Selective Inactivation of Glutaredoxin by Sporidesmin and Other Epidithiopiperazinediones^,

Abstract

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 analogs. Keywordsgliotoxin; glutaredoxin; mixed disulfides; sporidesmin; thioltransferase Glutaredoxin (GRx, also known as thioltransferase) is a member of the thiol-disulfide oxidoreductase enzyme family, which also includes thioredoxin and their corresponding reductase enzymes GSSG reductase and thioredoxin reductase, respectively. Mammalian NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 October 24. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptGRx1 is a 12 kDa cytosolic protein that has been characterized in vitro as a specific catalyst for the reduction of protein-glutathionyl mixed disulfides (protein-SSG) (1-6). The reaction catalyzed by GRx1 is also selective for GSH as the reducing substrate (4). This thioldisulfide interchange reaction is likely crucial for maintaining intracellular thiol status (2-7) under a variety of physiological and pathophysiological processes like aging, cardiovascular and neurodegenerative di...

Inhibition of Disulfide Reductases as a Therapeutic Strategy

“…The thiolcontaining mycotoxin sporidesmin (epidithiopiperazine-2,5-dione, #1456-55-9) inactivates human glutaredoxin in a time-and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductases such as glutathione reductase, thioredoxin, and thioredoxin reductase, are unaffected by sporidesmin [143]. The data are consistent with the formation of intermolecular disulfides containing one adducted toxin per glutaredoxin with elimination of two sulfur atoms from the detected product [143].…”

“…Under comparable conditions other thiol-disulfide oxidoreductases such as glutathione reductase, thioredoxin, and thioredoxin reductase, are unaffected by sporidesmin [143]. The data are consistent with the formation of intermolecular disulfides containing one adducted toxin per glutaredoxin with elimination of two sulfur atoms from the detected product [143]. Thiolcontaining compounds based on the quinol pharmacophore 4-(hydroxycyclohexa-2,5-dienone) exhibit potent and selective antitumor activity against colon, renal, and breast carcinoma cell lines in vitro [144].…”

Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry

“…Thus, the development of a methodology for the specific detection of gliotoxin, especially under aqueous conditions and without the requirement for prior organic extraction and solvent removal, is significant because it enables direct identification of gliotoxin without specimen pre-treatment which may otherwise lead to gliotoxin loss or modification. Moreover, as with alternative strategies for mycotoxin detection [34], the reductive alkylation approach developed for detection of gliotoxin will also be useful in the detection of related ETP toxins such as sporidesmin A, as we have demonstrated, and other disulphide-or thiol-containing molecules [35,36].…”

Resistance is not futile: gliotoxin biosynthesis, functionality and utility

“…Gliotoxin and related ETPs directly inactivate multiple enzymes and proteins in animal cells, including nuclear factor-kB (NF-kB), NADPH oxidase, and glutaredoxin, by conjugation to thiol groups [64][65][66]. It has been conclusively demonstrated that gliotoxin is taken up by animal cells in a GSH-dependent manner whereby this normally protective molecular species facilitates the concentration of gliotoxin within animal cells [59].…”