Author ORCID Identifier

https://orcid.org/0000-0003-2279-5863

Semester

Fall

Date of Graduation

2022

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Not Listed

Committee Chair

James Simpkins

Committee Co-Chair

David Smith

Committee Member

Visvanathan Ramamurthy

Committee Member

Aaron Robart

Committee Member

Gregory Konat

Committee Member

Werner Geldenhuys

Abstract

Aging is an inevitable process that occurs as humans grow older. It is characterized by the chronological accumulation of cellular damage over time leading to functional decline as an organism grows older. Several processes are thought to contribute to the aging phenomenon, but one of the most prolific of these is the disruption of protein homeostasis (proteostasis). The collapse of proteostasis can lead to accelerated aging and the development of age-related diseases including devastating neurodegenerative diseases (NDs) like Alzheimer and Parkinson disease. Virtually all NDs are characterized by the buildup of proteins in and around neurons resulting in neuronal death or loss of function. It is thought that this buildup of misfolded/damaged proteins is at least partially due to loss of protein degradation capacity of the ubiquitin proteasome system (UPS) as its impairment has been reported in essentially all NDs. It is hypothesized that the presence of specific protein oligomers in various NDs mediate this proteasomal inhibition. Determining the mechanism of oligomer mediated proteasome inhibition is the focus of the first study reported here. We show that three different proteins from Alzheimer, Parkinson, and Huntington disease that misfold and oligomerize into a shared three-dimensional structure potently impair the proteasome by stabilizing the closed-gate conformation. These oligomers were unable to inhibit a mutant proteasome construct with a constitutively open entry pore. Based on these findings, we sought to introduce the open-gate proteasome construct into a model organism, which is the focus of study two in this dissertation. Using CRISPR to introduce this mutation in the germline of Caenorhabditis elegans (C. elegans), we successfully generated the first animal model that endogenously expresses a hyperactive, open-gate proteasome. As expected, the proteasome showed enhanced degradation of peptides, unstructured proteins, and a folded ubiquitinated protein. Aside from a substantial decrease in fecundity, these nematodes showed significantly increased lifespan and a significant resistance to oxidative and proteotoxic stress. The results show that introducing a constitutively active proteasome into a multicellular organism is feasible and suggests targeting the proteasome gating mechanism as a valid approach for future age-related disease research efforts in mammals.

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