Redox control of Hsp90-client protein interactions

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S-glutathionylation is a physiological, reversible protein modification of cysteine residues with glutathione in response to mild oxidative stress. A method for the rapid determination of free and protein-bound glutathione levels in cultured cell lines has been developed based on the use of capillary electrophoresis with laser-induced fluorescence detection. In this approach, the samples were derivatized with 5-iodoacetamidofluorescein and analyzed. The results demonstrate that treatment of HepG2 cells with the compound pyrrolidine dithiocarbamate (PDTC) increased in a time-dependent manner the amount of protein-bound glutathione, which reflects the amount of protein S-glutathionylation. This approach should allow the measure of S-glutathionylated proteins as biomarkers of oxidative stress. Earlier work has showed that the transcription factor STAT3 is among the known client proteins interacting with Hsp90. STAT3 is central for the propagation of diverse cellular functions, ranging from differentiation and proliferation to migration and inflammation. Impairment in STAT3-Hsp90 interaction has been found to exert significant inhibition in STAT3 signaling. A study using PDTC and other oxidants was conducted in HepG2 cells and demonstrated that STAT3 was a S-glutathionylation target. This posttranslational modification had adverse effects with regard to STAT3s ability to become tyrosine phosphorylated and shuttle to the nucleus to activate expression of target genes in response to IL-6. The association of STAT3 with the molecular chaperone Hsp90 was dramatically reduced by PDTC, which could potentially explain the poor signaling potential of STAT3. The vulnerability of STAT3-Hsp90 interaction to S-thiolation represents a novel mode of regulation of IL-6 signaling. The ability of PDTC to dampen pro-inflammatory cytokine signaling both in cultured cell lines and in vivo has been reported and, yet, its effect on the complex profile of gene expression remains largely unknown. Using cDNA microarray analysis coupled with quantitative PCR assays, PDTC was found to alter time-dependently the expression of a number of target genes for the transcription factor heat shock factor 1 (HSF1) in HepG2 cells when compared to saline controls. Functional classification of these target genes demonstrated alterations in expression of several biological pathways, including response to unfolded protein and regulation of apoptosis. Experiments aimed at assessing the effects of knocking down HSF1 expression in HepG2 cells and administration of PDTC in a tumor xenograft model in mice have been conducted. Moreover, the mechanism by which PDTC activates HSF1 transcriptional activity was investigated by testing the hypothesis that modulation of the redox balance by PDTC could act on Hsp90, which is known to form a complex with HSF1 and maintain the transcription factor inactive in the cytosol. Work is underway to establish whether the reciprocal effects of PDTC on STAT3 and HSF1 transcriptional activities stem from its ability to disrupt the interactions between Hsp90 and these factors. This approach may ultimately lead to new therapies associated with Hsp90-client protein interactions.