Author ORCID Identifier

Date Available


Year of Publication


Degree Name

Master of Science in Medical Sciences (MSMS)

Document Type

Master's Thesis




Medical Sciences

First Advisor

Dr. Gregory A. Graf


Objective: To classify mutants of ABCG8 identified in subjects with clinically confirmed Sitosterolemia, a rare form of Familial Hypercholesterolemia distinguished by the accumulation of phytosterols in plasma and tissues and determine the effects of correctors and/or regulators of proteostasis on maturation of the ABCG5/ABCG8 sterol transporter.

Methods: Disease-causing missense mutants within the cytosolic domain of ABCG8 were generated through site-directed mutagenesis. Normal and mutant proteins were expressed in human hepatocytes. Cellular proteins were prepared, and maturation was assessed by SDS-PAGE and immunoblotting. Formation of the higher molecular weight, mature form of glycoproteins was used as a bioassay for trafficking the G5G8 complex beyond the Endoplasmic Reticulum. The impact of correctors and regulators of proteostasis on Class II mutant maturation was also determined.

Results: Approximately 44% of cytosolic, Sitosterolemia-associated mutants in ABCG8 are maturation incompetent. Of those which matured beyond the ER, about 60% were not able to traffic to the cell membrane. Of the mutants that did not mature, none were able to be rescued by small molecular chaperones (correctors).

Conclusion: In conclusion, HuH-7 cells are an efficiently transfected cell line that provides a system to manipulate ABCG5 and ABCG8 to make conclusions about protein maturation and trafficking to the cell surface. These experiments gave insight into the complexity of diseases caused by genetic mutations and the underlying mechanism of loss-of-function mutations. Further experimentation would be required to determine the fate of the CFTR correctors and/or regulators of proteostasis in the application in cases of Sitosterolemia.

Digital Object Identifier (DOI)