GSH Binding Research Articles

Molecular basis for the enzymatic inactivity of class III glutaredoxin ROXY9 on standard glutathionylated substrates

Class I glutaredoxins (GRXs) are nearly ubiquitous proteins that catalyse the glutathione (GSH)-dependent reduction of mainly glutathionylated substrates. In land plants, a third class of GRXs has evolved (class III). Class III GRXs regulate the activity of TGA transcription factors through yet unexplored mechanisms. Here we show that Arabidopsis thaliana class III GRX ROXY9 is inactive as an oxidoreductase on widely used model substrates. Glutathionylation of the active site cysteine, a prerequisite for enzymatic activity, occurs only under highly oxidizing conditions established by the GSH/glutathione disulfide (GSSG) redox couple, while class I GRXs are readily glutathionylated even at very negative GSH/GSSG redox potentials. Thus, structural alterations in the GSH binding site leading to an altered GSH binding mode likely explain the enzymatic inactivity of ROXY9. This might have evolved to avoid overlapping functions with class I GRXs and raises questions of whether ROXY9 regulates TGA substrates through redox regulation.

Structural Analysis of the Drosophila melanogaster GSTome.

Glutathione transferase (GST) is a superfamily of ubiquitous enzymes, multigenic in numerous organisms and which generally present homodimeric structures. GSTs are involved in numerous biological functions such as chemical detoxification as well as chemoperception in mammals and insects. GSTs catalyze the conjugation of their cofactor, reduced glutathione (GSH), to xenobiotic electrophilic centers. To achieve this catalytic function, GSTs are comprised of a ligand binding site and a GSH binding site per subunit, which is very specific and highly conserved; the hydrophobic substrate binding site enables the binding of diverse substrates. In this work, we focus our interest in a model organism, the fruit fly Drosophila melanogaster (D. mel), which comprises 42 GST sequences distributed in six classes and composing its GSTome. The goal of this study is to describe the complete structural GSTome of D. mel to determine how changes in the amino acid sequence modify the structural characteristics of GST, particularly in the GSH binding sites and in the dimerization interface. First, we predicted the 3D atomic structures of each GST using the AlphaFold (AF) program and compared them with X-ray crystallography structures, when they exist. We also characterized and compared their global and local folds. Second, we used multiple sequence alignment coupled with AF-predicted structures to characterize the relationship between the conservation of amino acids in the sequence and their structural features. Finally, we applied normal mode analysis to estimate thermal B-factors of all GST structures of D. mel. Particularly, we extracted flexibility profiles of GST and identify key residues and motifs that are systematically involved in the ligand binding/dimerization processes and thus playing a crucial role in the catalytic function. This methodology will be extended to guide the in silico design of synthetic GST with new/optimal catalytic properties for detoxification applications.

Ratiometric electrochemical biosensor based on Cu(II) modified covalent organic framework for the ultra-sensitive and specific detection of glutathione

As a vital biological polypeptide molecule, glutathione (GSH) is involved in the regulation of various life activities. Accurate and sensitive detection of GSH in human body is of great significance for the preliminary diagnosis of related diseases. In this research, Cu(II) modified TADH COF, which has been prepared by Cu2+ post-synthesis modification of the COF obtained through a condensation reaction of 1,3,5-(4-aminophenyl) benzene (TAPB) and 2,5-dihydroxy-1,4-benzenedicarboxaldehyde (DHTP), and methylene blue (MB) molecules are combined to construct a ratiometric electrochemical sensor for achieving specific and ultra-sensitive detection of GSH. As a stable and active covalent organic framework material, TADH COF possesses the special O(–OH) and N(CN) binding sites, which can load and coordinate with Cu2+ effectively. The obtained Cu(II)-TADH COF serves as a sensitive electrode material for sensing GSH due to “extrusion effect” caused CuCl solid-state electrochemical signal degeneration by the specific binding of Cu and GSH while MB provides a stable electrical signal in the electrochemical sensing process as an internal reference for eliminating errors and making the detection results more accurate. The constructed electrochemical sensing platform Cu(II)-TADH COF/MB/GCE exhibited an ultrahigh sensitivity with a limit of detection (LOD) of 0.0093 nM (S/N = 3), wide linear range of 0.05 nM–40 μM and high accuracy. For the detection of GSH in real samples including human blood, serum and urine samples, the sensor also showed excellent sensing performance. This is the first research on ratiometric electrochemical sensing platform for the ultra-sensitive detection of glutathione based on covalent organic frameworks, which provides an original and novel strategy for the electrochemical detection of GSH with COF materials.

Copper peroxide and cisplatin co-loaded silica nanoparticles-based trinity strategy for cooperative cuproptosis/chemo/chemodynamic cancer therapy

Cuproptosis, a novel identified regulated cell death modality, makes a great difference to known death mechanisms and offers encouraging opportunities for copper-based materials for cancer treatment. However, its capacity for killing cancer cells is restricted by the lack of tumor selectivity. Tumor microenvironment, characterized by hypoxia, acidosis and excessive glutathione (GSH), is another limitation of cuproptosis and cisplatin-based chemotherapy. Herein, a newly copper/cisplatin hybrid silica nanoplatform, denoted as CuO2/DDP@SiO2, is developed as copper metabolic disrupter and chemotherapy/chemodynamic therapy amplifier for synergistic cuproptosis/chemo/chemodynamic anticancer therapy. We imbued CuO2 with the rever-TME properties: O2 generation, increased pH value and oxidation of intracellular GSH. The depletion of GSH sensitizes cancer cells to the CuO2/DDP@SiO2-mediated cuproptosis, causing aggregation of lipoylated mitochondrial proteins, the target of copper-induced toxicity. In vitro experiments shown that reduced binding of GSH to cisplatin could largely increase intracellular cisplatin concentration. Simultaneously, CuO2 could significantly downregulate multidrug resistance-associated protein 2 (MRP2) by O2-dependent hypoxia-inducible factor 1 (HIF-1) inactivation. Thus, the entire cisplatin-efflux pathway was blocked, and the antitumor effect of cisplatin was enhanced both in vitro and in vivo. Collectively, both chemotherapy/chemodynamic therapy and cuproptosis were enhanced by TME modulation. Therefore, CuO2/DDP@SiO2 nanoparticle provides a new therapeutic modality paradigm to boost cuproptosis/chemo/chemodynamic therapies.

Probing the binding mode and interactions of proteinase K and glutathione: molecular simulation and experiments.

Proteinase K, a serine protease from Tritirachium album Limber, is crucial in research due to its potent proteolytic activity, which relies on conformational stability and substrate affinity. Glutathione (GSH), an essential intracellular antioxidant, regulates various physiological processes by interacting with proteins, influencing their stability and function. Despite the importance of both proteinase K and GSH, their potential interaction remains unexplored. Understanding this interaction could uncover new regulatory mechanisms affecting proteinase K, with significant implications for research and therapeutic applications. In this study, we systematically investigated the binding of GSH to proteinase K using a comprehensive approach in which theoretical and experimental methods mutually validate each other. Molecular docking determined the binding mode and the interaction mechanism of proteinase K and GSH. Molecular dynamics (MD) simulations revealed that GSH binding significantly improved the stability of proteinase K, affirming the binding process was spontaneous, with hydrogen bonds and van der Waals forces emerging as the predominant contributors throughout the interaction. At the same time, the fluorescence spectrum and circular dichroism spectrum confirmed the interaction mechanism between GSH and proteinase K, as well as the conformational changes of proteinase K induced by GSH binding. We believe this study could offer valuable insights for future research into the structure and binding dynamics of other protein-ligand complexes under physiological conditions.

Axial assembly of AuNR for tumor theranostics via Zn2+-GSH chelation induced degradation of AuNR@ZIF-8 heterostructures

Tumor microenvironment responsive photothermal ablation is a noninvasive and accurately targeted approach for cancer therapy. Herein, an intracellular directional assembly strategy for enhanced photothermal therapy (PTT) was realized by using ZIF-8 encapsulated Au nanorod (AuNR) heterostructure as the precursor of photothermal convertible material. The ZIF-8 shell selectively degraded in tumor cells upon the chelation between GSH and Zn2+, while the as-formed Zn(SG) connected the released AuNR in end-to-end fashion. The coating of ZIF-8 shell significantly improves the stability and targeting of AuNR, and the released Zn2+ shielded the GSH binding site on the lateral side of AuNR, increased the plasmonic coupling efficiency of AuNR assembly geometer. This design enabled atomic-economical, efficient and low-side effect targeted photothermal therapy through the effective integration of heterostructures.

Interaction of Glutathione with MMACHC Arginine-Rich Pocket Variants Associated with Cobalamin C Disease: Insights from Molecular Modeling.

Methylmalonic aciduria and homocystinuria type C protein (MMACHC) is required by the body to metabolize cobalamin (Cbl). Due to its complex structure and cofactor forms, Cbl passes through an extensive series of absorptive and processing steps before being delivered to mitochondrial methyl malonyl-CoA mutase and cytosolic methionine synthase. Depending on the cofactor attached, MMACHC performs either flavin-dependent reductive decyanation or glutathione (GSH)-dependent dealkylation. The alkyl groups of Cbl have to be removed in the presence of GSH to produce intermediates that can later be converted into active cofactor forms. Pathogenic mutations in the GSH binding site, such as R161Q, R161G, R206P, R206W, and R206Q, have been reported to cause Cbl diseases. The impact of these variations on MMACHC's structure and how it affects GSH and Cbl binding at the molecular level is poorly understood. To better understand the molecular basis of this interaction, mutant structures involving the MMACHC-MeCbl-GSH complex were generated using in silico site-directed point mutations and explored using molecular dynamics (MD) simulations. The results revealed that mutations in the key arginine residues disrupt GSH binding by breaking the interactions and reducing the free energy of binding of GSH. Specifically, variations at position 206 appeared to produce weaker GSH binding. The lowered binding affinity for GSH in the variant structures could impact metabolic pathways involving Cbl and its trafficking.

Structural conservation in the glutathione binding in Sphingomonas sp. glutaredoxin Grx3 and variations for cold adaptation

Glutaredoxin 3 (Grx3), a redox protein with a thioredoxin-fold structure, maintains structural integrity and glutathione (GSH) binding capabilities across varying habitat temperatures. The cis-Pro loop, essential for GSH binding, relies on the Arg-Asp salt bridge (α2-α3) and Gln-His hydrogen bond (β3-β4) for its conformation. In some psychrophilic Grx3 variants, Arg in α2 is replaced with Tyr, and His in β4 is replaced with Phe. This study examines the roles of these bonds in Grx3's structure, function, and cold adaptation, using SpGrx3 from the Arctic bacterium Sphingomonas sp. Despite its cold habitat, SpGrx3 maintains the Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond. The R51Y substitution disrupts the α2-α3 salt bridge, while the H63F and H63Y substitutions hinder the salt bridge through cation-π interactions with Arg51, involving Phe63/Tyr63, thereby enhancing flexibility. Conversely, mutations that disrupt the hydrogen bond (Q56A, H63A, and H63F) reduce thermal stability. In the psychrophilic Grx3 configuration A48T/R51Y/H63F, a Thr48-Gln56 hydrogen bond stabilizes the cis-Pro loop, enhancing flexibility by disrupting both bonds. Furthermore, all mutants exhibit reduced α-helical content and catalytic efficiency. In summary, the highly conserved Arg51-Asp69 salt bridge and Gln56-His63 hydrogen bond are crucial for stabilizing the cis-Pro loop and catalytic activity in SpGrx3. His63 is favored as it avoids cation-π interactions with Arg51, unlike Phe63/Tyr63. Psychrophilic Grx3 variants have adapted to cold environments by reducing GSH binding and increasing structural flexibility. These findings deepen our understanding of the structural conservation in Grx3 for GSH binding and the critical alterations required for cold adaptation.

Reconstructing hepatic metabolic profile and glutathione-mediated metabolic fate of acrylamide

The toxicity of acrylamide (AA) has continuously attracted wide concerns as its extensive presence from both environmental and dietary sources. However, its hepatic metabolic transformation and metabolic fate still remain unclear. This study aims to unravel the metabolic profile and glutathione (GSH) mediated metabolic fate of AA in liver of rats under the dose-dependent exposure. We found that exposure to AA dose-dependently alters the binding of AA and GSH and the generation of mercapturic acid adducts, while liver as a target tissue bears the metabolic transformation of AA via regulating GSH synthesis and consumption pathways, in which glutamine synthase (GSS), cytochrome P450 2E1 (CYP2E1), and glutathione S-transferase P1 (GSTP1) play a key role. In response to high- and low-dose exposures to AA, there were significant differences in liver of rats, including the changes in GSH and cysteine (CYS) activities and the conversion ratio of AA to glycidamide (GA), and liver can affect the transformation of AA by regulating the GSH-mediated metabolic pathway. Low-dose exposure to AA activates GSH synthesis pathway in liver and upregulates GSS activity and CYS content with no change in γ-glutamyl transpeptidase 1 (GGT1) activity. High-dose exposure to AA activates the detoxification pathway of GSH and increases GSH consumption by upregulating GSTP1 activity. In addition, molecular docking results showed that most of the metabolic molecules transformed by AA and GA other than themselves can closely bind to GSTP1, GSS, GGT1, N-acetyltransferase 8, and dimethyl sulfide dehydrogenase 1. The binding of AA-GSH and GA-GSH to GSTP1 and CYP2E1 enzymes determine the tendentiousness between toxicity and detoxification of AA, which exerts a prospective avenue for targeting protective role of hepatic enzymes against in vivo toxicity of AA.

Development and application of fragment-based de novo inhibitor design approaches against Plasmodium falciparum GST.

Modulation of disease progression is frequently started by identifying biochemical pathway catalyzed by biomolecule that is prone to inhibition by small molecular weight ligands. Such ligands (leads) can be obtained from natural resources or synthetic libraries. However, de novo design based on fragments assembly and optimization is showing increasing success. Plasmodium falciparum parasite depends on glutathione-S-transferase (PfGST) in buffering oxidative heme as an approach to resist some antimalarials. Therefore, PfGST is considered an attractive target for drug development. In this research, fragment-based approaches were used to design molecules that can fit to glutathione (GSH) binding site (G-site) of PfGST. The involved approaches build molecules from fragments that are either isosteric to GSH sub-moieties (ligand-based) or successfully docked to GSH binding sub-pockets (structure-based). Compared to reference GST inhibitor of S-hexyl GSH, ligands with improved rigidity, synthetic accessibility, and affinity to receptor were successfully designed. The method involves joining fragments to create ligands. The ligands were thenexplored using molecular docking, Cartesian coordinate's optimization, and simplified free energy determination as well as MD simulation and MMPBSA calculations. Several tools were used which includeOPENEYE toolkit, Open Babel, Autodock Vina, Gromacs, and SwissParam server, and molecular mechanics force field of MMFF94 for optimization and CHARMM27 for MD simulation. In addition,in-house scripts written in Matlab were used to control fragments connectionand automation of the tools.

Genome-wide identification and characterization of glutathione S-transferase gene family in quinoa (Chenopodium quinoa Willd.).

The present investigation was envisaged for large scale in-silico genome wide identification and characterization of glutathione S-transferases (GSTs) in Chenopodium quinoa. In this study, a total of 120 GST genes (CqGSTs) were identified and divided into 11 classes of which tau and phi were highest in numbers. The average protein length of protein was found to be 279.06 with their corresponding average molecular weight of 31,819.4kDa. The subcellular localization analysis results showed that proteins were centrally localized in the cytoplasm followed by chloroplast, mitochondria and plastids. Structural analysis revealed the presence of 2 -14 exons in CqGST genes. Most of the proteins possessed two exon one intron organization. MEME analysis identified 15 significantly conserved motifs with a width of 6-50 amino acids. Motifs 1, 3, 2, 5, 6, 8, 9 and 13 were found specifically in tau class family; motifs 3, 4, 5, 6, 7 and 9 were found in phi class gene family, while motifs 3, 4, 13 and 14 were found in metaxin class. Multiple sequence alignment revealed highly conserved N-terminus with active site serine (Ser; S) or cysteine (Cys; C) residue for the activation of GSH binding and GST catalytic activity. The gene loci were found to be unevenly distributed across 18 different chromosomes with a maximum of 17 genes located on chromosome number 7. Dominance of alpha helix was followed by coil, extended strand and beta turns. Gene duplication analysis revealed that segmental duplication and purifying type selection were highest in number and found to be main source of expansion of GST gene family. Cis acting regulatory elements analysis showed the presence of 21 different elements involved in stress, hormone and light response and cellular development. The evolutionary relationship of CqGST proteins carried out using maximum likelihood method revealed that all the tau and phi class GSTs were closely associated with those of G. max, O. sativa and A. thaliana. Molecular docking of GST molecules with the fungicide metalaxyl showed that the CqGSTF1 had the lowest binding energy. The comprehensive study of CqGST gene family in quinoa provides groundwork for further functional analysis of CqGST genes in the species at molecular level and has potential applications in plant breeding.

MOF-derived magnetic trimetallic oxide-carbon nanocomposites for modification-free and dual molecular events-dependent ratiometric electrochemical analysis of intracellular glutathione

The response of ratiometric electrochemical signals toward the targets of interest generally depends on specific molecules-mediated single molecular recognition event, which limits the application in analyzing the targets without specific recognition molecules. In this work, novel dual molecular recognition events were proposed for rapid and reliable electrochemical analysis of glutathione (GSH) using MOF-derived trimetallic oxide-carbon (Fe3O4/CuO/NiO-C) nanocomposites. The binding of GSH to CuO caused a decrease in the oxidation signal of Cu(Ⅱ) due to the formation of nonelectroactive complex, while NiO could electrocatalyze the oxidation of GSH. These GSH-involved dual molecular recognition events guaranteed the readout of ratiometric electrochemical signals with a complementary response range, allowing sensitive and wide-range analysis of GSH. Besides, the difference in spatial conformation gave rise to a different oxidation potential for different biothiols, which guaranteed selective analysis of GSH. The presence of Fe3O4 allowed modification-free detection within 5 min using a magnetic working electrode. Taking advantage of these merits, the developed electrochemical sensor could rapidly and reliably monitor the intracellular GSH.

Identification and characterization of ABCC gene family and their roles in the response to intraperitoneal injection of microcystin-LR in liver of silver carp (Hypophthalmichthys molitrix)

Silver carp (Hypophthalmichthys molitrix) is a widely distributed food resource and phytoplankton predator in China, which has been used to control algal blooms. Silver carp is quite resistant to microcystins (MCs), which are typical hepatotoxins generated by toxic algae. ATP-Binding Cassette, Subfamily C (ABCC) transporter are critical detoxification genes that could export toxic substances out of the cell, and with regard to ABCC transporters in H. molitrix, current studies are limited. In order to investigate the roles of ABCC genes in excretion of MCs, the full-length transcriptome of silver carp was analyzed to screen a total of nine ABCC transporter genes, which were expressed ubiquitously in silver carp tissues, especially in the liver. Furthermore, microcystin-LR (MC-LR) intraperitoneal injection was performed, and the results showed that, except abcc2, abcc10 and abcc12, the expression of other ABCC genes increased rapidly within 1 h and then began to decline after 6 h. Among these five genes, abcc3 and abcc9 exhibited the most pronounced response, indicating that these two were the most preeminent detoxification genes of microcystin in ABCC genes family in silver carp. Meanwhile, the activities of glutathione-S-transferase (GST) were increased significantly, while the content of glutathione (GSH) demonstrated a dramatic decrease, implying that GST catalyzed binding of GSH and MC-LR, and then excreted through ABCC transporters. The findings of this study will be useful in further investigating the fundamental characteristics of silver carp and provide new insights into the potential function of ABCC genes in the detoxication of MC-LR.

Enzymology of reactive intermediate protection: kinetic analysis and temperature dependence of the mesophilic membrane protein catalyst MGST1.

Glutathione transferases (GSTs) are a class of phase II detoxifying enzymes catalysing the conjugation of glutathione (GSH) to endogenous and exogenous electrophilic molecules, withmicrosomal glutathione transferase 1 (MGST1) being one of its key members. MGST1 forms a homotrimer displaying third-of-the-sites-reactivity and up to 30-fold activation through modification of its Cys-49 residue. It has been shown that the steady-state behaviour of the enzyme at 5 °C can be accounted for by its pre-steady-state behaviour if the presence of a natively activated subpopulation (~ 10%) is assumed. Low temperature was used as the ligand-free enzyme is unstable at higher temperatures. Here, we overcame enzyme lability through stop-flow limited turnover analysis, whereby kinetic parameters at 30 °C were obtained. The acquired data are more physiologically relevant and enable confirmation of the previously established enzyme mechanism (at 5 °C), yielding parameters relevant for in vivo modelling. Interestingly, the kinetic parameter defining toxicant metabolism, kcat /KM , is strongly dependent on substrate reactivity (Hammett value 4.2), underscoring that glutathione transferases function as efficient and responsive interception catalysts. The temperature behaviour of the enzyme was also analysed. Both the KM and KD values decreased with increasing temperature, while the chemical step k3 displayed modest temperature dependence (Q10 : 1.1-1.2), mirrored in that of the nonenzymatic reaction (Q10 : 1.1-1.7). Unusually high Q10 values for GSH thiolate anion formation (k2 : 3.9), kcat (2.7-5.6) and kcat /KM (3.4-5.9) support that large structural transitions govern GSH binding and deprotonation, which limits steady-state catalysis.

Differentially-expressed genes related to glutathione metabolism and heavy metal transport reveals an adaptive, genotype-specific mechanism to Hg2+ exposure in rice (Oryza sativa L.)

Rice consumption is an essential cause of mercury (Hg) exposure for humans in Asia. However, the mechanism of Hg transport and accumulation in rice plants (Oryza sativa L.) remains unclear. Here, rice genotypes with contrasting Hg uptake and translocation abilities, i.e. H655 (high Hg-accumulator) and H767 (low Hg-accumulator), were selected from 261 genotypes. Through comparative physiological and transcriptome analyses, we investigated the processes responsible for the relationship between Hg accumulation, transport and tolerance. The results showed significant stimulation of antioxidative metabolism, particularly glutathione (GSH) accumulation, and up-regulated expression of regulatory genes of glutathione metabolism for H655, but not for H767. In addition, up-regulated expression of GSH S-transferase (GST) and OsPCS1 in H655 that catalyzes the binding of Hg and GSH, enhances the Hg detoxification capacity, while high-level expression of YSL2 in H655 enhances the transport ability for Hg. Conclusively, Hg accumulation in rice is a consequence of enhanced expression of genes related to Hg binding with GSH and Hg transport. With these results, the present study contributes to the selection of rice genotypes with limited Hg accumulation and to the mitigation of Hg migration in food chains thereby enhancing nutritional safety of Hg-polluted rice fields.

Combination with vorinostat enhances the antitumor activity of cisplatin in castration-resistant prostate cancer by inhibiting DNA damage repair pathway and detoxification of GSH.

Like DNA methylation, histone modifications are considered important processes for epigenetic alterations in gene function, and abnormally high expression of histone deacetylases (HDACs) plays a key role in many human diseases. In addition to regulating the acetylation levels of histone and non-histone proteins and gene transcription, HDAC inhibitors as antitumor drugs can also affect the DNA damage repair (DDR) pathway in tumor cells. Prostate cancer (PCa) is one of the most heritable malignancies in which DDR pathway defects can be detected in a considerable proportion of cases. Such defects are more prevalent in castration-resistant prostate cancer (CRPC) and are highly enriched in metastatic lesions. There is currently evidence that DDR pathway-deficient PCa is associated with high-risk biological behaviors and response sensitivity to platinum-based chemotherapy. Platinum-based drugs have been used in multiple clinical trials as monotherapy or in combination with other chemotherapeutic agents for the treatment of CRPC. This study evaluated the combined anticancer effect of (cisplatin) CDDP and the HDAC inhibitors vorinostat (SAHA) on three androgen-dependent cell lines PC-3, DU-145, and C4-2B in vitro. The efficacy and safety of SAHA combined with CDDP in the treatment of CRPC were further verified through animal experiments. The combination of the two drugs increases cytotoxic effects by increasing DNA damage. Our results showed that the SAHA could not only reduce the expression of homologous recombinant repair proteins BRCA2, BRCA1, PARP1, and RAD51, but also decrease enzymes that Reduce the key enzymes of GSH biosynthesis, GSS and GCLC, and GSTP1 which can catalyze the binding of GSH to cisplatin. The intracellular GSH level also decreased with the increase of SAHA concentration, at the same time, the content of intracellular Pt element. The combination of CDDP and SAHA can produce synergistic anticancer effects in androgen-independent PCa cells in vitro and in vivo. Our results open up a new avenue for the effective treatment of CRPC. To optimize the chemotherapy regimen for patients with advanced PCa, it is necessary to further study the molecular mechanism of platinum drugs, HDAC inhibitors, and their combined action.

The 2-Oxoglutarate Carrier Is S-Nitrosylated in the Spinal Cord of G93A Mutant hSOD1 Mice Resulting in Disruption of Mitochondrial Glutathione Transport.

Mitochondrial oxidative stress and dysfunction are strongly implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Glutathione (GSH) is an endogenous antioxidant that exists as distinct cytosolic and mitochondrial pools. The status of the mitochondrial GSH pool is reliant on transport from the cytosol through the 2-oxoglutarate carrier (OGC), an inner membrane anion carrier. We have previously reported that the outer mitochondrial membrane protein, Bcl-2, directly binds GSH and is a key regulator of OGC-dependent mitochondrial GSH transport. Here, we show that G93A mutant SOD1 (Cu, Zn-superoxide dismutase) reduces the binding of GSH to Bcl-2 and disrupts mitochondrial GSH uptake in vitro. In the G93A mutant hSOD1 mouse model of ALS, mitochondrial GSH is significantly depleted in spinal cord of end-stage mice. Finally, we show that OGC is heavily S-nitrosylated in the spinal cord of end-stage mice and consequently, the GSH uptake capacity of spinal cord mitochondria isolated from these mutant mice is significantly diminished. Collectively, these findings suggest that spinal cord GSH depletion, particularly at the level of the mitochondria, plays a significant role in ALS pathogenesis induced by mutant SOD1. Furthermore, the depletion of mitochondrial GSH in the G93A mutant hSOD1 mouse model may be caused by the S-nitrosylation of OGC and the capacity of mutant SOD1 to disrupt the Bcl-2/GSH interaction, resulting in a disruption of mitochondrial GSH transport.

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

Observing How Glutathione and S-Hexyl Glutathione Bind to Glutathione S-Transferase from Rhipicephalus (Boophilus) microplus.

Rhipicephalus (Boophilus) microplus is one of the most widespread ticks causing a massive loss to livestock production. The long-term use of acaracides rapidly develops acaracide resistance. In R. microplus, enhancing the metabolic activity of glutathione S-transferase (RmGST) is one of the mechanisms underlying acaracide resistance. RmGST catalyzes the conjugation of glutathione (GSH) to insecticides causing an easy-to-excrete conjugate. The active RmGST dimer contains two active sites (hydrophobic co-substrate binding site (H-site) and GSH binding site (G-site)) in each monomer. To preserve the insecticide efficacy, s-hexyl glutathione (GTX), a GST inhibitor, has been used as a synergist. To date, no molecular information on the RmGST-GSH/GTX complex is available. The insight is important for developing a novel RmGST inhibitor. Therefore, in this work, molecular dynamics simulations (MD) were performed to explore the binding of GTX and GSH to RmGST. GSH binds tighter and sits rigidly inside the G-site, while flexible GTX occupies both active sites. In GSH, the backbone mainly interacts with W8, R43, W46, K50, N59, L60, Q72, and S73, while its thiol group directs to Y7. In contrast, the aliphatic hexyl of GTX protrudes into the H-site and allows a flexible peptide core to form various interactions. Such high GTX flexibility and the protrusion of its hexyl moiety to the H-site suggest the dual role of GTX in preventing the conjugation reaction and the binding of acaracide. This insight can provide a better understanding of an important insecticide-resistance mechanism, which may in turn facilitate the development of novel approaches to tick control.

Introduction of a More Glutaredoxin-like Active Site to PDI Results in Competition between Protein Substrate and Glutathione Binding.

Proteins in the thioredoxin superfamily share a similar fold, contain a -CXXC- active site, and catalyze oxidoreductase reactions by dithiol-disulfide exchange mechanisms. Protein disulfide isomerase (PDI) has two -CGHC- active sites. For in vitro studies, oxidation/reduction of PDI during the catalytic cycle is accomplished with glutathione. Glutathione may act as electron donor/acceptor for PDI also in vivo, but at least for oxidation reactions, GSSG probably is not the major electron acceptor and PDI may not have evolved to react with glutathione with high affinity, but merely having adequate affinity for both glutathione and folding proteins/peptides. Glutaredoxins, on the other hand, have a high affinity for glutathione. They commonly have -CXFC- or -CXYC- active site, where the tyrosine residue forms part of the GSH binding groove. Mutating the active site of PDI to a more glutaredoxin-like motif increased its reactivity with glutathione. All such variants showed an increased rate in GSH-dependent reduction or GSSG-dependent oxidation of the active site, as well as a decreased rate of the native disulfide bond formation, with the magnitude of the effect increasing with glutathione concentration. This suggests that these variants lead to competition in binding between glutathione and folding protein substrates.