Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects.

Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting.

Hsp104 is a hexameric AAA ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'--(thiotriphosphate)).

Potentiating Hsp104 activity via phosphomimetic mutations in the middle domain.

Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo.

Disparate Mutations Confer Therapeutic Gain of Hsp104 Function.

Hsp104, a protein disaggregase from yeast, can be engineered and potentiated to counter TDP-43, FUS, or α-synuclein misfolding and toxicity implicated in neurodegenerative disease. Here, we reveal that extraordinarily disparate mutations potentiate Hsp104. Remarkably, diverse single missense mutations at 20 different positions interspersed throughout the middle domain (MD) and small domain of nucleotide-binding domain 1 (NBD1) confer a therapeutic gain of Hsp104 function.

Isolating potentiated Hsp104 variants using yeast proteinopathy models.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and α-synuclein, implicated in Parkinson's disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders.