Abstract

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 -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. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress -synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, -synuclein-specific Hsp104 variants emerge that mitigate -synuclein toxicity via distinct ATPase-dependent mechanisms, involving -synuclein disaggregation or detoxification of -synuclein conformers without disaggregation. Importantly, both types of -synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in a C. elegans model of Parkinsons disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.