# Engineering and evolving substrate-specific Hsp104 variants

> **NIH NIH F31** · WASHINGTON UNIVERSITY · 2024 · $22,930

## Abstract

Project Summary
Protein misfolding underpins numerous fatal neurodegenerative diseases including amyotrophic lateral sclerosis
(ALS), Parkinson's disease (PD), and Alzheimer’s disease. Currently there are no effective treatments for protein
misfolding diseases. Each of these disorders is linked to the accumulation of disordered aggregates, toxic pre-
amyloid oligomers, and amyloid or amyloid-like conformers. While amyloid is typically implicated in disease, the
amyloid fold has also been employed for beneficial purposes, and so regulatory pathways have evolved to
promote amyloid disassembly. Yeast have evolved to employ amyloid for specific roles. Hsp104, a conserved
hexameric AAA+ protein-remodeling factor from yeast, solubilizes disordered aggregates and amyloid but has
only limited activity against human neurodegenerative disease proteins. While Hsp104 only has limited ability to
rescue proteins that aggregate in human cells, it can be re-engineered to solubilize disease-associated
aggregates and amyloid. Numerous potentiated Hsp104 variants have been discovered that harbor mutations to
both conservative and non-conservative residues throughout the middle domain of Hsp104. Application of
Hsp104 variants in animal models has been stalled due to the toxicity of Hsp104 variants in neurons. In pilot
studies, I have developed new screening approaches to isolate two Hsp104 variants that rescue the toxicity of
proteins implicated in ALS and PD without conferring off-target effects. In addition, I have developed and
employed new selection strategies to isolate Hsp104 variants with improved properties. My next steps are to
comprehensively survey the collection of these variants to determine the correlation between potentiation and
the selection trends from our screens. I will also employ computational approaches to better understand the
basis for Hsp104 potentiation. Finally, I will establish techniques that enable high-throughput analysis of large
libraries of Hsp104 variants to better understand the mechanism of Hsp104 and the basis for Hsp104 substrate-
specificity. I will then assess the therapeutic potential of top variants in an α-syn FRET biosensor cell line of
HEK293T cells and a primary neuron model of α-syn aggregation. Specifically, I will address two aims: 1)
Engineer and evolve substrate-specific Hsp104 variants and 2) Assess the efficacy of newly developed Hsp104
variants in mammalian cells. Ultimately, we anticipate that finely-tuned protein disaggregases could be
developed to reverse the misfolding of proteins that underpin diverse protein-misfolding disorders and could
simultaneously counter both a loss or gain of function mechanism. Regardless of the therapeutic potential of
these variants, they will serve as useful probes for testing what happens when misfolding is reversed, helping to
delineate the therapeutic goals for targeting these disorders.

## Key facts

- **NIH application ID:** 10748384
- **Project number:** 5F31GM140622-03
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Jeremy Ryan
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $22,930
- **Award type:** 5
- **Project period:** 2021-12-01 → 2024-01-26

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10748384

## Citation

> US National Institutes of Health, RePORTER application 10748384, Engineering and evolving substrate-specific Hsp104 variants (5F31GM140622-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10748384. Licensed CC0.

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