Project 1: Does the lipid composition of lipid droplets influence the number and type of proteins that bind?
Our experiments suggest that the phospholipid composition of the lipid droplet surface influences the numbers and types of proteins that associate with the lipid droplets. This has implications for alcoholic fatty liver disease (AFLD), a condition in which the lipid droplets that accumulate in the liver show difference surface phospholipids and associated proteins. Our lab has developed a cell culture system that models the changes to the lipid droplet phospholipids that occur with AFLD. We have used this system to show changes in relative levels of select lipid droplet proteins. Our next experiments will look at whether DGAT2 localization is influenced by lipid droplet phospholipids. This protein is of interest, in part, because it interacts with lipid droplets through a hydrophobic hairpin loop. We will attempt to overexpress this protein in cultured cell lines and isolate lipid droplets from cultured cells in presence and absence of choline, and use SDS PAGE and western blotting to identify and quantify DGAT2.
Project 2: Protein binding to model lipid droplets.
The goal of this project is to explore how changes to the phospholipid surface of the lipid droplet impact protein binding. We have previously developed an in vitro binding assay to measure perilipin 2 (a ubiquitous lipid droplet protein) binding to phospholipid vesicles. We have used this assay to show that perilipin 2 binding is sensitive to relative levels of phosphatidylcholine(PC) and phosphatidylethanolamine(PE). These are the two most prevalent classes of phospholipids in cells, and their relative levels change with the onset of AFLD. In the past year, we have modified the in vitro assay to test binding to model lipid droplets rather than phospholipid vesicles. We will use this improved in vitro binding assay to determine if perilipin 2 binding prefers PC or PE, and whether other proteins are sensitive to similar changes in phospholipid composition.
Project 3: Lipid droplets in Tetrahymena
A bioinformatics search of human lipid droplet proteins find few obvious homologues in Tetrahymena thermophila. Even so, lipid droplets are abundant in Tetrahymena. This is especially true under conditions of starvation when significant membrane remodeling occurs. Given the timing, it is reasonable to hypothesize that lipid droplets may act as a source of lipid for membrane biogenesis or for catabolism. Very little is known about lipid droplet biology in Tetrahymena. We will apply the expertise of our lab to Tetrahymena in order to explore cellular mechanisms of lipid storage and utilization in this ciliate.