Developing therapeutic protein disaggregases for Neurodegenerative Disease

The accumulation of abnormal protein aggregates in human brain is connected with neurodegenerative diseases. The mammalian protein‐disaggregase system comprised of Hsp110, Hsp70, and Hsp40 can protect cells from proteinopathies. However, this disaggregase system has limitations and its activity declines with age. As a therapeutic strategy for neurodegenerative disease we propose to introduce an exogenous, synthetic disaggregase machinery, based on Hsp104 that converts amyloid to soluble, functional protein. Hsp104 is a hexameric AAA+ ATPase that is critical for stress tolerance in yeast by facilitating the resolubilization of stress‐damaged, aggregated proteins. The homologues of Hsp104 are highly conserved in bacteria, fungi, and plants, but absent from metazoa. The disaggregase activity of Hsp104 can be enhanced by the presence Hsp70 and Hsp40. Hsp104 can dissolve α‐synuclein, β‐amyloid, and TDP‐43 aggregates, but impractically high Hsp104 concentrations are needed. We aim to rationally design potentiated Hsp104 variants that can reverse protein misfolding at low concentrations. One potential problem for the currently discovered potentiated Hsp104 variants is their “off‐target” effects, where expression potentiated Hsp104 variant in ∆hsp104 yeast causes cell death possibly by unfolding metastable proteins at 37°C. By investigating the mechanism of Hsp104 disaggregation and its structural basis, we will engineer and evolve potentiated Hsp104 variants with minimized “off‐target” effect that eradicate α‐synuclein, β‐amyloid, or TDP‐43 misfolding and toxicity. We have exploited new cryo‐EM structures of Hsp104 to engineer key interfaces to yield several novel Hsp104 variants with tunable activity, which can reverse protein aggregation in the presence or absence of human Hsp70 and Hsp40. These novel Hsp104 variants effectively rescue the aggregation and toxicity of diverse human neurodegenerative disease proteins and could have therapeutic utility. Moreover, our engineered Hsp104 variants provide key mechanistic insights into disaggregase function.