Systematic Study of Cellular Cholesterol Homeostasis
Date
2022-12-14
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
Type
Thesis
Degree Level
Masters
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.
Description
Keywords
Cholesterol, membrane homeostasis, phospholipids, trafficking
Citation
Degree
Master of Science (M.Sc.)
Department
Anatomy, Physiology, and Pharmacology
Program
Anatomy, Physiology, and Pharmacology