-apoptotic functions but the molecular mechanisms responsible for these opposite effects are not fully understood. The action of NO occurs mainly through redox changes in target proteins, particularly by S-nitrosylation of reactive cysteine residues. Thioredoxin (Trx) and glutaredoxin (Grx) systems are the main cellular controllers of the thiolic redox state of proteins exerting controversial effects on apoptosis with consequences for the resistance to or the development of cancer. The aim of this study was to ascertain whether Trx and/or Grx systems mediate the antiproliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase-3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO. Enzyme levels and cysteine redox state at several metabolic checkpoints were consistent with prominence of the pentose phosphate pathway to direct the metabolic flux toward NADPH for antioxidant defense and lowering of nucleotide biosynthesis and hence proliferation. Proteins involved in cell survival pathways, proteins of the redoxin systems and phosphorylation of MAPK were all significantly increased accompanied by a shift of the thiolic redox state of Akt1, Trx1 and Grx1 to more oxidized. Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and -8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells. These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new antitumoral therapies.

Zebrafish heart development is regulated via glutaredoxin 2 dependent migration and survival of neural crest cells.

Glutaredoxin 2 is a vertebrate specific oxidoreductase of the thioredoxin family of proteins modulating the intracellular thiol pool. Thereby, glutaredoxin 2 is important for specific redox signaling and regulates embryonic development of brain and vasculature via reversible oxidative posttranslational thiol modifications. Here, we describe that glutaredoxin 2 is also required for successful heart formation. Knock-down of glutaredoxin 2 in zebrafish embryos inhibits the invasion of cardiac neural crest cells into the primary heart field. This leads to impaired heart looping and subsequent obstructed blood flow. Glutaredoxin 2 specificity of the observed phenotype was confirmed by rescue experiments. Active site variants of glutaredoxin 2 revealed that the (de)-glutathionylation activity is required for proper heart formation. Our data suggest that actin might be one target during glutaredoxin 2 regulated cardiac neural crest cell migration and embryonic heart development. In summary, this work represents further evidence for the general importance of redox signaling in embryonic development and highlights additionally the importance of glutaredoxin 2 during embryogenesis.

Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases.

Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson׳s disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Akt plays, including in the Akt-mTOR signaling cascade.

Redox modulation of endothelial nitric oxide synthase by glutaredoxin-1 through reversible oxidative post-translational modification.

S-Glutathionylation is a redox-regulated modification that uncouples endothelial nitric oxide synthase (eNOS), switching its function from nitric oxide (NO) synthesis to (•)O2(-) generation, and serves to regulate vascular function. While in vitro or in vivo eNOS S-glutathionylation with modification of Cys689 and Cys908 of its reductase domain is triggered by high levels of glutathione disulfide (GSSG) or oxidative thiyl radical formation, it remains unclear how this process may be reversed. Glutaredoxin-1 (Grx1), a cytosolic and glutathione-dependent enzyme, can reverse protein S-glutathionylation; however, its role in regulating eNOS S-glutathionylation remains unknown. We demonstrate that Grx1 in the presence of glutathione (GSH) (1 mM) reverses GSSG-mediated eNOS S-glutathionylation with restoration of NO synthase activity. Because Grx1 also catalyzes protein S-glutathionylation with an increased [GSSG]/[GSH] ratio, we measured its effect on eNOS S-glutathionylation when the [GSSG]/[GSH] ratio was >0.2, which can occur in cells and tissues under oxidative stress, and observed an increased level of eNOS S-glutathionylation with a marked decrease in eNOS activity without uncoupling. This eNOS S-glutathionylation was reversed with a decrease in the [GSSG]/[GSH] ratio to <0.1. Liquid chromatography and tandem mass spectrometry identified a new site of eNOS S-glutathionylation by Grx1 at Cys382, on the surface of the oxygenase domain, without modification of Cys689 or Cys908, each of which is buried within the reductase. Furthermore, Grx1 was demonstrated to be a protein partner of eNOS in vitro and in normal endothelial cells, supporting its role in eNOS redox regulation. In endothelial cells, Grx1 inhibition or gene silencing increased the level of eNOS S-glutathionylation and decreased the level of cellular NO generation. Thus, Grx1 can exert an important role in the redox regulation of eNOS in cells.

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1.

CONTEXT: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. OBJECTIVE: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. MATERIALS AND METHODS: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. RESULTS: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. DISCUSSION: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. CONCLUSION: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.

Glutaredoxin is involved in the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate.

Clopidogrel is a thienopyridine antiplatelet agent that is converted to the active metabolite, R-361015, in vivo. Clopidogrel is first oxidized to a thiolactone intermediate R-115991. R-115991 is thought to be metabolized to a GSH conjugate of R-361015 (R-361015-SG) and then is reduced to R-361015 in the presence of GSH. In this study, we investigated the enzyme-mediated formation of R-361015 from R-361015-SG in human liver microsomes and cytosols. After incubation of R-115991 in human liver microsomes, the formation of R-361015-SG, and subsequently of R-361015, was observed. The apparent formation rate of R-361015-SG was markedly decreased when human liver cytosols were added. Fitting the data to the kinetic model showed that the rate constant of R-361015-SG reduction to R-361015 in human liver microsomes was approximately 20-fold higher in the presence of human liver cytosols (6.56 min⁻¹) than in the absence of cytosols (0.326 min⁻¹). In addition, the formation rate of R-361015 from R-361015-SG was higher in human liver cytosols (2843 ± 1176 pmol · min⁻¹ · mg⁻¹) compared with in human liver microsomes (508 ± 396 pmol · min⁻¹ · mg⁻¹). The formation of R-361015 from R-361015-SG in human liver microsomes or cytosols was inhibited by anti-human glutaredoxin antibody in a concentration-dependent manner. Recombinant human glutaredoxin mediated the formation of R-361015 from R-361015-SG with the K(m) and V(max) values of 30.0 ± 1.3 µM and 381.6 ± 209.8 pmol · min⁻¹ · µg⁻¹, respectively. The intrinsic clearance value (V(max)/K(m)) was 12.9 ± 7.5 µl · min⁻¹ · µg⁻¹. In conclusion, we found that human glutaredoxin is a main contributor to the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate in human liver.

Glutaredoxin s12: unique properties for redox signaling.

AIMS: Cysteines (Cys) made acidic by the protein environment are generally sensitive to pro-oxidant molecules. Glutathionylation is a post-translational modification that can occur by spontaneous reaction of reduced glutathione (GSH) with oxidized Cys as sulfenic acids (-SOH). The reverse reaction (deglutathionylation) is strongly stimulated by glutaredoxins (Grx) and requires a reductant, often GSH. RESULTS: Here, we show that chloroplast GrxS12 from poplar efficiently reacts with glutathionylated substrates in a GSH-dependent ping pong mechanism. The pK(a) of GrxS12 catalytic Cys is very low (3.9) and makes GrxS12 itself sensitive to oxidation by H(2)O(2) and to direct glutathionylation by nitrosoglutathione. Glutathionylated-GrxS12 (GrxS12-SSG) is temporarily inactive until it is deglutathionylated by GSH. The equilibrium between GrxS12 and glutathione (E(m(GrxS12-SSG))= -315 mV, pH 7.0) is characterized by K(ox) values of 310 at pH 7.0, as in darkened chloroplasts, and 69 at pH 7.9, as in illuminated chloroplasts. INNOVATION: Based on thermodynamic data, GrxS12-SSG is predicted to accumulate in vivo under conditions of mild oxidation of the GSH pool that may occur under stress. Moreover, GrxS12-SSG is predicted to be more stable in chloroplasts in the dark than in the light. CONCLUSION: These peculiar catalytic and thermodynamic properties could allow GrxS12 to act as a stress-related redox sensor, thus allowing glutathione to play a signaling role through glutathionylation of GrxS12 target proteins.

Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase.

BACKGROUND: Cellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network. RESULTS: Stable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins. CONCLUSIONS: Our results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.

Glutaredoxin and thioredoxin can be involved in producing the pharmacologically active metabolite of a thienopyridine antiplatelet agent, prasugrel.

A thienopyridine antiplatelet agent, prasugrel, is rapidly hydrolyzed to a thiolactone metabolite (R-95913, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone). R-95913 is oxidized by hepatic cytochromes P450 to the pharmacologically active metabolite R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid). One possible intermediate in the in vitro bioactivation pathway is a glutathione conjugate, R-133490, which could be reduced to generate R-138727 in the presence of a reducing agent such as glutathione. In this study, enzymes in human liver cytosols were found to accelerate reduction of R-133490 leading to the formation of R-138727. To explore the possible reductive enzymes, we separated the various proteins in human liver cytosol based on size using gel filtration chromatography. Two active peaks were detected and found to contain thioredoxin and glutaredoxin, respectively. In addition, recombinant human glutaredoxin and thioredoxin promoted the formation of R-138727 from R-133490 with much higher activity for glutaredoxin than for thioredoxin. This study is the first in vitro observation indicating that glutaredoxin and thioredoxin in human liver are active in reducing the mixed disulfide formed between xenobiotics and glutathione.