ascorbic-acid and geldanamycin

Morphogenetic transitions of the opportunistic fungal pathogen Candida albicans are influenced by temperature changes, with induction of filamentation upon a shift from 30 to 37°C. Hsp90 was identified as a major repressor of an elongated cell morphology at low temperatures, as treatment with specific inhibitors of Hsp90 results in elongated growth forms at 30°C. Elongated growth resulting from a compromised Hsp90 is considered neither hyphal nor pseudohyphal growth. It has been reported that ascorbic acid (vitamin C) interferes with the yeast-to-hypha transition in C. albicans. In the present study, we show that ascorbic acid also antagonizes the morphogenetic change caused by hampered Hsp90 function. Further analysis revealed that Upc2, a transcriptional regulator of genes involved in ergosterol biosynthesis, and Erg11, the target of azole antifungals, whose expression is in turn regulated by Upc2, are required for this antagonism. Ergosterol levels correlate with elongated growth and are reduced in cells treated with the Hsp90 inhibitor geldanamycin (GdA) and restored by cotreatment with ascorbic acid. In addition, we show that Upc2 appears to be required for ascorbic acid-mediated inhibition of the antifungal activity of fluconazole. These results identify Upc2 as a major regulator of ascorbic acid-induced effects in C. albicans and suggest an association between ergosterol content and elongated growth upon Hsp90 compromise.

Topics: Ascorbic Acid; Benzoquinones; Candida albicans; Cytochrome P-450 Enzyme System; Drug Resistance, Fungal; Ergosterol; Fluconazole; Fungal Proteins; Gene Expression Regulation, Fungal; HSP90 Heat-Shock Proteins; Hyphae; Lactams, Macrocyclic; Morphogenesis; Temperature; Trans-Activators

Heat shock transcription factor A2 (HsfA2) is induced under environmental stress and regulates transcription of various defense-related genes. Thus HsfA2 plays an important role in induction of defenses against different types of environmental stress, but its mode of regulation remains unknown. To clarify the signal transduction pathway involved in the regulation of HsfA2 expression, we investigated the effect of MG132, a 26S proteasome inhibitor, or geldanamycin (GDA), a heat shock protein 90 (Hsp90) inhibitor, on the transcription of HsfA2 and its targets, Hsp18.1-CI and ascorbate peroxidase 2 (Apx2), in Arabidopsis T87 cells. The levels of transcripts were significantly increased by treatment with MG132 or GDA. Overexpression of a dexamethazone-inducible dominant-negative form of Hsp90.2 in Arabidopsis plants caused significant expression of HsfA2 and its target gene on treatment with the compound. Treatment with MG132 or GDA had no effect on intracellular levels of reactive oxygen species (ROS). Interestingly, the levels of polyubiquitinated proteins as well as the levels of HsfA2 transcript were rapidly increased under oxidative stress derived from treatment with H2O2 or methylviologen, while they were completely suppressed by pre-treatment with ascorbate, a scavenger of ROS, under oxidative stress. The present findings suggest that the inhibition of 26S proteasome function and/or Hsp90 activity is involved in the induction of HsfA2 expression in response to oxidative stress.

Topics: Arabidopsis; Arabidopsis Proteins; Ascorbic Acid; Benzoquinones; Cells, Cultured; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Gene Expression Regulation, Plant; Heat Shock Transcription Factors; Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Hydrogen Peroxide; Lactams, Macrocyclic; Leupeptins; Oxidative Stress; Paraquat; Plant Proteins; Proteasome Endopeptidase Complex; Reactive Oxygen Species; Signal Transduction; Transcription Factors; Ubiquitinated Proteins

The ansamycin antibiotic geldanamycin has frequently been used as an inhibitor of heat shock protein 90 (Hsp90), and this agent has been widely employed as a probe to examine the interactions of Hsp90 with endothelial nitric-oxide synthase. Geldanamycin contains a quinone group, which may participate in redox cycling. When geldanamycin was exposed to the flavin-containing enzyme cytochrome P-450 reductase, both semiquinone and superoxide (O(2)(*)(-)) radicals were detected using electron spin resonance. The treatment of endothelial cells with geldanamycin resulted in a dramatic increase in O(2)(*)(-) generation, which was independent of endothelial nitric-oxide synthase, because it was not inhibited by N-nitro-l-arginine methyl ester and also occurred in vascular smooth muscle cells. Diphenylene iodinium inhibited this increase in O(2)(*)(-) by 50%, suggesting that flavin-containing enzymes are involved in geldanamycin-induced O(2)(*)(-) generation. In the absence of cells, geldanamycin directly oxidized ascorbate, consumed oxygen, and produced O(2)(*)(-). Geldanamycin decreased the bioavailable nitric oxide generated by 3,4-dihydrodiazete 1,2-dioxide in smooth muscle cells by 50%, whereas pretreatment with superoxide dismutase inhibited the effect of geldanamycin. These findings demonstrate that geldanamycin generates O(2)(*)(-), which scavenges nitric oxide, leading to loss of its bioavailability. This effect is independent of the inhibition of Hsp90 and indicates that geldanamycin cannot be used as a specific inhibitor of Hsp90. In light of these findings, the studies using geldanamycin as an inhibitor of Hsp90 should be interpreted with caution.

Topics: Ascorbic Acid; Benzoquinones; Cells, Cultured; Electron Spin Resonance Spectroscopy; HSP90 Heat-Shock Proteins; Humans; Hydroxylamine; Lactams, Macrocyclic; Muscle, Smooth, Vascular; NADPH-Ferrihemoprotein Reductase; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Quinones; Superoxides