# Defining the mechanistic basis of a prion disaggregase

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2020 · $339,308

## Abstract

Project summary: Our research objective is to define the mechanistic basis of Hsp104, a protein
disaggregase and hexameric AAA+ (ATPases Associated with diverse Activities) protein from yeast, which
remains poorly understood. Hsp104 couples ATP hydrolysis to the dissolution and reactivation of diverse
proteins trapped in disordered aggregates, toxic preamyloid oligomers, amyloids, and prions. Hsp104 is the
only factor known to dissociate α-synuclein (α-syn) oligomers and amyloids connected with Parkinson's
disease (PD) and rescue α-syn-induced neurodegeneration in the substantia nigra of a rat PD model.
However, Hsp104 activity is limited against α-syn and very high Hsp104 concentrations are needed for optimal
effects. Thus, we engineered potentiated Hsp104 variants, which dissolve fibrils formed by neurodegenerative
disease proteins such as TDP-43, FUS, and α-syn, and mitigate neurodegeneration in the metazoan nervous
system at concentrations where Hsp104 is inactive. Curiously, Hsp104 is absent from metazoa. Thus, Hsp104
and potentiated variants could represent a disruptive technology to enhance proteostasis to counter
neurodegenerative disease and enable purification of irksome, aggregation-prone proteins for valuable basic or
pharmaceutical purposes. However, these endeavors are frustrated by a limited mechanistic understanding of
Hsp104, which despite intense investigation remains stalled at a low level of resolution. Three critical barriers
impede our understanding of Hsp104. First, we do not understand how Hsp104 selects clients for
disaggregation, which limits our ability to tailor Hsp104 activity for specific substrates. This issue is pernicious
because potentiated Hsp104 variants can have damaging, off-target effects due to promiscuous activity, which
could restrict therapeutic or biotechnological applications. Second, Hsp104 sequence space remains largely
unexplored. It is unclear whether natural Hsp104 orthologues exist with divergent enhanced or selective
activity against neurodegenerative disease substrates. Third, there is no atomic structure of the Hsp104
hexamer and conflicting cryo-electron microscopy reconstructions have confused the field. Based on our
preliminary data, we hypothesize that: (1) potentiated Hsp104 variants can be engineered to be more
substrate specific to avoid damaging off-target effects; (2) natural Hsp104 orthologues exist with
enhanced activity against neurodegenerative disease substrates and minimal off-target effects; and (3)
large structural changes in Hsp104 hexamers upon ATP hydrolysis drive protein disaggregation. Thus,
we will meet three aims: (1) Define potentiated Hsp104 variants with enhanced substrate selectivity; (2) Define
conserved and divergent activities of natural Hsp104 orthologues; (3) Define high-resolution structural changes
in Hsp104 and potentiated variants that drive protein disaggregation. In this way, we will secure a high-
resolution mechanistic view of Hsp104, which will empower...

## Key facts

- **NIH application ID:** 9831085
- **Project number:** 5R01GM099836-08
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** James Shorter
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $339,308
- **Award type:** 5
- **Project period:** 2013-01-01 → 2021-06-03

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9831085

## Citation

> US National Institutes of Health, RePORTER application 9831085, Defining the mechanistic basis of a prion disaggregase (5R01GM099836-08). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9831085. Licensed CC0.

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