Browsing Theses and Dissertations School of Pharmacy by Title "Molecular Mechanisms of Intestinal Bile Acid Transport and Immunomodulatory Potential of Bile Acids"
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Molecular Mechanisms of Intestinal Bile Acid Transport and Immunomodulatory Potential of Bile AcidsBile acids are catabolic products of cholesterol that play an important role in the digestion of dietary facts, lipid soluble vitamins, and drugs, as well as a role in immune regulation and glucose homeostasis. They function as complex signaling molecules to prevent intracellular accumulation of bile acids and modulate bile acid and cholesterol homeostasis via activation of a nuclear receptor, Farnesoid X receptor (FXR), to repress bile acid uptake transporters and enhance bile acid efflux transporters. The human sodium dependent bile acid transporter (ASBT) is a highly regulated intestinal uptake transporter that acts as the rate limiting step in bile acid transport in the enterohepatic circulation. Targeted ASBT inhibition is currently being investigated for use in cholestasis, hyperlipidemia, chronic idiopathic constipation, and type 2 diabetes. While studies on post-translational modifications (PTMs) have revealed N-linked glycosylation and phosphorylation as regulators of ASBT, ASBT regulation is still poorly understood. The lipid-based PTM, S-acylation, is the reversible addition of an acyl chain, via a labile thioester linkage, onto cysteine residues, thereby increasing the affinity of proteins to cellular membranes. In this work, we show that human ASBT is S-acylated and that S-acylation is vital for ASBT function, cell surface expression, substrate transport kinetics, and protein stability. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Mutation of cytosolic tyrosine residues resulted in decreased ASBT S-acylation suggestive of crosstalk between both PTMs and the existence of multiple PTM-based proteoforms. Finally, we investigate functional implications of the potential acyl transferases responsible for ASBT acylation. Overall, we have provided valuable insight on human ASBT regulation and highlighted the necessity for further investigation of the impact of PTM proteoform in drug development. While understanding ASBT regulation is vital in addressing intestinal and hepatobiliary disease states, the extent as to which its substrate, bile acids, play in other molecular processes is important in fully understanding the broader relevance of intestinal bile acid transport and bile acid signaling. Bile acids have emerged as complex signaling molecules in glucose homeostasis and immune regulation where they can activate specific receptors to increase insulin secretion and exert anti-inflammatory responses from mucosal immune cells, respectively. Using immunological approaches, we provide preliminary evidence and scientific perspective on the use of bile acids in nanoformulation that aims to exploit the immunomodulatory potential of bile acids.