Engineering Hsp104 Variants to Counter Protein Misfolding

There are no therapies that reverse the proteotoxic misfolding events that underpin fatal neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). Hsp104, a conserved hexameric AAA+ protein from yeast, solubilizes disordered aggregates and amyloid but has no metazoan homolog and only limited activity against human neurodegenerative disease proteins. Here, we reprogram Hsp104 to rescue TDP‐43, FUS, and α‐synuclein proteotoxicity by mutating single residues in the middle domain or nucleotide‐binding domain 1 of Hsp104. Strikingly, we have uncovered numerous missense mutations throughout these regions that potentiate Hsp104. Hsp104 potentiation stems from loss of amino acid identity rather than introduction of specific interactions. Potentiated Hsp104 variants enhance aggregate dissolution, restore proper protein localization, suppress proteotoxicity, and in a C. elegans PD model attenuate dopaminergic neurodegeneration. Potentiating mutations reconfigure how Hsp104 subunits collaborate, desensitize Hsp104 to inhibition, obviate any requirement for Hsp70, and enhance ATPase, translocation, and unfoldase activity. Our work establishes that disease‐associated aggregates and amyloid are tractable targets and that enhanced disaggregases can restore proteostasis and mitigate neurodegeneration. Support or Funding Information Target ALS Springboard Fellowship