Abstract
Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic.
Highlights
Normal eukaryotic cells depend on the molecular chaperone heat shock protein 90 (Hsp90) to maintain protein homeostasis [1,2]
Consistent with a recent study [12], we found that repeated challenge of a tumor cell protein lysate with GA- or PU-conjugated agarose beads could not capture the entire Hsp90 population, PU-beads were able to capture a larger fraction of Hsp90 compared to GA-beads (Figure 1A)
To discern whether the Hsp90 populations isolated by each inhibitor were mutually exclusive, we subjected a tumor cell lysate to several rounds of GA-agarose followed by several rounds of PUagarose, and vice versa
Summary
Normal eukaryotic cells depend on the molecular chaperone heat shock protein 90 (Hsp90) to maintain protein homeostasis [1,2]. After initial affinity pulldown of purified recombinant Hsp90 protein with drug-bound agarose, we incubated samples with increasing concentrations of trypsin and we visualized the resultant protein fragments by Western blotting with Hsp90 antibodies recognizing either N- or C-terminal epitopes.
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