Monday, June 30, 2025 6/30/2025

Engineering and evolving substrate-specific Hsp104 variants

Award Number: F31GM140622
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 12/01/2021
PERIOD OF PERFORMANCE END DATE: 01/26/2024

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Engineering and evolving substrate-specific Hsp104 variants - 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$68,856 )
2025	2024	WASHINGTON UNIVERSITY, THE	1 BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	002	3	12/20/2024	NON-COMPETING CONTINUATION	-$22,930
2025	2024	WASHINGTON UNIVERSITY, THE	1 BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	3	10/28/2024	NON-COMPETING CONTINUATION	-$9,557
2025	2023	WASHINGTON UNIVERSITY, THE	1 BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	2	12/20/2024	NON-COMPETING CONTINUATION	-$36,369
														Subtotal = -$68,856

Issue Date FY: 2024 ( Subtotal = $32,487 )
2024	2024	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	000	3	12/1/2023	NON-COMPETING CONTINUATION	$31,847
2024	2024	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63110	SAINT LOUIS CITY	USA	Biomedical Research and Research Training	001	3	6/11/2024	NON-COMPETING CONTINUATION	$640
														Subtotal = $32,487

Issue Date FY: 2023 ( Subtotal = $47,694 )
2023	2023	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	2	3/1/2023	NON-COMPETING CONTINUATION	$1,658
2023	2023	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	2	12/5/2022	NON-COMPETING CONTINUATION	$46,036
														Subtotal = $47,694

Issue Date FY: 2021 ( Subtotal = $46,036 )
2021	2021	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	000	1	8/1/2021	NEW	$46,036
2021	2021	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	001	1	8/1/2021	NEW	$0
2021	2021	WASHINGTON UNIVERSITY, THE	ONE BROOKINGS DR	SAINT LOUIS	MO	63130	SAINT LOUIS	USA	Biomedical Research and Research Training	002	1	8/1/2021	NEW	$0
														Subtotal = $46,036

Grand Total All Awards = $57,361

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Engineering and evolving substrate-specific Hsp104 variants

Award Number: F31GM140622

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 12/01/2021

PERIOD OF PERFORMANCE END DATE: 01/26/2024

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