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PreprintCited by 6Heat shock proteins researchReceptor Mechanisms and SignalingGenomics and Chromatin Dynamics

The interplay between Bag-1, Hsp70, and Hsp90 reveals that inhibiting Hsp70 rebinding is essential for Glucocorticoid Receptor activity

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Kirschke, Elaine, Roe-Zurz, Zygy, Noddings, Chari, Agard, David·2020·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2020.05.03.075523
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Abstract

The glucocorticoid receptor (GR), like many signaling proteins requires Hsp90 for sustained activity. Previous biochemical studies revealed that the requirement for Hsp90 is explained by its ability to reverse Hsp70-mediated inactivation of GR through a complex process requiring both cochaperones and Hsp90 ATP hydrolysis. How ATP hydrolysis on Hsp90 enables GR reactivation is unknown. The canonical mechanism of client release from Hsp70 requires ADP:ATP exchange, which is normally rate limiting. Here we show that independent of ATP hydrolysis, Hsp90 acts as an Hsp70 nucleotide exchange factor (NEF) to accelerate ADP dissociation, likely coordinating GR transfer from Hsp70 to Hsp90. As Bag-1 is a canonical Hsp70 NEF that can also reactivate Hsp70:GR, the impact of these two NEFs was compared. Simple acceleration of Hsp70:GR release was insufficient for GR reactivation as Hsp70 rapidly re-binds and re-inactivates GR. Instead, inhibition of GR re-inactivation by Hsp70 is critical. This can be accomplished by high non-physiological Bag-1 concentrations, which also inhibit Hsp70:ATP binding. In contrast, in an ATP-hydrolysis dependent process, Hsp90 plays a unique role by kinetically partitioning GR into a state that can bind ligand, but is protected from Hsp70 inactivation, thus allowing GR to be activated by its ligand but still able to re-enter the chaperone cycle. At physiologic concentrations, Bag-1 works synergistically with Hsp90 to accelerate the first rate-limiting step in GR reactivation. The net effect is that the chaperone machinery cyclically dictates the on and off rates for GR ligand, providing a timer controlling the persistence of activated GR. Significance Statement: The glucocorticoid receptor (GR) is an essential transcription regulatory factor. Like many signaling proteins, GR activity is regulated by two essential molecular chaperones: Hsp90 and Hsp70. Functioning like a toggle switch, Hsp70 first inactivates GR, and then Hsp90 reactivates GR in an Hsp90 ATP hydrolysis dependent manner. Here, an intricate set of biochemistry experiments uncover fundamental principles governing how these chaperone systems collaboratively regulate GR activity. While Hsp90 promotes GR release from Hsp70 by modulating Hsp70’s nucleotide state, this occurs independently of Hsp90 ATP hydrolysis. Instead, ATP hydrolysis on Hsp90 facilitates a second essential reactivation step resulting in an Hsp90-bound GR state that protects GR from Hsp70 re-inactivation. A kinetic partitioning model best describes chaperone modulation of GR’s activity.

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  • In the loading complex, Hsp70NBD-IIAs deviate from the crystal structure and Hsp90MD interacts with Hsp70 residues R171, N174 and T177 in the ATP catalytic motif, suggesting how Hsp90 enhances nucleotide exchange on Hsp70 during the GR-chaperoning cycle.

    “Of note, in the loading complex, Hsp70NBD-IIAs deviate from the crystal structure (Supplementary Fig. 3a, b) and Hsp90MD interacts with Hsp70 (R171, N174, T177) in the ATP catalytic motif (Fig. 1e, Supplementary Fig. 3c), suggesting how Hsp90 enhances the nucleotide exchange on Hsp70 during the GR-chaperoning cycle30.”

    From Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism (2022) · cites this paper as [30]

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  • The canonical NEF-binding sites on Hsp70NBD-IIB remain available in the loading complex, explaining how the NEF Bag-1 can accelerate GR maturation and how the NEF Hsp110 can participate in GR maturation.

    “The canonical nucleotide exchange factor (NEF)-binding sites10,14 on Hsp70NBD-IIB remain available, explaining how the NEF Bag-1 can accelerate GR maturation30 (Supplementary Fig. 4a, b) and also how the NEF Hsp110 can be involved in GR maturation31 (Supplementary Fig. 4c, d).”

    From Structure of Hsp90–Hsp70–Hop–GR reveals the Hsp90 client-loading mechanism (2022) · cites this paper as [30]

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Authors
Kirschke, E., Roe-Zurz, Z., Noddings, C., & Agard, D.
Affiliations
University of California, San Francisco (US)
Corresponding author
Agard, David
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2020-05-05
DOI
10.1101/2020.05.03.075523
OpenAlex
W3022297640
Cited by
6
References
58
Citations by year
2025: 1 · 2023: 1 · 2021: 2 · 2020: 2
Type
posted content
Language
en
Open access
Licence
www.biorxiv.org/about/FAQ#license (vor)
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
Funders
  • National Science Foundation
  • Howard Hughes Medical Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Heat shock proteins research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
glucocorticoid receptor, BAG-1, HSP70, HSP90, molecular chaperones, nucleotide exchange factor, ADP/ATP exchange, receptor resensitization, chaperone cycle, ligand binding, transcriptional regulation, chaperone co-chaperone

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