We propose to investigate the processes of apolipoprotein B (apo B) biogenesis and lipoprotein particle formation. Apolipoprotein B, synthesized in liver and intestine, plays a major role in cholesterol transport. Hypercholesterolemia is a major risk factor for atherosclerosis and coronary artery disease. A subset of patients with elevated serum cholesterol have defects in apo B structure, while many others have uncharacterized aberrations in metabolism and clearance of apo B containing lipoproteins. A molecular understanding of these defects will require detailed knowledge of the mechanism by which a normal lipoprotein particle is assembled. Despite major progress in understanding the general mechanisms of protein biogenesis, the specific process of lipoprotein assembly remains largely unknown. We will express partial and full-length cloned cDNAs encoding apo B in both membrane-supplemented cell-free systems and in living cells (Xenopus oocytes). Intermediates in apo B biogenesis, identified by pulse-chase immunoprecipitation, will be related to stages in apo B translocation across the endoplasmic reticulum membrane and lipoprotein particle assembly. These pilot studies will initiate a program aimed at dissection and reconstitution of apo B translocation, assembly, intracellular transport and secretion. Through this work will emerge a molecular description of events in the biogenesis of apo B and the lipoprotein particles with which it is associated. This understanding will ultimately provide insight into possible roles for aberrant particle assembly in lipoprotein disorders of humans.
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