Defining the role of PLA2G12B on apoB-containing lipoprotein assembly, plasma levels, metabolism and cardiovascular disease risk - PROJECT SUMMARY- Triglyceride (TG)-rich ApoB-containing lipoproteins (BLP) are micelle-like particles that enable efficient transport of lipids through the bloodstream, but also promote atherosclerotic cardiovascular disease (CVD), the leading cause of death worldwide. Although the lipid content of BLP is known to be a strong determinant of CVD risk, very little is known about how ER lipids are loaded onto nascent BLP prior to entering the secretory pathway. Whereas invertebrate lipoproteins predominantly transport cholesterol and are quite small, vertebrate lipoproteins evolved the additional capacity to transport TGs in much larger BLP. Neither the mechanistic basis nor the physiological implications of TG incorporation into vertebrate lipoproteins have been well defined. In this Multi-PI proposal, we leverage our most recent findings that identify Phospholipase A2 Group XIIB (PLA2G12B), a catalytically inactive phospholipase gene with no known function, as a vertebrate-specific protein responsible for channeling TG to nascent BLP. Our preliminary data indicates that PLA2G12B interacts with ApoB and recruits microsomal triglyceride transfer protein (MTP) to the lumenal face of the ER membrane. We show that PLA2G12B deficiency selectively disrupts TG transfer to BLP, and in a mouse model of CVD, leads to significant reductions in plasma lipid levels and atherosclerosis. We find corresponding BLP phenotypes in cultured human liver and intestinal cells deficient in PLA2G12B, providing direct relevance of our findings to human physiology. These data suggest that incorporation of TG into BLP via PLA2G12B and MTP is a key driver of delayed lipoprotein turnover and hypercholesterolemia in vertebrate animals. We have developed a suite of novel tools to measure digestive organ lipid uptake, transport, and storage in zebrafish. These include in vivo reporter lines to quantify BLP size, numbers, and turnover, as well as lipid droplet protein dynamics. In Aim 1, we will use these new tools along with genetic epistasis analyses to define the position of PLA2G12B along the BLP assembly pathway. We specifically are focusing on proteins that are implicated in BLP synthesis and/or interact with PLA2G12B (e.g., TM6SF2, ERLIN1/2, and OIT3/LZP). We will also define the PLA2G12B interactome and create zebrafish knockouts of these interacting proteins and explore the degree to which they alter ApoB and MTP binding. In Aim 2, we will (a) address the role of intestinal and hepatic PLA2G12B in driving proatherogenic phenotypes; (b) explore the cellular response to loss of PLA2G12B and how this alters ApoB degradation; and (c) explain the role of polymorphisms in the promoter of human PLA2G12B and the role of some transcription factors in regulating PLA2G12B expression and plasma lipid levels. The experiments proposed leverage a long-standing partnership between two field-leading labs to shed new light on the poorly understood process of BLP expansion, ascribe function to the previously uncharacterized gene PLA2G12B, and reveal a potentially promising new strategy to remodel serum lipoproteins to prevent disease.
