Abstract
Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes.
Highlights
Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; little is known how these conformations affect interactions with co-chaperones and client proteins
Identification of Novel HSP90 Interactions and Those Influenced by Nucleotides and Pharmacological Inhibition—Nand C-terminal TAP-tagged human HSP90␣ cDNAs were stably expressed in the human kidney epithelial cell line HEK293T, and TAP was performed on HSP90 complexes in the presence of 5 mM ATP, 5 mM ADP, or 5 M GA
USP19 did not bind the D93N mutant, which is in accordance with the prediction that this CS domain-containing co-chaperone is predicted to bind the N-terminal ATPase domain [45]. These results suggest that the effects of geldanamycin on HSP90 complexes are intrinsic to HSP90 and are dependent upon a full ATPase activity and/or the C-terminal MEEVD motif
Summary
Plasmids—Expression vectors for proteomics analysis were derived from the parental TAP1 expression vector pGlue [28]. Subsequent coexpression analysis was performed using pCS2ϩ parental vectors for all C-terminal HSP90-TAP constructs and pSPORT6 (Invitrogen) for expression of HA-tagged CHORDC1 constructs in HEK293T cells. Peptide spectra counts for each protein were calculated by CPAS as the total number of individual spectra (unique or non-unique sequences) matched to a protein using ProteinProphet in which the PeptideProphet scores for individual spectra exceeded a 5% FDR. The spectral abundance factor (SAF) was determined by dividing the normalized spectra count for an individual protein by the length of the protein in amino acids [37, 38]. Real Time RT-PCR—Total RNA was isolated from HEK293T cells expressing N-terminal TAP-tagged HSP90 either treated for 2 h with 5 M geldanamycin or DMSO alone. Data were analyzed by REST software (Corbett Lifescience) using -actin as a reference gene
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