The Glutathione System of Aspergillus nidulans Involves a Fungus-specific Glutathione S-Transferase

Abstract

The tripeptide glutathione is involved in cellular defense mechanisms for xenobiotics and reactive oxygen species. This study investigated glutathione-dependent mechanisms in the model organism Aspergillus nidulans. A recombinant dimeric protein of A. nidulans glutathione reductase (GR) contained FAD and reduced oxidized glutathione (GSSG) using NADPH as an electron donor. A deletion strain of the GR gene (glrA) accumulated less intracellular reduced glutathione (GSH), indicating that the fungal GR contributes to GSSG reduction in vivo. Growth of the deletion strain of glrA was temperature-sensitive, and this phenotype was suppressed by adding GSH to the medium. The strain subsequently accumulated more intracellular superoxide, and cell-free respiration activity was partly defective. Growth of the strain decreased in the presence of oxidants, which induced glrA expression 1.5-6-fold. These results indicated that the fungal glutathione system functions as an antioxidant mechanism in A. nidulans. Our findings further revealed an initial proteomic differential display on GR-depleted and wild type strains. Up-regulation of thioredoxin reductase, peroxiredoxins, catalases, and cytochrome c peroxidase in the glrA-deletion strain revealed interplay between the glutathione system and both the thioredoxin system and hydrogen peroxide defense mechanisms. We also identified a hypothetical, up-regulated protein in the GR-depleted strains as glutathione S-transferase, which is unique among Ascomycetes fungi.

Glutathione Reductase/Glutathione Is Responsible for Cytotoxic Elemental Sulfur Tolerance via Polysulfide Shuttle in Fungi

“…Thioredoxin reductase was assayed in a mixture comprising crude extract, 100 mM potassium phosphate (pH 7.5), 0.2 mM NADPH, 0.34 mM recombinant human insulin solubilized by the method of Holmgren et al (22) and 20 M recombinant thioredoxin (rTrxA) of Aspergillus nidulans (20,23). The reaction was started by adding rTrxA and the decrease in NADPH was measured at absorbance 340 nm.…”

“…of PS at 30°C for 12 h, and total RNA was purified using the RNeasy plant mini kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions. First-strand cDNA was synthesized by incubating total RNA (10 g) in 10 l of reaction buffer comprising Oligo (dT) 20 (Toyobo, Osaka, Japan), 5 ϫ reverse transcriptase buffer and reverse transcriptase M-MLV (200 units) (Takara Bio, Kyoto, Japan) at 42°C for 90 min. Firststrand cDNA (330 ng) synthesized in the reaction was analyzed by quantitative PCR using iQ TM SYBR Green Supermix (BioRad) and MiniOpticon TM version 3.1 (Bio-Rad) according to the manufacturer's instructions.…”

Further characterization of the role of the mitochondrial high-mobility group box protein in the intracellular redox environment of Aspergillus nidulans

“…The explanation for the intriguing downregulation of gstB may be connected to the elevated glrA transcription. A study by Sato et al (2009) has shown that gstB is upregulated in a glrAD genetic background. In an hmbBD strain, the transcription of glrA is upregulated by 2.8-fold and gstB is downregulated by 2.9-fold, which would be consistent with downregulation of glrA upon gstA overexpression.…”

“…the glutathione redox couple (Sato et al, 2009). The absence of glrA leads to the upregulation of the expression of trxR [thioredoxin reductase (TR), also known as trrA ], catB (CAT) and gstB which offsets the increased oxidative redox state due to GlrA depletion (Sato et al, 2009). The interplay between the glutathione couple system and other redox systems led us to the study of trxR and catB expression together with that of glrA.…”

“…Some GSTs, however, oxidize GSH to GSSG and act as GPxs and disulfide reductases (glutaredoxins). We monitored the relative mRNA levels of gstA (Fraser et al, 2002), gstB (Sato et al, 2009), five putative GST genes (AN10273, AN0815, AN2948, AN5831 and AN6158) and four putative glutaredoxin genes (gpxA, AN3255, AN4215 and AN4304). gstA, gpxA, AN3255, AN4304, GstB and AN10273 were found to be oxidative stress-induced genes/proteins in previous high-throughput studies (Fraser et al, 2002;Pó csi et al, 2005;Pusztahelyi et al, 2011), so we selected them for our investigation.…”