Sunday, January 29, 2023
1/29/2023
Cholesterol is continually acquired by de novo synthesis or from the diet and must be metabolized or excreted in order to maintain cholesterol homeostasis. In vertebrate animals, cholesterol can be actively excreted via members of the ATP-binding cassette G (ABCG) subfamily. In the liver, cholesterol is secreted into bile by a heterodimer of ABCG5 and ABCG8 (G5G8). In the intestine, G5G8 limits the absorption of dietary sterols. Inactivation of G5 or G8 causes sitosterolemia, a recessive disorder associated with sterol accumulation and premature atherosclerosis. Another ABCG family member, ABCG1 (G1) also transports cholesterol. Unlike G5/G8, G1 functions as a homodimer and promotes the export of phospholipids and sphingomyelin, as well as cholesterol, from cells. G1 participates in the transport of cholesterol from peripheral tissues, where is it synthesized, through the circulation to the liver for excretion into bile. The overall goal of our research program is to determine how G5G8 and G1 promote the translocation of neutral sterols across biological membranes. In the last funding period, we used X-ray crystallography and cryo-electron microscopy (EM) to obtain high-resolution structures of nucleotide-free G5G8 with its cholesterol substrate at 2.7-3.0 Å resolution and both nucleotide-free and ATP-bound G1 at 3.1-3.7Å resolution. These results provided the first insights into the basic structure-function mechanisms of ABCG transporters and formed the basis of this grant application, which is designed to address 3 fundamental questions: 1) How do the conformations of G1 and G5G8 change during the transport cycle, and how do these changes relate to sterol translocation? 2) How do G1 and G5G8 recognize highly insoluble neutral sterols that are embedded in lipid bilayers and what accounts for the substrate specificity of the two transporters? And 3) How is substrate binding related to ATPase activity, and what is the structural basis for substrate-stimulated ATPase activity? Each of these questions is organized as a Specific Aim. The studies proposed take advantage of new developments in cryo-EM, our expertise in functional reconstitution of polytopic membrane proteins in vivo and in vitro, and our discovery of missense mutations in G5 and G8 with very specific effects on G5G8 and G1 function (e.g. large changes in substrate specificity or ATPase activity) that provide unique mechanistic insights. We have marshalled the reagents and expertise required to ensure successful completion of the studies proposed. Elucidation of the transport mechanisms of G1 and G5G8 will reveal how cells and organisms efflux lipids and maintain sterol balance, thus preventing two common disorders: coronary artery disease (CAD) and gallstones.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
