ORCID

http://orcid.org/0000-0002-4178-8055

Date of Award

Spring 5-15-2020

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cholesterol is an essential mammalian lipid. It is a major component of cellular membranes, a precursor molecule for the synthesis of hormones and bile acids, and a regulator of protein function. Although cholesterol is synthesized, de novo, in the endoplasmic reticulum, cells principally meet cholesterol requirements through uptake of lipoprotein particles. Lipoprotein-derived cholesterol is transported to the lysosome where it is transferred from the soluble lysosomal protein, NPC2, to limiting-lysosomal membrane protein NPC1. Cholesterol is then re-distributed to other cellular membranes in order to fulfill organellar cholesterol requirements; however, the cellular machineries involved in coordinating this distribution are poorly characterized. In the absence of NPC proteins, cholesterol is inefficiently redistributed beyond the lysosome, and instead accumulates, leading to lysosomal dysfunction and eventual cell death. NPC protein deficiency underlies Niemann-Pick Type C disease, a fatal neurodegenerative condition for which there is no FDA-approved treatment. While some potential therapeutics have been identified, their mechanisms remain unknown. NPC1-dependent and independent post-lysosomal cholesterol trafficking has been difficult to study, in part, because existing cholesterol probes either fail to mimic authentic cholesterol trafficking or cannot be efficiently used to identify candidate trafficking proteins. In this thesis, I describe new tools and methodologies for the study of post-lysosomal cholesterol trafficking and demonstrate their utility in NPC1-dependent and -independent cell models. First, through rigorous biochemical characterization, I evaluate a series of cholesterol probes that are functionalized to enable tagging and enrichment of protein binding partners for identification by mass spectrometry. By screening many probes, I identify permissible and non-permissible sites for functional modification. Next, I develop a strategy for delivering cholesterol probe to the lysosome in order to identify the post-lysosomal trafficking interactome. Finally, I use isotopically labeled cholesterol to investigate drug-induced cholesterol trafficking in NPC1-deficient cell models. The new tools developed in these studies will advance our understanding of post-lysosomal cholesterol trafficking and may provide mechanistic insight into therapeutics for NPC disease.

Language

English (en)

Chair and Committee

Daniel S. Ory

Committee Members

Jason M. Held, Babak Razani, Jean E. Schaffer, Paul H. Schlesinger,

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