Our long-term goal is to understand lipoprotein assembly for which microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) are vital. We have shown that apoB and MTP bind with high affinity and that lipids modulate this binding. Lysine and arginine residues in apoB are critical for MTP binding. Inhibition of apoB-MTP binding results in decreased apoB secretion by HepG2 cells. Amino acids 270-570 in apoB bind MTP, whereas, amino acids 777-977 inhibit apoB-MTP binding. Here, we will understand the role of apoB-MTP binding and dissociation in lipoprotein biogenesis.
Aim I. We propose to identify amino acids in apoB critical for MTP binding by site-directed mutagenesis. We will then study the effect of decreasing MTP binding on the synthesis, translocation, association with hsp70, hsp90 and ubiquitin, intracellular degradation, and secretion of apoB48. These experiments will provide a fine structural map of the binding site and show that MTP binding helps in apoB translocation and in the prevention of proteosomal degradation.
Aim lI. A sequence within apoB: 777-977 that inhibits apoB-MTP binding will be identified by N- and C-terminal deletions, and critical amino acids that decrease apoB-MTP binding will be defined by site-directed mutagenesis. We will determine whether the inhibitory motif binds to apoB and inhibits MTP binding by competition.
Aim III. To define the physiologic role of the inhibitory peptide, we will study the effect of expression of the inhibitory sequence on apoB-lipoprotein assembly in HepG2 cells. To determine the importance of the inhibitory motif in apoB secretion, we will study the rate of association and dissociation of normal and mutant apoB48 with MTP. We will examine whether MTP sequences homologous to the inhibitory motif bind apoB. These studies may identify a novel apoB-binding site in MTP. The proposed studies should identify critical steps in lipoprotein assembly dependent on apoB-MTP binding and show that the inhibitory motif plays a role in rendering apoB secretion-competent. The knowledge about the inhibitory mechanisms may lead to the identification of novel therapeutic agents controlling plasma lipid levels.
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