Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

James Shorter

Abstract

Hsp104 is a AAA+ protein disaggregase found in yeast that employs energy from ATP hydrolysis to disassemble various forms of aggregated protein substrates. Hsp104 collaborates with Hsp70 and Hsp40 to remodel amorphous aggregates and amyloid fibrils. Since humans lack an Hsp104 homolog, we view Hsp104 as a potential therapeutic candidate for targeting toxic, misfolded proteins in neurodegenerative disease. Protein misfolding is a contributor to several neurodegenerative diseases, including Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease (AD). Since no therapies directly target underlying protein misfolding, Hsp104 may be a novel strategy for reversing aggregated proteins and restoring native function. We previously engineered potentiated variants of Hsp104 capable of suppressing toxicity of a variety of neurodegenerative disease-associated substrates. These Hsp104 variants do not have specificity for substrates, and can exhibit unfavorable off-target effects. Additionally, it is unknown if potentiated Hsp104 variants can suppress toxicity of substrates in different cellular compartments. As such, the goals of this thesis were two-fold. First, we sought to develop substrate-specific Hsp104 variants to address the off-target, toxic effects observed in generally potentiated Hsp104 variants. Second, we aimed to harness previously-engineered, potentiated Hsp104 variants to suppress toxicity of substrates in different cellular compartments. To the first aim, we tuned the substrate repertoire of Hsp104 and thus engineered a set of substrate-specific Hsp104 variants that selectively suppressed α-syn toxicity (involved in PD). These α-syn-specific Hsp104 variants likely suppressed toxicity through different mechanisms, and a set of variants protected against neurodegeneration in a C. elegans PD model. To the second aim, we found potentiated Hsp104 variants suppress toxicity of various forms of misfolded amyloid-β (Aβ), involved in AD, in yeast. Cytoplasmic, potentiated Hsp104 variants effectively suppressed Aβ toxicity even though Aβ is localized to the secretory pathway in yeast. A set of Hsp104 variants also protected against neurodegeneration in a C. elegans model of Aβ toxicity. Furthermore, potentiated Hsp104 variants suppressed toxicity of another substrate localized to the secretory pathway, Transmembrane Protein 106B (TMEM106B), a genetic risk factor for frontotemporal lobar degeneration with TDP-43 protein aggregates. Taken together, these findings deepen our understanding of the utility of enhanced disaggregases to counter toxic misfolding events in neurodegenerative disease.

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