Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2–3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.热休克蛋白（Heat shock proteins, Hsps）已知可与雌激素受体（Estrogen Receptor, ER）结合，并调控雌激素受体介导的细胞增殖。长期以来，该领域的研究多聚焦于热休克蛋白90（Heat shock protein 90, Hsp90）。然而，热休克蛋白与雌激素受体α（Estrogen Receptor α, ERα）之间的相互作用仍存在诸多未阐明的关键环节。例如，目前尚不清楚哪些热休克蛋白是与ERα结合的主要或次要相互作用伴侣，也不清楚不同的热休克蛋白亚型是否能与ERα等量结合。本研究通过定量蛋白质组学方法，在培养于含细胞增殖必需成分培养基的人源293T细胞中，发现共有21种热休克蛋白与3种热休克蛋白共伴侣蛋白（heat shock protein cochaperones, Hsp cochaperones）可与ERα结合。在已鉴定的可与ERα结合的热休克蛋白中，4种热休克蛋白70家族成员（Heat shock protein 70, Hsp70）——Hsp70-1、Hsc70、Grp75及Grp78——丰度最高，其次为2种热休克蛋白90家族成员（Hsp90α与Hsp90β）以及3种热休克蛋白110家族成员（Hsp105、HspA4及HspA4L）。在ERα结合复合物中，Hsp90α的丰度约为Hsp90β的2至3倍。在已报道的热休克蛋白共伴侣蛋白中，本研究检测到前列腺素E合酶3（prostaglandin E synthase 3, p23）、肽基脯氨酰顺反异构酶FKBP5（peptidyl-prolyl cis-trans isomerase FKBP5, FKBP51）以及E3泛素蛋白连接酶CHIP（E3 ubiquitin-protein ligase CHIP, CHIP）。针对两种丰度最高的ERα结合热休克蛋白——Hsp70-1与Hsc70，本研究使用人乳腺癌MCF7细胞开展实验，结果显示：当细胞在添加胎牛血清与酚红的培养基中培养时，这两种热休克蛋白可在细胞质与细胞核内与ERα发生相互作用。值得注意的是，在激素饥饿培养条件下，仅能在细胞质中检测到ERα与Hsp70-1/Hsc70的相互作用，而细胞核中未检测到该相互作用；且用17β-雌二醇（17β-estradiol, E2）刺激处于饥饿状态的细胞后，这一现象并未发生改变。此外，17β-雌二醇处理会削弱ERα与Hsc70之间的相互作用，但对ERα与Hsp70-1的相互作用无显著影响。进一步研究表明，在MCF7细胞中，Hsp70-1与Hsc70的相当一部分组分可分别与转录活性染色质及非活性染色质结合，且这两种热休克蛋白均可在两类染色质上与ERα发生相互作用。