Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions

Abstract

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Keywords: DIA-MS; client-specific effects; cochaperone; molecular chaperone.

Fig. 1.

Model of the Hsp90 folding cycle and the steps affected by each Hsc82 mutant. Model of Hsp90-client interaction. Client is targeted to Hsp90 by Hsp70 and Hop/Sti1. Hsp90 adopts the closed conformation characterized by interaction with Cyp40/Cpr6 and p23/Sba1. ATP hydrolysis results in return to the open conformation and client release. Mutants in the loading group disrupt Hsp70–Hsp90 interaction. Mutants in the closing group disrupt formation of the closed conformation and interaction with Sba1 and Cpr6. Mutations in the reopening group disrupt ATP hydrolysis and/or associated conformational changes. The W296A and G424D mutants do not fit within those groups and thus are marked “miscellaneous”.

Fig. 2.

Summary of proteomic changes in the Hsc82 mutant strains. a) Out of 350 total, the number of proteins that exhibited a log₂ fold change ≥ 1.5 (P ≤ 0.055) in the presence of each mutant relative to cells expressing wild-type Hsc82 (Supplementary Table 2, sheet 1). b) The relative proportions of proteins with increased or decreased abundance in the presence of each mutant. c) Comparison of hits to proteins linked to Hsp90 in high-throughput studies (see text for details). Left side shows 204 shared hits with studies identifying genetic or physical interactors of Hsp90. These were grouped together since they are likely clients due to evidence that they physically interact with Hsp90 and/or their function is decreased when Hsp90 function is decreased. Including studies identifying impacts of altered Hsp90 function using microarray analysis (right side), a total of 257 shared hits were identified. Hits affected at the mRNA level are likely due to indirect effects, such as downstream targets of Hsp90 clients. Hypergeometric distribution was used to calculate the significance of overlap between the current and prior studies (***P < 0.001; **P < 0.01; *P < 0.05).

Fig. 3.

Analysis of proteomic changes in W296A. a) Heat map showing log₂FC of 43 proteins with increased abundance (green) in W296A and a corresponding increase in mRNA levels using published microarray data [W296A (m)] (Flom et al. 2012). Hbt1 protein is highlighted in red.b) β-Galactosidase activity of cells expressing STRE-lacZ reporter plasmid in indicated Hsc82 mutant. P = 0.05*, 0.005**, 0.0005***. c) Heat map showing log₂FC of 62 proteins with significantly decreased abundance in W296A. In heat maps, green represents increased abundance in the mutant and red indicated decreased abundance in the mutant. Potential clients shown in red and/or bold in c). Proteins for which there was no significant change in mRNA levels in prior study are marked in red (Flom et al. 2012). Proteins that crosslinked to Hsp90 shown in bold (Girstmair et al. 2019; Kolhe et al. 2023).

Fig. 4.

Functional clustering of the Hsc82 mutants. a) Principal component analysis (PCA) of the entire dataset of 2,482 proteins shows distinct effects. b) PCA analysis of the dataset lacking W296A separates the remainder of the mutants in to 3 groups: (1) G309S and S481Y, (2) Q380K, and (3) the remainder of the mutants. c) The top 70 loading scores explaining variability along the 1st component axis in b) were extracted. Heat map showing log₂FC of the extracted 70 proteins showing all mutants. Green represents increased abundance in the mutant and red indicated decreased abundance in the mutant. Since this analysis was performed on the entire set of 2,482 proteins, some proteins shown are not in the top 350 hits.

Fig. 5.

Co-expression of Hsc82 mutants with distinct effects restores growth. An hsc82hsp82 strain expressing either G424D a) or W296A b) was transformed with vector, a plasmid expressing wild-type HSC82, or plasmids expressing either W296A or G424D. Cells were grown overnight in selective media, serially diluted 10-fold, then grown for 2 days at 37°C. c) An hsc82hsp82 strain expressing G309S was transformed with vector, a plasmid expressing wild-type HSC82, or plasmids expressing the indicated mutant. Cells were grown overnight in selective media, serially diluted 10-fold, then grown for 2 days at 37°C.

Fig. 6.

Model of effect of Hsp90 conformation on client specificity. Our prior grouping of Hsc82 mutants identified 4 groups: loading, closing, reopening, and other. Based on these studies, a refined model is shown. The W296A mutant is proposed to be in an extended conformation (Peng et al. 2023), G309S and S481Y are proposed to favor open conformations, and S25P and G424D are proposed to be in various closed conformations (Mercier et al. 2023). These conformation groups fit well with the clusters identified by principal component analysis (PCA) and our prior groupings. According to this model, Hsc82 mutants that affect steps prior to adoption of the closed conformation impact the most proteins.