Cholesterol Regulation Through Retriever-Dependent LDL Receptor Recycling

PROJECT SUMMARY Elevated plasma LDL cholesterol is a potent risk factor for atherosclerosis and cardiovascular complications. LDL is cleared from circulation by LDL receptors (LDLR). Importantly, the balance between internalization, recycling, and intracellular degradation of LDLR is critical in determining surface expression of LDLR, LDL clearance, and plasma LDL concentrations. A pivotal regulator of endosomal recycling of LDLR is the trimeric protein complex known as Retriever. Comprised of VPS35L, VPS26C, and VPS29, Retriever is essential for identifying cargo proteins in the endosomal compartment to initiate their recycling to the plasma membrane (PM). Over the past year, our multi-PI group leading this application has published the first high-resolution structural model of Retriever using cryogenic electron microscopy (cryo-EM), revealing unique features of this complex. Recycling of LDLR and other cargoes requires Retriever binding with several crucial ligands, including sorting nexin SNX17, the COMMD/CCDC22/CCDC93 (CCC) complex, and the WASH complex. Mutations in Retriever, CCC and WASH complex subunits are associated with hypercholesterolemia in humans. However, the molecular mechanisms linking Retriever to CCC and WASH complexes remain incompletely understood, representing a major knowledge gap in the field, hindering our mechanistic understanding of how Retriever regulates cholesterol homeostasis. Our hypothesis is that by using structural insights to precisely uncouple Retriever from these critical ligands, we can achieve a comprehensive understanding of their functional contributions to endosomal recycling and cholesterol homeostasis. Our team will combine expertise in biochemistry, structural biology, cell biology, and animal models to obtain a detailed molecular understanding of Retriever function through 3 aims: (1) Structural and biochemical analysis of Retriever binding with key ligands, (2) Functional characterization of Retriever-ligand interactions in cargo recycling using cell models, and, (3) Studies on the physiological significance of Retriever-ligand interactions in cholesterol homeostasis using in vivo models of Retriever deficiency. Altogether, completion of this work will greatly advance our mechanistic understanding of Retriever, a critical regulator of endosomal protein recycling. Furthermore, by taking advantage of our unique structural, biochemical, cellular, and animal model approaches, we will identify molecular pathways regulated by Retriever-dependent recycling that play meaningful roles in determining circulating LDL levels. These studies are of high relevance to cardiovascular health and the mission of NHLBI.