p23 and Aha1: Distinct Functions Promote Client Maturation.

Hsp90 is a conserved molecular chaperone regulating the folding and activation of a diverse array of several hundreds of client proteins. The function of Hsp90 in client processing is fine-tuned by a cohort of co-chaperones that modulate client activation in a client-specific manner. They affect the Hsp90 ATPase activity and the recruitment of client proteins and can in addition affect chaperoning in an Hsp90-independent way. p23 and Aha1 are central Hsp90 co-chaperones that regulate Hsp90 in opposing ways. While p23 inhibits the Hsp90 ATPase and stabilizes a client-bound Hsp90 state, Aha1 accelerates ATP hydrolysis and competes with client binding to Hsp90. Even though both proteins have been intensively studied for decades, research of the last few years has revealed intriguing new aspects of these co-chaperones that expanded our perception of how they regulate client activation. Here, we review the progress in understanding p23 and Aha1 as promoters of client processing. We highlight the structures of Aha1 and p23, their interaction with Hsp90, and how their association with Hsp90 affects the conformational cycle of Hsp90 in the context of client maturation.

NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle.

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.

Hsp90 Co-chaperones Form Plastic Genetic Networks Adapted to Client Maturation.

Heat shock protein 90 (Hsp90) is a molecular chaperone regulating the activity of diverse client proteins together with a plethora of different co-chaperones. Whether these functionally cooperate has remained enigmatic. We analyze all double mutants of 11 Saccharomyces cerevisiae Hsp90 co-chaperones in vivo concerning effects on cell physiology and the activation of specific client proteins. We find that client activation is supported by a genetic network with weak epistasis between most co-chaperones and a few modules with strong genetic interactions. These include an epistatic module regulating protein translation and dedicated epistatic networks for specific clients. For kinases, the bridging of Hsp70 and Hsp90 by Sti1/Hop is essential for activation, whereas for steroid hormone receptors, an epistatic module regulating their dwell time on Hsp90 is crucial, highlighting the specific needs of different clients. Thus, the Hsp90 system is characterized by plastic co-chaperone networks fine-tuning the conformational processing in a client-specific manner.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

Structure, Function, and Regulation of the Hsp90 Machinery.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the maturation of a plethora of substrates ("clients"), including protein kinases, transcription factors, and E3 ubiquitin ligases, positioning Hsp90 as a central regulator of cellular proteostasis. Hsp90 undergoes large conformational changes during its ATPase cycle. The processing of clients by cytosolic Hsp90 is assisted by a cohort of cochaperones that affect client recruitment, Hsp90 ATPase function or conformational rearrangements in Hsp90. Because of the importance of Hsp90 in regulating central cellular pathways, strategies for the pharmacological inhibition of the Hsp90 machinery in diseases such as cancer and neurodegeneration are being developed. In this review, we summarize recent structural and mechanistic progress in defining the function of organelle-specific and cytosolic Hsp90, including the impact of individual cochaperones on the maturation of specific clients and complexes with clients as well as ways of exploiting Hsp90 as a drug target.

The HSP90 chaperone machinery.

The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.