Post-translational Control of Triglyceride and Cholesterol Metabolism by ANGPTL3 & ANGPTL8 in ApoBCL Clearance

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PROJECT 3: Post-translational Control of Triglyceride and Cholesterol Metabolism by ANGPTL3 & ANGPTL8 in ApoBCL Clearance PROJECT SUMMARY The goal of this grant is to elucidate the molecular basis of the lipid-lowering effects of inactivating ANGPTL3 (A3) and ANGPTL8 (A8). Previously we discovered that inactivating mutations in A3 in humans are associated with reduced circulating levels of triglycerides (TGs). We showed that A3 forms a complex with a related protein, A8, to inactivate lipoprotein lipase (LPL), an intravascular enzyme in peripheral tissues that hydrolyzes circulating TG. Inactivation of either A3 or A8 reduced plasma TG levels by increasing LPL activity. Despite these advances in our understanding, fundamental questions remain about how A3 and A8 interact to inhibit LPL in vivo. Moreover, A3 has activities that are independent of A8. Inactivation of A3 dramatically lowers plasma cholesterol, as well as TG levels. This activity does not require A8 or any of the known hepatic lipoprotein clearance pathways. Our group, and that of Dan Rader’s, has shown that endothelial lipase (EL) is needed for the cholesterol-lowering effects of A3 inactivation. The scientific premise of this application is that elucidating the molecular mechanisms underlying the lipid-lowering effects of A3 and A8 inhibition will lead to new strategies for the prevention and treatment of cardiometabolic disorders. In AIM 1 we will determine the forms of A3/A8 and A3 that are operative in vivo in inhibiting LPL and EL, respectively. Both proteins are subject to post-translational modifications, including proteolytic cleavage and oligomerization. We will define the basic properties of the active forms of the native proteins with respect to cleavage (Aim 1A) and stoichiometry (Aim 1A), before purifying the active complexes and determining their high resolution structures (Aim 1C). In AIM 2 we will characterize molecularly the noncanonical pathway for hepatic clearance of ApoB-containing lipoproteins (ApoBL) in A3 deficiency and test 3 hypotheses regarding the molecular basis for this pathway: First, that EL-stimulated ApoBL uptake is a receptor-mediated endocytic process (Aim 2A); Second, that EL alters the composition of nascent VLDL, exposing a new ligand for ApoBL uptake by the liver (Aim 2B). Finally, we will establish an unbiased genetic screen in cultured hepatocytes to identify the receptor that mediates ApoBL uptake by liver and validate receptor activity in vivo (Aim 2C). Elucidating the molecular mechanisms by which A3 and A8 alter lipid metabolism will provide new insights into key metabolic pathways. They will also provide a mechanistic basis for a new generation of lipid-lowering agents. These goals complement the aims of Research Project 1 and Project 2 to understand what regulates the formation, location, and metabolic fate of lipids so as to develop new agents for the treatment of cardiometabolic disorders.