Heat shock protein 90α (Hsp90α) is a molecular chaperone that has been targeted for the development of new anticancer therapies. To date, co-immunoprecipitation (IP) has been primarily used to identify novel client proteins. We now report an alternative approach in which Hsp90α has been immobilized onto the surface of silica-based magnetic beads. The beads were used to isolate known Hsp90α ligands from a mixture containing ligands and nonligands. In addition, they were also used to isolated proteins from a mixture of proteins, as well as a cellular extract. The results indicate that the Hsp90α coated magnetic beads can be used to "fish" from complex chemical and biological mixtures for new lead drug candidates and client proteins.Heat shock protein 90 (Hsp90) is a family of cellular proteins (Hsp90α, Hsp90β, Grp94, Trap1) that act as molecular chaperones, which guide the normal folding, intracellular disposition, and proteolytic turnover of many key regulators of cell growth and survival. 1 It is one of the most abundant molecular chaperones in eukaryotes 2 and is essential in the activation of over 100 client proteins, including receptors, protein kinases, and transcription factors. 1,3 Increased expression and activity of Hsp90 have been observed in human cancers, and this protein has become a therapeutic target for the development of new anticancer agents. 4 The intrinsic ATPase activity of Hsp90 is also a target in drug discovery as ATPase activity is a key component in the chaperone function. 5Although a large number of Hsp90 client proteins have been identified, it is likely that there are many as of yet unidentified client proteins whose function could help elucidate novel oncogenic signaling pathways. At the present time, co-immunoprecipitation (IP) is the primary method used to identify client proteins. 6 However, in this approach an antibody raised against the coprecipitating protein is required for identification by Western blot analysis. Thus, it is difficult to find new proteins without first knowing what you are looking for. Additional problems with this technique include antibody contamination, nonspecific binding interactions, and leaching contamination that can result from protein A or protein G immobilization on agarose.We now report an alternative approach to the isolation of Hsp90 client proteins based upon the immobilization of Hsp90α on the surface of magnetic beads. In a previous study, we demonstrated that Hsp90 could be immobilized on a silica-based stationary phase through
The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRα and ERRγ proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC50 and IC50 values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRα and ERRγ are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.
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