Post-translational regulation of hepatic uptake transporters in health and disease

PROJECT SUMMARY Given the growing prevalence of metabolic syndrome, obesity, and nonalcoholic fatty liver disease (NAFLD), we need to know how these disease states affect the function and expression of the hepatocellular uptake transporters NTCP, OATP1B1, and OCT1. While it is known that these disease states lead to increased amounts

of free cholesterol and palmitoylated proteins in the liver, it remains unclear how these alterations influence the function and post-translational regulation of clinically significant hepatic membrane proteins, such as NTCP, OATP1B1, and OCT1. Our long-term goal is to understand how these transporters are expressed and regulated

in human hepatocytes. Our overall objective in this application is to elucidate how changes in membrane composition due to obesity, with respect to cholesterol and lipids, affect the function and expression of NTCP, OATP1B1, and OCT1. Our central hypothesis is that increased free cholesterol and the palmitoylation state

modulate the function and expression of NTCP, OATP1B1, and OCT1. The rationale for the proposed research is that understanding the mechanisms by which transporter activity is regulated by changes in membrane composition and lipid modifications will explain pathological observations, optimize drug response, and lead to

information that could inform drug safety profiles. The central hypothesis will be tested by pursuing two specific aims: 1) Identify the mechanisms by which free cholesterol affects the function and expression of NTCP, OATP1B1, and OCT1; and 2) Elucidate the modulatory effects of protein palmitoylation on NTCP, OATP1B1,

and OCT1 function and expression. Under the first aim, HEK293 and HepG2-CRISPRa cell lines stably expressing the transporters, freshly isolated human hepatocytes, and mouse models of NAFLD will be used to examine to what extent free cholesterol regulates these. For the second aim, the same model systems will be

used along with MS proteomics and site-directed mutagenesis to assess how palmitoylation affects the transporter's function and expression. The research proposed in this application is innovative because it represents a substantive departure from the status quo by focusing on how free cholesterol and palmitoylation

affect the hepatic uptake transporters. The proposed research is significant because it will clarify the basic molecular mechanism(s) of the post-translational regulation of NTCP, OATP1B1, and OCT1 function and expression and help ultimately explain alterations in bile acid and drug uptake in patients with obesity.