Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.     

Role of vicinal protein thiols in radiation and cytotoxic responses

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, beta-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD+ (K1) and G6PD- (E89) cells treated with L-buthionine sulfoximine (L-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of L-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.     

The phenotype and genotype of rheumatoid arthritis in the Democratic Republic of Congo

Introduction

Little is known about rheumatoid arthritis in the black, particularly in Congolese, populations. Our objective was to describe the phenotype and genotype of rheumatoid arthritis (RA) in Congolese.

Methods

All consecutive rheumatoid arthritis (RA) patients attending Kinshasa University Hospital in a three-year time period were included. Demographics, clinical features and tobacco consumption were noted. Disease Activity Score (DAS)-28 based on the erythrocyte sedimentation rate (ESR), Health Assessment Questionnaire (HAQ), anti-citrullinated peptide antibodies (CCP) antibodies and rheumatoid factor (RF) were determined. Radiographs were scored according to Sharp-van der Heijde. On a subset of patients and controls HLA-DRB1 typing was performed.

Results

A total of 114 females and 14 males aged 51.2 ± 14.9 were included. Mean duration of symptoms was four years. Moderate tobacco consumption was reported in a minority of patients. DAS-28 at first visit was >5.1 and HAQ ≥0.5 in all patients. X-rays showed joint erosions and/or joint space narrowing, mostly of a moderate grade in 55.8% of patients. Anti-CCP and/or RF were present in 48.6% of patients with available data (n = 72) and in 3.0% of controls (n = 67). Radiographic changes and nodules were more frequent in RF or anti-CCP positive patients. One copy of the shared epitope was found in 13 patients (35.1%) and 3 controls (12.5%). Two copies were found in one patient (2.7%) and in one control (4.2%).

Conclusion

Congolese patients with RA consult long after disease onset. Despite this delay, the majority presents without major damage and is RF, anti-CCP and SE negative. We put forward the hypothesis that besides different environmental factors there is probably also a particular genetic risk profile in Congolese patients, different from the HLA-DRB1 shared epitope.     

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.     

Reversible glutathionylation regulates actin polymerization in A431 cells.

In response to growth factor stimulation, many mammalian cells transiently generate reactive oxygen species (ROS) that lead to the elevation of tyrosine-phosphorylated and glutathionylated proteins. While investigating EGF-induced glutathionylation in A431 cells, paradoxically we found deglutathionylation of a major 42-kDa protein identified as actin. Mass spectrometric analysis revealed that the glutathionylation site is Cys-374. Deglutathionylation of the G-actin leads to about a 6-fold increase in the rate of polymerization. In vivo studies revealed a 12% increase in F-actin content 15 min after EGF treatment, and F-actin was found in the cell periphery suggesting that in response to growth factor, actin polymerization in vivo is regulated by a reversible glutathionylation mechanism. Deglutathionylation is most likely catalyzed by glutaredoxin (thioltranferase), because Cd(II), an inhibitor of glutaredoxin, inhibits intracellular actin deglutathionylation at 2 microM comparable with its IC(50) in vitro. Moreover, mass spectral analysis showed efficient transfer of GSH from immobilized S-glutathionylated actin to glutaredoxin. Overall, this study revealed a novel physiological relevance of actin polymerization regulated by reversible glutathionylation of the penultimate cysteine mediated by growth factor stimulation.