Engineering Potentiated Hsp104 Variants With Enhanced Substrate‐Specificity to Counter Neurodegeneration

Hsp104 is a hexameric AAA+ ATPase and disaggregase found in yeast. Together with the Hsp70 chaperone system, Hsp104 uses energy from ATP hydrolysis to reactivate and refold protein substrates. Hsp104 eliminates aggregates that accumulate after environmental stress, and with the exception of the Hsp110 system, is the only protein known to rapidly disassemble amyloid. Interestingly, metazoa lack an Hsp104 homolog, and as such, humans have limited ability to eradicate amorphous aggregates and amyloid. This shortcoming is problematic, as protein misfolding underlies several neurodegenerative diseases. As such, Hsp104 is an appealing exogenous candidate for therapeutic targeting of toxic misfolded proteins. We previously engineered potentiated Hsp104 variants that robustly disaggregate and suppress the toxicity of neurodegenerative disease substrates α‐synuclein (implicated in Parkinson's disease) and TDP‐43 and FUS (implicated in amyotrophic lateral sclerosis). The potentiated variants also restore proper cellular localization of these neurodegenerative disease substrates. Yet, potentiated Hsp104 variants lack strong substrate specificity, an important attribute needed to prevent nonspecific unfolding of essential proteins. Here, we developed substrate‐specific variants by rationally tuning conserved Hsp104 pore loop residues, Y257 and Y662, which are known to contact substrate. In altering the pore loops in potentiated backgrounds, we discovered variants specific for α‐synuclein. In yeast proteinopathy models, the α‐synuclein‐specific variants suppress toxicity of α‐synuclein, and only minimally suppress toxicity of TDP‐43 and FUS. The α‐synuclein‐specific variants are inherently less toxic to yeast than generally potentiated variants, and prevent α‐synuclein‐induced neurodegeneration in a C. elegans model of Parkinson's disease. Interestingly, we observed differences between the α‐synuclein‐specific variants in their ability to suppress toxicity and restore proper cellular localization of α‐synuclein. We have also uncovered a new set of generally potentiated Hsp104 variants that strongly suppress toxicity of α‐synuclein, TDP‐43, and FUS. Overall, our findings expose crucial insights to tailoring Hsp104 to selectively and therapeutically target toxic misfolded species. Support or Funding Information NSF Graduate Research Fellowship (DGE‐1321851)