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Title

The Role of Oxysterol-Membrane Effects on Cholesterol Activity and Homeostasis

Date of Award

Spring 5-15-2014

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

Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis, which feedback on a number of cholesterol-regulatory pathways to lower cellular cholesterol levels. Growing evidence suggests that the lipid-disordering membrane properties of 25-HC contribute to its cholesterol-regulatory actions. In order to determine how the lipid-disordering properties of 25-HC may be coupled with downstream cholesterol-regulatory effects, I examined the role of oxysterol-membrane effects on "cholesterol activity", or the cholesterol accessibility within the membrane.

For these studies, perfringolysin O (PFO), a cholesterol-dependent cytolysin that binds only to active cholesterol, was used as a sensor of membrane cholesterol activity. With this assay, I showed that 25-HC increases cholesterol activation, and that saturated lipids blunt membrane cholesterol activity, as well as the ability of 25-HC to activate cholesterol. To further examine the role of oxysterol-membrane effects on cholesterol homeostasis, I synthesized the natural and enantiomeric forms of the oxysterol-antagonist LY295427. Using these molecular probes, it was determined that LY295427 decreases 25-HC-mediated cholesterol activation in a non-enantioselective manner, suggesting that LY295427 antagonizes the cholesterol homeostatic effects of 25-HC by abrogating its properties in the membrane. I also synthesized 25-hydroxycholestatrienol (25-HCTL), a fluorescent analogue of 25-HC, which can be used as a molecular probe to help determine the cellular distribution of 25-HC.

These studies demonstrate that oxysterols regulate cholesterol activity - and thus the proportion of cholesterol available to be sensed and trafficked throughout the cell - by modulating the membrane environment. This work provides further insight into how alterations in membrane structure can be used to relay cholesterol-regulatory signals.

Language

English (en)

Chair and Committee

Daniel S Ory

Committee Members

Douglas F Covey, Katherine Henzler-Wildman, Linda Pike, Paul Schlesinger, Dwight Towler

Comments

Permanent URL: https://doi.org/10.7936/K7W37T8H

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