Redox control of Hsp90-client protein interactions

S谷胱甘肽基化是谷胱甘肽对半胱氨酸残基的一种生理性、可逆性的蛋白质修饰，以应对轻微的氧化应激。基于毛细管电泳法和激光诱导荧光检测法，建立了一种快速测定培养细胞中游离谷胱甘肽和蛋白质结合谷胱甘肽的方法。在该方法中，样品用5-碘乙酰氨基荧光素衍生化并进行分析。结果表明，复方吡咯烷二硫代氨基甲酸酯处理肝癌细胞后，蛋白结合型谷胱甘肽含量呈时间依赖性增加，这反映了蛋白质S-谷胱甘肽基化的量。这种方法应该允许将S谷胱甘肽基化的蛋白质作为氧化应激的生物标记物进行测量。早期的工作表明，转录因子STAT3是已知的与Hsp90相互作用的客户蛋白之一。STAT3是多种细胞功能传播的中心，从分化和增殖到迁移和炎症。STAT3-Hsp90相互作用的损伤被发现在STAT3信号转导中起到显著的抑制作用。利用PDTC和其他氧化剂在HepG2细胞中进行的研究表明，STAT3是S谷胱甘肽基化的靶标。这种翻译后修饰对STAT3的酪氨酸磷酸化能力产生了不利影响，并穿梭到细胞核以激活靶基因的表达，以响应IL-6。PDTC显著降低了STAT3与分子伴侣Hsp90的结合，这可能解释了STAT3信号转导能力较差的原因。STAT3-Hsp90相互作用对S-硫基化的易感性代表了一种新的IL-6信号调节模式。 已经报道了PDTC在培养的细胞系和体内抑制促炎细胞因子信号的能力，但它对复杂的基因表达谱的影响在很大程度上仍不清楚。利用基因芯片分析和定量聚合酶链式反应分析发现，与生理盐水对照组相比，PDTC可以时间依赖性地改变HepG2细胞中转录因子热休克因子1(HSF1)的一些靶基因的表达。这些靶基因的功能分类表明，几种生物学途径的表达发生了变化，包括对未折叠蛋白的反应和对细胞凋亡的调控。在小鼠肿瘤异种移植模型中，进行了旨在评估下调HSF1在HepG2细胞中的表达和给予PDTC的影响的实验。此外，通过检验PDTC调节氧化还原平衡可能作用于Hsp90的假设，研究了PDTC激活HSF1转录活性的机制。Hsp90与HSF1形成复合体，并保持转录因子在胞浆中的失活。目前正在研究PDTC对STAT3和HSF1转录活动的相互影响是否源于其破坏Hsp90和这些因子之间的相互作用的能力。这种方法可能最终导致与Hsp90-客户蛋白质相互作用相关的新疗法。

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.