Systematic Proteomic Identification of the Heat Shock Proteins and Biochemical Characterization of the ER[alpha]-Hsp70 Interaction

• Published in: PloS one Vol. 11; no. 8; p. e0160312
• Main Authors: Dhamad, Ahmed E, Zhou, Zhenqi, Zhou, Jianhong, Du, Yuchun
• Format: Journal Article
• Language: English
• Published: Public Library of Science 02.08.2016
• Subjects: Cell proliferation; Chemical properties; Chromatin; Estrogen receptors; Heat shock proteins; Physiological aspects; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0160312

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-ER[alpha] interactions remain unclear. For example, we do not know which Hsps are the major or minor ER[alpha] interactants and whether or not different Hsp isoforms associate equally with ER[alpha]. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ER[alpha] 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 ER[alpha], followed by two Hsp90s (Hsp90[alpha] and Hsp90[beta]) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90[alpha] was found to be 2-3 times more abundant than Hsp90[beta] in the ER[alpha]-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 ER[alpha]-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ER[alpha] in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ER[alpha]-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 17[beta]-estradiol (E2) did not change this. In addition, E2-treatment weakened the ER[alpha]-Hsc70 interaction but had no effect on the ER[alpha]-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 ER[alpha] in both forms of the chromatins in MCF7 cells.