Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

James Shorter

Abstract

Protein aggregation is a hallmark of neurodegenerative disease where the accumulation of insoluble protein aggregates within neurons is often associated with degenerating brain regions. Hsp70, Hsp40, and Hsp110 form a human protein-disaggregase system capable of robust protein disaggregation in vitro against various substrates, including aggregated luciferase and α-synuclein (αSyn) amyloid fibrils, which are associated with Parkinson’s disease and related α-synucleinopathies. However, the presence of these human chaperones in patient neurons is clearly insufficient to maintain protein homeostasis in disease, and thus could benefit from pharmacological stimulation. We tested a library of dihydropyrimidines, a scaffold known to modulate Hsp70 ATPase activity, and discovered several drug-like, small molecules that stimulate the disaggregase activity of the Hsp70 system against aggregated luciferase. We show that the lead compound can also stimulate the disaggregase activity of the Hsp70 system against αSyn amyloid fibrils. We found that certain members of the Hsp70 and Hsp40 chaperone families were unable to promote disaggregation and reactivation of soluble luciferase aggregates while all members of the Hsp110 family tested were. Our lead compound was able to stimulate the activity of three-component chaperone combinations with a class B Hsp40 but not combinations with a class A Hsp40, suggesting selectivity for certain Hsp40 proteins. We also uncovered that the disaggregase activity of the Hsp70 system is very sensitive to the relative stoichiometry of Hsp70, Hsp40, and Hsp110. We propose that perturbations in this stoichiometry may contribute to the deficiency of the Hsp70 system in protein aggregation disorders. Importantly, our lead compound stimulates disaggregase activity across the stoichiometric landscape and shifts the peak activity such that it activates compositions that were otherwise inactive. Our results suggest that a fine-tuned balance between Hsp40 and Hsp110 activity is required for optimal disaggregase activity by the Hsp70 system. Moreover, we discover first-in-class compounds that are able to stimulate the disaggregase activity of the three-component system. These findings suggest that the human Hsp70 disaggregase system is a promising molecular target for pharmacologically bolstering the proteostasis machinery, which could be leveraged to combat protein aggregation in disease.

Embargoed

Available to all on Tuesday, January 02, 2024

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS