Mechanisms of energy storage in lipid droplets

Project Summary/Abstract In eukaryotes, energy is stored primarily as highly reduced triacylglycerols (TG) in membrane-less organelles called lipid droplets (LD). A central question for the field is what protein machinery evolved to efficiently and reproducibly make these emulsion particles in cells for storing lipids. Starting with our screens to identify genetic components of this process, our work has helped to shape the current understanding of LD formation, including elucidating the molecular structure and mechanism for a TG-synthesis enzyme (DGAT1), the components and structural information for seipin and the ER-based LD assembly complex (LDAC), and molecular insights into ER enzymes that modify this process, such as FIT2. The current proposal builds on our progress and proposes to overcome key knowledge gaps for each of these aspects of LD formation. We will extend our studies of TG synthesis to elucidate the structure, biochemical function and cell biological functions of the DGAT2 enzyme, which is closely linked to de novo fatty acid synthesis and a drug target that is in phase 2 trials for fatty liver disease. We will obtain a structure of the complete LDAC, including the key protein LDAF1, and utilize biochemical and molecular dynamics simulations studies to test models of how the LDAC facilitates LD formation. Finally, we will study the LD modifier, FIT2, to test the hypothesis that this enzyme plays a key functional role in providing CoA for mitochondrial metabolism. Achieving our goals will provide leading edge data to unravel the molecular machinery that underlies lipid and energy storage in cells and will help to elucidate the pathogenesis of diseases with alterations in lipid storage.