Glutathione-dependent Oxidoreductase Research Articles

Pericyte loss via glutaredoxin2 downregulation aggravates diabetes-induced microvascular dysfunction

Diabetic retinopathy (DR) is the leading cause of vision loss and blindness among working-age adults. Pericyte loss is an early pathological feature of DR. Under hyperglycemic conditions, reactive oxygen species (ROS) production increases, leading to oxidative stress and subsequent mitochondrial dysfunction and apoptosis. Dysfunctional pericyte can cause retinal vascular leakage, obliteration, and neovascularization. Glutaredoxin 2 (Grx2) is a mitochondrial glutathione-dependent oxidoreductase which protects cells against oxidative insults by safeguarding mitochondrial function. Whether Grx2 plays a protective role in diabetes-induced microvascular dysfunction remains unclear. Our findings revealed that diabetes-related stress reduced Grx2 expression in pericytes, but not in endothelial cells. Grx2 knock-in ameliorated diabetes-induced microvascular dysfunction in vivo DR models. Decreased Grx2 expression led to significant pericyte apoptosis, and pericyte dysfunction, namely reduced pericyte recruitment towards endothelial cells and increased endothelial cell permeability. Conversely, upregulating Grx2 reversed these effects. Furthermore, Grx2 regulated pericyte apoptosis by modulating complex I activity, which is crucial for pericyte mitochondrial function. Overall, our study uncovered a novel mechanism whereby high glucose inhibited Grx2 expression in vivo and in vitro. Grx2 downregulation exacerbated pericyte apoptosis, pericyte dysfunction, and retinal vascular dysfunction by inactivating complex I and mediating mitochondrial dysfunction in pericytes.

Glutaredoxin 2 Protein (Grx2) as an Independent Prognostic Factor Associated with the Survival of Colon Adenocarcinoma Patients.

Glutaredoxin 2 (Grx2; Glrx2) is a glutathione-dependent oxidoreductase located in mitochondria, which is central to the regulation of glutathione homeostasis and mitochondrial redox, and plays a crucial role in highly metabolic tissues. In response to mitochondrial redox signals and oxidative stress, Grx2 can catalyze the oxidation and S-glutathionylation of membrane-bound thiol proteins in mitochondria. Therefore, it can have a significant impact on cancer development. To investigate this further, we performed an immunohistochemical analysis of Grx2 protein expression in colon adenocarcinoma samples collected from patients with primary colon adenocarcinoma (stage I and II) and patients with metastasis to regional lymph nodes (stage III). The results of our study revealed a significant relationship between the immunohistochemical expression of Grx2 and tumor histological grade, depth of invasion, regional lymph node involvement, angioinvasion, staging, and PCNA immunohistochemical expression. It was found that 87% of patients with stage I had high levels of Grx2 expression. In contrast, only 33% of patients with stage II and 1% of patients with stage III had high levels of Grx2 expression. Moreover, the multivariate analysis revealed that the immunohistochemical expression of Grx2 protein apart from the grade of tumor differentiation was an independent prognostic factors for the survival of patients with colon adenocarcinoma. Studies analyzing Grx2 levels in patients' blood confirmed that the highest levels of serum Grx2 protein was also found in stage I patients, which was reflected in the survival curves. A higher level of Grx2 in the serum has been associated with a more favorable outcome. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western Blot.

Glutaredoxin 2 protects lens epithelial cells from epithelial-mesenchymal transition by suppressing mitochondrial oxidative stress-related upregulation of integrin-linked kinase

Glutaredoxin 2 (Grx2), a mitochondrial glutathione-dependent oxidoreductase, is crucial for maintaining redox homeostasis and cellular functions in the lens. The oxidative stress-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is related to posterior capsule opacification. In this study, we investigated the effects of Grx2 on oxidative stress-induced EMT in LECs during posterior capsule opacification. We found that Grx2 expression was substantially decreased during the EMT of LECs and in a mouse model of cataract surgery. Deletion of Grx2 aggravated the generation of reactive oxygen species, including those that are mitochondria-derived, and promoted the proliferation and EMT of the LECs. This was reversed by Grx2 overexpression. In vivo, proteomic liquid chromatography-mass spectrometry analysis showed that integrin-linked kinase (ILK) was significantly upregulated in the lens posterior capsule of a Grx2 knockout (KO) mouse model. Compared with that of the wild-type group, the expression of ILK and EMT markers was increased in the Grx2 KO group which was reversed in the Grx2 knock-in group. Inhibition of ILK partially blocked Grx2 knockdown-induced EMT and prevented the increased phosphorylation of Akt and GSK-3β and the nuclear translocation of β-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/β-catenin pathway partially blocked the Grx2 knockdown-induced EMT. In conclusion, we demonstrated that Grx2 protects LECs from oxidative stress-related EMT by regulating the ILK/Akt/GSK-3β axis.

Glutaredoxin 2 in the mud crab Scylla paramamosain: Identification and functional characterization under hypoxia and pathogen challenge.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge.

Expression Analysis of a Novel Oxidoreductase Glutaredoxin 2 in Black Tiger Shrimp, Penaeus monodon.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that is an important component of the redox system in organisms. However, there is a serious lack of sequence information and functional validation related to Grx in crustaceans. In this study, a novel Grx was identified in Penaeus monodon (PmGrx2). The full-length cDNA of PmGrx2 is 998 bp, with an open reading frame (ORF) of 441 bp, encoding 119 amino acids. Sequence alignment showed that PmGrx2 had the highest identity with Grx2 of Penaeus vannamei at 96.64% and clustered with Grx2 of other crustaceans. Quantitative real-time PCR (qRT-PCR) analysis showed that PmGrx2 was expressed in all examined tissues, with higher expression levels in the stomach and testis. PmGrx2 was continuously expressed during development and had the highest expression level in the zygote stage. Both ammonia-N stress and bacterial infection could differentially induce the expression of PmGrx2 in hepatopancreas and gills. When PmGrx2 was inhibited, the expression of antioxidant enzymes was suppressed, the degree of apoptosis increased, and the GSH content decreased with the prolongation of ammonia-N stress. Inhibition of PmGrx2 resulted in shrimp being exposed to a greater risk of oxidative damage. In addition, an SNP locus was screened on the exons of PmGrx2 that was significantly associated with an ammonia-N-stress-tolerance trait. This study suggests that PmGrx2 is involved in redox regulation and plays an important role in shrimps’ resistance to marine environmental stresses.

Molecular Characterization and Expression Analysis of Glutaredoxin 5 in Black Tiger Shrimp (Penaeus monodon) and Correlation Analysis Between the SNPs of PmGrx5 and Ammonia-N Stress Tolerance Trait

Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin (Trx) superfamily and are an essential part of the redox system in living organisms. However, there is a serious lack of sequence information and functional validation associated with Grxs in crustaceans. In this study, a new Grx gene (PmGrx5) was identified and characterized in black tiger shrimp (Penaeus monodon). The full-length cDNA of PmGrx5 is 787 bp and consists of 114 bp 5′-UTR, 232 bp 3′-UTR, and 441 bp ORF, encoding a hypothetical protein of 146 amino acids. The putative PmGrx5 protein is 16.27 kDa with a theoretical isoelectric point of 5.90. Sequence alignment showed that PmGrx5 had the highest amino acid sequence homology with Grx5 from Penaeus vannamei at 98.63% and clustered with Grx5 from other crustaceans. Quantitative real-time PCR (qRT-PCR) analysis showed that PmGrx5 was expressed in all tissues examined, with a higher expression in the testis, stomach, lymphoid organ, and gill. PmGrx5 was continuously expressed during development, with the highest expression in zoea I. Ammonia-N stress and bacterial infection both differentially upregulated PmGrx5 expression in the hepatopancreas and gill. In addition, when PmGrx5 was inhibited, the expression of some other antioxidant enzymes was upregulated at the beginning of ammonia-N stress, but as the stress time increased, the expression of antioxidant enzymes was inhibited, the expression of apoptotic genes was increased, and the GSH content was significantly reduced. Inhibition of PmGrx5 led to a greater risk of oxidative damage in shrimp. In addition, the relationship between SNPs in exons of the PmGrx5 gene and tolerance to ammonia-N stress was identified and analysed. A total of nine SNPs were successfully identified, eight of which were significantly associated with ammonia and nitrogen stress tolerance trait in shrimp (P < 0.05). The present study shows that PmGrx5 is involved in redox regulation and plays an important role in shrimp resistance to marine environmental stresses. Meanwhile, this study will provide a basis for molecular marker breeding in shrimp.

Mitochondrial depletion of glutaredoxin 2 induces metabolic dysfunction-associated fatty liver disease in mice

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that facilitates glutathionylation/de-glutathionylation of target proteins. The main variants of Grx2 are the mitochondrial Grx2a and the cytosolic Grx2c. The aim of this study was to investigate the specific role of mitochondrial Grx2 in vivo using a mitochondrial Grx2 depleted (mGD) mouse model. mGD mice displayed an altered mitochondrial morphology and functioning. Furthermore, the lack of Grx2 in the mitochondrial compartment is responsible for increased blood lipid levels under a normal diet, a metabolic dysfunction-associated fatty liver disease (MAFLD) phenotype and a decreased glycogen storage capacity. In addition, depleting Grx2a leads to an alteration in abundance and in glutathionylation pattern of different mitochondrial enzymes, highlighting the selective role of Grx2 in the regulation of metabolic pathways. Overall, our findings identify the involvement of mitochondrial Grx2a in the regulation of cell metabolism and highlight a previously unknown association between Grx2 and MAFLD.

Insights into biodegradation mechanisms of triphenyl phosphate by a novel fungal isolate and its potential in bioremediation of contaminated river sediment

In this study, Aspergillus sydowii FJH-1 isolated from soil was verified to be a novel triphenyl phosphate (TPhP) degrader. Biodegradation efficiency of TPhP by Aspergillus sydowii FJH-1 exceeded 90% within 6 days under the optimal conditions (pH 4–9, 30 ℃, initial concentration less than 20 mg/L). Proteomics analysis uncovered the proteins perhaps involved in hydrolysis, hydroxylation, methylation and sulfonation of TPhP and the primary intracellular adaptive responses of Aspergillus sydowii FJH-1 to TPhP stress. The expression of carboxylic ester hydrolase along with several thioredoxin- and glutathione-dependent oxidoreductases were induced to withstand the toxicity of TPhP. The presence of TPhP also caused obvious upregulation of proteins concerned with glycolysis, pentose phosphate pathway and tricarboxylic acid cycle. Data from toxicological tests confirmed that the cytotoxicity and phytotoxicity of TPhP was effectively decreased after treatment with Aspergillus sydowii FJH-1. Additionally, bioaugmentation with Aspergillus sydowii FJH-1 was available for promoting TPhP removal in real water and water-sediment system. Collectively, the present study offered a deeper insight into the biodegradation mechanism and pathway of TPhP by a newly screened fungal strain Aspergillus sydowii FJH-1 and validated the feasibility of applying this novel degrader in the bioremediation of TPhP-polluted matrices.

An intracellular assay for activity screening and characterization of glutathione-dependent oxidoreductases

The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

Grx2 Regulates Skeletal Muscle Mitochondrial Structure and Autophagy.

Glutathione is an important antioxidant that regulates cellular redox status and is disordered in many disease states. Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that plays a pivotal role in redox control by catalyzing reversible protein deglutathionylation. As oxidized glutathione (GSSG) can stimulate mitochondrial fusion, we hypothesized that Grx2 may contribute to the maintenance of mitochondrial dynamics and ultrastructure. Here, we demonstrate that Grx2 deletion results in decreased GSH:GSSG, with a marked increase of GSSG in primary muscle cells isolated from C57BL/6 Grx2−/− mice. The altered glutathione redox was accompanied by increased mitochondrial length, consistent with a more fused mitochondrial reticulum. Electron microscopy of Grx2−/− skeletal muscle fibers revealed decreased mitochondrial surface area, profoundly disordered ultrastructure, and the appearance of multi-lamellar structures. Immunoblot analysis revealed that autophagic flux was augmented in Grx2−/− muscle as demonstrated by an increase in the ratio of LC3II/I expression. These molecular changes resulted in impaired complex I respiration and complex IV activity, a smaller diameter of tibialis anterior muscle, and decreased body weight in Grx2 deficient mice. Together, these are the first results to show that Grx2 regulates skeletal muscle mitochondrial structure, and autophagy.

Glutaredoxin-2 and Sirtuin-3 deficiencies impair cardiac mitochondrial energetics but their effects are not additive

Altered redox biology and oxidative stress have been implicated in the progression of heart failure. Glutaredoxin-2 (GRX2) is a glutathione-dependent oxidoreductase and catalyzes the reversible deglutathionylation of mitochondrial proteins. Sirtuin-3 (SIRT3) is a class III histone deacetylase and regulates lysine acetylation in mitochondria. Both GRX2 and SIRT3 are considered as key in the protection against oxidative damage in the myocardium. Knockout of either contributes to adverse heart pathologies including hypertrophy, hypertension, and cardiac dysfunction. Here, we created and characterized a GRX2 and SIRT3 double-knockout mouse model, hypothesizing that their deletions would have an additive effect on oxidative stress, and exacerbate mitochondrial function and myocardial structural remodeling. Wildtype, single-gene knockout (Sirt3−/−, Grx2−/−), and double-knockout mice (Grx2−/−/Sirt3−/−) were compared in heart weight, histology, mitochondrial respiration and H2O2 production. Overall, the hearts from Grx2−/−/Sirt3−/− mice displayed increased fibrosis and hypertrophy versus wildtype. In the Grx2−/− and the Sirt3−/− we observed changes in mitochondrial oxidative capacity, however this was associated with elevated H2O2 emission only in the Sirt3−/−. Similar changes were observed but not worsened in hearts from Grx2−/−/Sirt3−/− mice, suggesting that these changes were not additive. In human myocardium, using genetic and histopathological data from the human Genotype-Tissue Expression consortium, we confirmed that SIRT3 expression correlates inversely with heart pathology. Altogether, GRX2 and SIRT3 are important in the control of cardiac mitochondrial redox and oxidative processes, but their combined absence does not exacerbate effects, consistent with the overall conclusion that they function together in the complex redox and antioxidant systems in the heart.

Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

Over-expression of chickpea glutaredoxin (CaGrx) provides tolerance to heavy metals by reducing metal accumulation and improved physiological and antioxidant defence system

Glutaredoxins (Grxs) are small multifunctional redox proteins. Grxs have glutathione-dependent oxidoreductase activity in the presence of glutathione reductase and NADPH. The role of Grxs is well studied in heavy metal tolerance in prokaryotic and mammalian systems but not in plant genera. In the present study, a chickpea glutaredoxin (CaGrx) gene (LOC101493651) has been investigated against metal stress based on its primary screening in chickpea which revealed higher up-regulation of CaGrx gene under various heavy metals (AsIII-25μM, AsV-250μM, Cr(VI)-300μM, and Cd-500μM) stress. This CaGrx gene was overexpressed in Arabidopsis thaliana and investigated various biochemical and physiological performances under each metal stress. Transgenic plants showed significant up-regulation of the CaGrx gene during qRT-PCR analysis as well as longer roots, higher seed germination, and survival efficiency during each metal stress. The levels of stress markers, TBARS, H2O2, and electrolyte leakage were found to be less in transgenic lines as compared to WT revealed less toxicity in transgenics. The total accumulation of AsIII, AsV, and Cr(VI) were significantly reduced in all transgenic lines except Cd, which was slightly reduced. The physiological parameters such as net photosynthetic rate (PN), stomatal conductance (gs), transpiration (E), water use efficiency (WUE), photochemical quenching (qP), and electron transport rate (ETR), were maintained in transgenic lines during metal stress. Various antioxidant enzymes such as glutaredoxin (GRX), glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-transferase (GST), ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), antioxidant molecules (ascorbate, GSH) and stress-responsive amino acids (proline and cysteine) levels were significantly increased in transgenic lines which provide metal tolerance. The outcome of this study strongly indicates that the CaGrx gene participates in the moderation of metal stress in Arabidopsis, which can be utilized in biotechnological interventions to overcome heavy metal stress conditions in different crops.

Abstract AP18: PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3

Abstract Cancer cells are embedded within a microenvironment populated with cancer associated fibroblasts (CAFs) with whom they establish two-way communications through paracrine factors and physical interactions. Through these interactions, CAFs play pivotal roles in cancer and have emerged as promising therapeutic target. To unravel key contributors of CAF-tumour/stroma cell interactions, we have developed mass spectrometry (MS)-based approaches to map proteins secreted in condition medium and extracellular matrix (ECM). Through an extensive MS-proteomic comparative analysis of CAFs with their normal fibroblasts (NFs) counterpart, we have identified the chloride intracellular channel protein 3 (CLIC3), a protein previously considered an intracellular chloride channel (regulator), as one of the most upregulated proteins in CAFs and deposited in the ECM. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive blood vessels growth and increases invasiveness of cancer cells, both in vivo and in 3D cell culture models, via activation of the tissue transglutaminase-2 (TGM2). We found that CLIC3 is a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels in primary tumours and omental metastases correlate with poor clinical outcome. Our work has unraveled an unprecedented mechanism of cell invasion to be explored for targeting in ovarian cancer. Citation Format: Juan R Hernandez-Fernaud, Elena Ruengeler, Lisa J Neilson, Shehab Ismail, Iain McNeish, Samuel Atkinson, Sergio Lilla, Alice Santi, Stella M Valenzuela, Karen Blyth, Huabing Yin, Massimiliano Mazzone, Jim C Norman and Sara Zanivan. PRO-INVASIVE TUMOUR-STROMA INTERACTIONS: ROLE OF THE SECRETED OXIDOREDUCTASE CLIC3 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP18.

Glutaredoxin-2 controls cardiac mitochondrial dynamics and energetics in mice, and protects against human cardiac pathologies

Glutaredoxin 2 (GRX2), a mitochondrial glutathione-dependent oxidoreductase, is central to glutathione homeostasis and mitochondrial redox, which is crucial in highly metabolic tissues like the heart. Previous research showed that absence of Grx2, leads to impaired mitochondrial complex I function, hypertension and cardiac hypertrophy in mice but the impact on mitochondrial structure and function in intact cardiomyocytes and in humans has not been explored. We hypothesized that Grx2 controls cardiac mitochondrial dynamics and function in cellular and mouse models, and that low expression is associated with human cardiac dysfunction. Here we show that Grx2 absence impairs mitochondrial fusion, ultrastructure and energetics in primary cardiomyocytes and cardiac tissue. Moreover, provision of the glutathione precursor, N-acetylcysteine (NAC) to Grx2-/- mice did not restore glutathione redox or prevent impairments. Using genetic and histopathological data from the human Genotype-Tissue Expression consortium we demonstrate that low GRX2 is associated with fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, GRX2 is important in the control of cardiac mitochondrial structure and function, and protects against human cardiac pathologies.

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity.

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

A Cyanobacterium <i>Synechocystis</i> sp. PCC 6803 Glutaredoxin Gene (<i>slr1562</i>) Protects <i>Escherichia coli</i> against Abiotic Stresses

Problem statement: Glutaredoxins (GRXs) are ubiquitous small heat stable glutathione-dependent oxidoreductase enzymes that play a crucial role in plant development and response to oxidative stress. Approach: Cyanobacterium Synechocystis strain PCC 6803 contains two genes (slr1562 and ssr2061) encoding glutaredoxins. In the present investigation the slr1562 gene (grxC) was isolated and characterized. Results: The results revealed that the amino acid sequence deduced from GrxC protein share high identity with those of GRXs from other organisms and contain the consensus GRX family domain with a CPFC active site. Northern blotting analysis revealed that the expression of slr1562 gene could be induced by oxidative and salt stresses. Moreover, the protein GrxC was successfully overexpressed as a soluble fraction in Escherichia coli JM109. The over-expression of GrxC in Escherichia coli cells significantly increased resistance of cells to oxidative, drought and salt stresses. Conclusion/Recommendations: These results suggest that the slr1562 gene could play an important role in regulating abiotic tolerance against oxidative, drought and salt stresses in different organisms.

The ROXY1 C-terminal L**LL motif is essential for the interaction with TGA transcription factors.

Glutaredoxins (GRXs) are small, ubiquitous, glutathione-dependent oxidoreductases that participate in redox-regulated processes associated with stress responses. Recently, GRXs have been shown to exert crucial functions during flower developmental processes. GRXs modulate their target protein activities by the reduction of protein disulfide bonds or deglutathionylation reactions. The Arabidopsis (Arabidopsis thaliana) GRX ROXY1 participates in petal primordia initiation and further petal morphogenesis. ROXY1 belongs to a land plant-specific class of GRXs with a CC-type active site motif, deviating from the ubiquitously occurring CPYC and CGFS GRX classes. ROXY1 was previously shown to interact with floral TGA transcription factors in the nucleus, and this interaction is a prerequisite for ROXY1 to exert its activity required for Arabidopsis petal development. Deletion analysis further identified the importance of the ROXY1 C terminus for the ROXY1/TGA protein interactions and for the ROXY1 function in petal development. Here, by dissecting the ROXY1 C terminus, an α-helical L**LL motif immediately adjacent to the ROXY1 C-terminal eight amino acids was identified that is essential for the interaction with TGA transcription factors and crucial for the ROXY1 function in planta. Similar to the α-helical L**LL motifs binding to transcriptional coactivators with liganded nuclear receptors in animals, a hydrophobic face formed by the conserved leucines in the L**LL motif of ROXY1 possibly mediates the interaction with TGA transcription factors. Thus, the α-helical L**LL sequence is a conserved protein-protein interaction motif in both animals and plants. Furthermore, two separate TGA domains were identified by deletion experiments as being essential for mediating TGA protein interactions with ROXYs.

Genome-Wide Survey and Expression Analysis Suggest Diverse Roles of Glutaredoxin Gene Family Members During Development and Response to Various Stimuli in Rice

Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.