Lipid droplet regulation and proteome dynamics

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PROJECT SUMMARY / ABSTRACT Lipid droplets (LDs) are neutral lipid storage organelles that act as cellular hubs of lipid homeostasis. Dysregulation in LD function has been implicated in prevalent metabolic diseases such as obesity, diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD). Indeed, the pathological hallmark of NAFLD is the accumulation of large hepatic LDs. In addition to metabolic diseases, LDs have also been implicated in cancer proliferation and survival, host-pathogen interactions, and neurodegeneration. Thus, understanding how LDs are regulated has the potential to broadly impact our understanding of human health and disease. LDs are ER-derived organelles that have a unique ultrastructure, consisting of a core of neutral lipid surrounded by a phospholipid monolayer decorated with integral and peripheral proteins. While recent findings have advanced our understanding of LD biogenesis, how LDs are regulated under different metabolic conditions and how the composition of the LD proteome remain poorly understood. To overcome these critical gaps in knowledge and define the mechanisms that regulate neutral lipid storage, we performed a series of CRISPR-Cas9 screen in human cells using a fluorescence-based neutral lipid reporter under different metabolic conditions. We also employed genetic screens to examine the mechanisms that regulate PLIN2, a near ubiquitous Class II LD protein that plays important roles in regulating LD stability. Our findings establish a compendium of neutral lipid storage regulators, revealing interesting novel regulators that are condition specific. Furthermore, we identify several ubiquitination factors that influence neutral lipid storage and the stability of PLIN2. The current proposal aims to build on the foundation provided by our extensive preliminary data to characterize new mechanisms of LD regulation. In aim 1, we will complete our validation experiments to establish an extensive, phenotypic-rich resource for the community that is hypothesis generating. We will also examine the concept that metabolic state-dependent regulation of LDs is a significant contributor to cellular lipid homeostasis. Finally, we will characterize high priority candidates in iPSC-derived hepatocytes and examine the hypothesis that a subset of regulators governs LD stability as part of a host response to pathogens. In Aim 2, we will define the role of new ubiquitination pathways in regulating lipid homeostasis, examining the hypothesis that the identified factors regulate LD stability by controlling the degradation of PLIN2 during lipolysis. These findings will provide new global and mechanistic insights in to LD proteome remodeling and regulation under different metabolic conditions.