# Triage mechanisms for directing protein refolding and degradation

> **NIH NIH R35** · MIAMI UNIVERSITY OXFORD · 2020 · $359,327

## Abstract

PROJECT SUMMARY
Cellular protein quality control systems enable the robust response to protein misfolding that is essential for
normal cellular function. Numerous pathologies including neurodegenerative disorders, ataxias, and cancers
can result from defects in the protein quality control system formed by the interaction of the E3 ubiquitin ligase
CHIP (C-terminus of Hsp70 Interacting Protein) and the ATP-dependent chaperone Hsp70 (70 kilodalton heat
shock protein). Despite the importance of the CHIP/Hsp70 protein quality control complex, the triage
mechanism used by the CHIP/Hsp70 complex to target misfolded proteins for either Hsp70-mediated refolding
or ubiquitin proteasome-mediated degradation is unknown. Within the triage mechanism, several key areas
remain unexplored. These include: (1) How does the CHIP/Hsp70 complex execute the observed triage
mechanism that ubiquitinates misfolded clients first, then Hsp70, then CHIP? (2) What is the role played by
nucleotide occupancy of Hsp70 and how do the conformational changes in Hsp70 throughout the catalytic
cycle dictate the ability of CHIP to ubiquitinate misfolded clients? (3) What are the determinants that control
whether a misfolded client becomes ubiquitinated, or is refolded? The proposed research program will examine
these issues using nuclear magnetic resonance (NMR), small angle X-ray scattering (SAXS), electron
paramagnetic resonance (EPR), and a suite of biophysical and biochemical techniques. NMR data will provide
high-resolution information on dynamics within CHIP/Hsp70/client complexes and enable a detailed dissection
of the role that dynamics play in dictating the CHIP/Hsp70 triage mechanism. SAXS and EPR will be used
together in a hybrid methods approach to determine structural ensembles for dynamic CHIP/Hsp70 complexes
and connect dynamic conformational changes to alterations in CHIP-mediated ubiquitination of Hsp70 and
misfolded clients. The proposed development of a SAXS/EPR hybrid methods approach to structure
determination will produce an approach that will be applicable to many protein/protein interactions regulated by
dynamic conformational changes. Structural data from NMR, SAXS, and EPR will be complemented with
biophysical and biochemical approaches that enable real-time monitoring of ubiquitination and directly assay
the competition between CHIP-mediated ubiquitination and Hsp70-mediated protein refolding. These assays
will allow for rapid testing of mechanistic hypotheses generated from the NMR and SAXS/EPR structural
studies. The proposed research program is set up to evolve by expanding to examine the role that clients play
in the triage mechanism. These efforts will be extended to soluble protein clients with either perturbed refolding
capabilities, or controlled numbers or locations of ubiquitination sites.

## Key facts

- **NIH application ID:** 9989130
- **Project number:** 5R35GM128595-03
- **Recipient organization:** MIAMI UNIVERSITY OXFORD
- **Principal Investigator:** Richard C Page
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $359,327
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9989130

## Citation

> US National Institutes of Health, RePORTER application 9989130, Triage mechanisms for directing protein refolding and degradation (5R35GM128595-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9989130. Licensed CC0.

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