Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.

Glutaredoxins catalyze glutathione-dependent disulfide oxidoreductions, particularly reduction of glutathione (GSH)-protein mixed disulfides. Mammalian glutaredoxins are present in the cytosol/nucleus as Grx1 or in mitochondria as Grx2a. Here we describe di-eosin-glutathione disulfide (Di-E-GSSG) as a new tool to study glutaredoxin (Grx) activity. Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. Di-E-GSSG was a very poor substrate for glutathione reductase, but we discovered that the molecule was an excellent substrate for glutaredoxin in a coupled assay system with GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase or with lipoamide, NADH, and lipoamide dehydrogenase. In addition, Di-E-GSSG was used to glutathionylate the free SH group of bovine serum albumin (BSA), yielding eosin-glutathionylated BSA (E-GS-BSA) readily observed in ultraviolet (UV) light. E-GS-BSA also displayed a quenched fluorescence, and its Grx-catalyzed reduction could be followed by the formation of E-GSH by fluorescence emission using microtiter plates. This way of measuring Grx activity provided an ultrasensitive method that detected Grx1 and Grx2 at picomolar levels. Human Grx1 was readily quantified in 40 µl of plasma and determined to be 680 ± 208 pM in healthy controls.

Glutaredoxin regulates vascular development by reversible glutathionylation of sirtuin 1.

Embryonic development depends on complex and precisely orchestrated signaling pathways including specific reduction/oxidation cascades. Oxidoreductases of the thioredoxin family are key players conveying redox signals through reversible posttranslational modifications of protein thiols. The importance of this protein family during embryogenesis has recently been exemplified for glutaredoxin 2, a vertebrate-specific glutathione-disulfide oxidoreductase with a critical role for embryonic brain development. Here, we discovered an essential function of glutaredoxin 2 during vascular development. Confocal microscopy and time-lapse studies based on two-photon microscopy revealed that morpholino-based knockdown of glutaredoxin 2 in zebrafish, a model organism to study vertebrate embryogenesis, resulted in a delayed and disordered blood vessel network. We were able to show that formation of a functional vascular system requires glutaredoxin 2-dependent reversible S-glutathionylation of the NAD(+)-dependent protein deacetylase sirtuin 1. Using mass spectrometry, we identified a cysteine residue in the conserved catalytic region of sirtuin 1 as target for glutaredoxin 2-specific deglutathionylation. Thereby, glutaredoxin 2-mediated redox regulation controls enzymatic activity of sirtuin 1, a mechanism we found to be conserved between zebrafish and humans. These results link S-glutathionylation to vertebrate development and successful embryonic angiogenesis.

Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

The crystal structure of human GLRX5: iron-sulfur cluster co-ordination, tetrameric assembly and monomer activity.

Human GLRX5 (glutaredoxin 5) is an evolutionarily conserved thiol-disulfide oxidoreductase that has a direct role in the maintenance of normal cytosolic and mitochondrial iron homoeostasis, and its expression affects haem biosynthesis and erythropoiesis. We have crystallized the human GLRX5 bound to two [2Fe-2S] clusters and four GSH molecules. The crystal structure revealed a tetrameric organization with the [2Fe-2S] clusters buried in the interior and shielded from the solvent by the conserved ß1-α2 loop, Phe69 and the GSH molecules. Each [2Fe-2S] cluster is ligated by the N-terminal activesite cysteine (Cys67) thiols contributed by two protomers and two cysteine thiols from two GSH. The two subunits co-ordinating the cluster are in a more extended conformation compared with iron-sulfur-bound human GLRX2, and the intersubunit interactions are more extensive and involve conserved residues among monothiol GLRXs. Gel-filtration chromatography and analytical ultracentrifugation support a tetrameric organization of holo-GLRX5, whereas the apoprotein is monomeric. MS analyses revealed glutathionylation of the cysteine residues in the absence of the [2Fe-2S] cluster, which would protect them from further oxidation and possibly facilitate cluster transfer/acceptance. Apo-GLRX5 reduced glutathione mixed disulfides with a rate 100 times lower than did GLRX2 and was active as a glutathione-dependent electron donor for mammalian ribonucleotide reductase.

Glutaredoxin mediated redox effects of coenzyme Q10 treatment in type 1 and type 2 diabetes patients.

The possible beneficial effects of coenzyme Q10 (CoQ10) supplementation on disease progression and oxidant status in diabetes remains debated. In the present study, patients with type 1 and type 2 diabetes were treated with oral CoQ10, 100 mg twice daily for 12 weeks. We assessed total antioxidant capacity, intra- and extracellular levels of the redox regulating protein glutaredoxin 1 (Grx1), CoQ10, oxidized LDL-cholesterol, lipid profile and HbA1c. We have previously shown that extracellular Grx1 is increased in patients with type 2 diabetes compared to healthy subjects. In the present study, CoQ10 treatment significantly decreased serum Grx1 activity as well as total antioxidant capacity independent of type of diabetes, indicating an improvement to a less oxidized extracellular environment. The effect on serum Grx1 activity was more prominent in patients not on statin treatment. Conversely, intracellular Grx1 activity as well as mRNA levels increased independent of statin treatment. There was a significant improvement in oxidized LDL-cholesterol and lipid profile, with a tendency to improved metabolic control (HbA1c). Additionally, we describe for the first time that CoQ10 is a direct substrate for glutathione, and that Grx1 catalyzes this reaction, thus presenting a novel mechanism for CoQ10 reduction which could explain our findings of an increased intracellular Grx1. In conclusion, 12 weeks CoQ10 treatment significantly improved the extracellular redox balance and lipid profile, indicating that prolonged treatment may have beneficial effects also on clinical outcome in diabetes.

Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.

Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.