Selective Uptake and Hydrolysis of Cholesteryl Ester by SR-BI

PROJECT SUMMARY The receptor-ligand complex of scavenger receptor class B type I (SR-BI) and HDL is responsible for cholesterol disposal from the body via reverse cholesterol transport (RCT) and is critical in the prevention of atherosclerosis. The long-term objective of our research is to understand the mechanisms that regulate SR-BI-mediated delivery of cholesteryl ester (CE) from HDL to the liver for excretion. The scientific premise of this application is based on a growing body of literature that suggests CVD risk can be reduced by strategies that promote cholesterol clearance through enhanced cholesterol efflux and RCT. The premise is also supported by mutations in SCARB1 (the human SR-BI gene), identified in patients with high HDL-C, that prevent the selective uptake of HDL-CE and increase the risk of CVD. While efficient clearance of HDL-C hinges on the last steps of RCT, studies on the importance of the SR-BI/HDL interaction that triggers selective uptake of HDL-CE remain limited. In the previous funding cycle, our biggest discovery was the first high-resolution NMR structure of a region of SR-BI that encompasses the C-terminal transmembrane (TM) domain and contributes to SR-BI oligomerization, in addition to an adjacent extracellular region that harbors a short alpha helix with unique hydrophobic properties. In this application, we build on these exciting novel findings and additional promising preliminary data to test the overall hypothesis that cholesterol flux via RCT is driven by structural features of SR-BI that are important for membrane association and receptor oligomerization. In Aim 1, we will use structure-guided mutagenesis and a series of innovative biophysical techniques to determine whether receptor/membrane interactions involving a putative extracellular juxtamembrane helix are required to mediate the cholesterol transport functions of SR-BI. Next, we will test the in vivo importance of the juxtamembrane helix by assessing macrophage-to-feces RCT in mice expressing mutant SR-BI receptors where helix hydrophobicity has been altered. In Aim 2, we will use cutting-edge NMR strategies and paramagnetic relaxation experiments, as well as structure-guided mutagenesis, to identify the organization of the SR-BI oligomer, a complex deemed essential for the movement of cholesterol from HDL to cells. Further, we will map the precise binding interfaces between TM domains of SR-BI that are likely critical in mediating the selective uptake of HDL-CE into the plasma membrane. Finally, in Aim 3, using a non- oligomerizing mutant of SR-BI and relevant controls, we will perform macrophage-to-feces RCT studies, as well as atherosclerosis studies, to define the in vivo functional relevance of SR-BI oligomerization in murine models. The combined use in vitro and in vivo studies, together with innovative and state-of-the-art technologies, will advance our knowledge of the molecular architecture of SR-BI. Importantly, the outcomes of our studies will identify future therapies aimed at preventing hypercholesterolemia and its associated pathologies such as atherosclerosis, by pinpointing the underlying structural mechanisms that drive SR-BI-mediated selective uptake of HDL-CE and net cholesterol excretion from the body.