Hepatic apolipoprotein B (apoB)-containing lipoproteins are the predominant carriers of plasma cholesterol and, in elevated concentrations, represent a positive risk factor for the development of premature coronary artery atherosclerosis. Despite its importance of numerous aspects of lipoprotein metabolism, including the prevention and treatment of hyperlipidemias and their complications, the mechanism underlying the hepatic assembly of apoB-containing lipoprotein remains poorly defined. The overall goal of the proposed research is to characterized apoB's interactions with lipids, membranes and the microsomal triglyceride transfer protein (MTP) and to understand how these interactions results in the assembly and regulation of apoB-containing lipoproteins. The following specific aims will be addressed: I. Define how the amino-terminal domain of apoB functions to initiate lipoprotein formation in the hepatic endoplasmic reticulum (ER). To define how this domain functions, its properties will be analyzed using several in vitro assays designed to reflect potential functions during lipoprotein assembly in vivo. For each assay the behavior of native and chemically reduced apoB17 (amino-terminal 17% of apoB) was well as apoB17 harboring specific cysteine to serine mutations will be studied. The critical function of this domain will emerge by identifying which assay(s) displays a structure-function profile similar to that observed for apoB17's capacity to initiate lipoprotein assembly in vivo; II. Establish whether the initial co-translational phase of lipoprotein assembly requires recruitment by apoB of specific lipid classes. This question will be addressed by determining the relative lipid compositions of a series of C- terminally truncated, epitope-tagged forms of apoB that are though to represent static intermediates in the normal process of co-translational lipoprotein formation; III. Identify and characterize intermediates in the post-translational folding and assembly of apoB28. Since the post- translational assembly of apoB28 can be readily synchronized and manipulated, intermediates in this process can be identified as can the roles of cellular factors with known or proposed roles in the process of lipoprotein assembly; IV. Unequivocally establish the topology of apoB within the hepatic ER. The prevailing, through controversial view of the regulation of apoB secretion, involves the capacity of the hepatocyte to regulate the translocation of apoB across the R membrane in response to lipid availability. To establish unequivocally whether cytosolic, translocation arrested forms of apoB exist within the hepatic ER, the extent of glycosylation at specific sites within the C-terminal 50% of the protein will be monitored.
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