Systematic Study of Cellular Cholesterol Homeostasis
| dc.contributor.advisor | Widenmaier, Scott B | |
| dc.contributor.committeeMember | Ianowski, Juan P | |
| dc.contributor.committeeMember | Eames, Brian F | |
| dc.contributor.committeeMember | Harkness, Troy A | |
| dc.contributor.committeeMember | Eskiw, Christopher | |
| dc.creator | McDonald, Sherin | |
| dc.date.accessioned | 2022-12-14T16:27:13Z | |
| dc.date.available | 2022-12-14T16:27:13Z | |
| dc.date.copyright | 2022 | |
| dc.date.created | 2022-12 | |
| dc.date.issued | 2022-12-14 | |
| dc.date.submitted | December 2022 | |
| dc.date.updated | 2022-12-14T16:27:14Z | |
| dc.description.abstract | Cholesterol is a vital nutrient/lipid with irreplaceable function and is intricately linked to membrane physiology in mammals. Its unique biophysical properties (size, hydrophobicity) enable rapid intercalation into membranes where it exerts strong ordering effects which facilitates the organization and functionality of membrane-bound proteins critical for cell survival. Individual cells maintain tight homeostatic control over intracellular cholesterol metabolism as abnormal levels are cytotoxic and can lead to an array of pathological states. Different membrane compartments (e.g., plasma versus other organelle membranes) within a cell require distinct cholesterol concentration for proper function. Mechanisms that sense and constrain cholesterol level within these membranes are incompletely understood and likely involve a complex array of membrane-localized proteins to co-ordinate processes that maintain these distinct ranges. To identify these membrane localized proteins, we altered membrane cholesterol levels in Hep3B cells with methyl-β-cyclodextrin. Hep3B cells were demonstrated as a suitable cell culture model system for our study. The cells not only exhibited predicted transcriptional and metabolic responses to cholesterol loading and/or depletion but were also adaptive to this challenge, as they sustained cell growth throughout a 7-day treatment period. Proteomics analysis of biochemically purified endoplasmic reticulum and plasma membranes revealed an abundance of more than 50 proteins residing on endoplasmic reticulum-enriched or plasma membranes were altered by cholesterol excess or insufficiency. Novel findings include proteins involved in membrane tethering, lipid transport and intracellular trafficking. Using both shRNA- and CRISPR/Cas9- based genetic screen approaches, 900 genes were identified to defend the viability of cells chronically challenged with cholesterol excess or with cholesterol insufficiency. Interestingly, proteins involved in unfolded protein response; endoplasmic reticulum associated degradation were identified as essential for modulating cholesterol-induced endoplasmic reticulum stress – a major driver but poorly characterized phenomenon underlying several metabolic disorders. While the endoplasmic reticulum is a highly adaptive organelle intricately linked to a wide spectrum of cellular functions and serves as an integrative platform for nutrient sensing and maintaining metabolic health, prolonged cholesterol overload can affect membrane fluidity, offset membrane properties, and disrupt its adaptive capacity. Therefore, continued study of these molecules may reveal insight as to how cells coordinate constraints on cholesterol and membrane homeostasis with necessary adaptations to changes in the cellular environment that can pose a pathological risk. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/14368 | |
| dc.language.iso | en | |
| dc.subject | Cholesterol | |
| dc.subject | membrane homeostasis | |
| dc.subject | phospholipids | |
| dc.subject | trafficking | |
| dc.title | Systematic Study of Cellular Cholesterol Homeostasis | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Anatomy, Physiology, and Pharmacology | |
| thesis.degree.discipline | Anatomy, Physiology, and Pharmacology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |