Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen

Abstract

Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein–protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans . In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.

Protein-Protein Interactions in Candida albicans

“…Prof. Liu’s lab has also used a TAP approach to identify 103 interacting proteins for the Wor1 protein (Alkafeef et al, 2018) while the labs of Profs. Emili, Gingras, and Cowen found more than 250 PPIs when studying Hsp90 (O’Meara et al, 2019). The labs of Profs.…”

“…Different versions of the TAP-tag were later designed without the TEV protease site (Xu et al, 2010). The TAP technique has the advantage that it can be used in a large-scale high-throughput setup where protein complexes are purified in two steps followed by protein identification with MS (Xu et al, 2010; Rao et al, 2014; Podobnik et al, 2016; O’Meara et al, 2019). It has, for example, been proven to be very efficient to perform large-scale screenings to identify PPIs and protein complexes in S. cerevisiae (Gavin et al, 2002; Krogan et al, 2006).…”

“…Whether this difference is caused by the use of the pre-purification step is unclear. More recently O’Meara et al (2019) applied the TAP-tag to identify well over 250 PPIs.…”

“…In C. albicans the technique was first used in 2018 to perform a quantitative proteomic analysis of Cdc14 (Ong et al, 2002; Kaneva et al, 2018). Later the technique was used to study the proteome changes while transcriptionally repressing or pharmacologically inhibiting Hsp90 (O’Meara et al, 2019).…”

DeORFanizingCandida albicansGenes using Co-Expression

“…In many cases, gene annotation information comes from inferred orthology with the model yeast S. cerevisiae, but these organisms diverged approximately 150 MYA (10), and even gene essentiality is not necessarily conserved across these species (8,11). Current high-throughput approaches for gene function analysis include transposon insertion screens (11,12), functional genomic screens of mutant libraries (8,(13)(14)(15)(16), genetic interaction screens (17), yeast-two-hybrid approaches (18), and other protein-protein interaction screens (19). However, there are also many transcriptomic analyses of C. albicans available through NCBI SRA, suggesting that it is now feasible to create a co-expression network for C. albicans as a complementary approach for predicting gene function.…”