| 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. |
