Apo-B Domains and Lipoprotein Structure and Assembly

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The assembly of triadyglycerol-rich lipoproteins (TAG-LP) involves the initial formation of a small HDL sized primordial particle with a neutral lipid core and a later process which adds TAG and phospholipid (PL) to create nascent VLDL. The N-terminal 20.5% of apoB(B0.5) has homology to lipovitellin (LV) that consists of an N-terminal beta barrel, a region of 17 amphipathic alpha helices (AaH) and 2 beta sheet domains, the A and C sheets. Proper folding of the N-terminal is necessary for secretion since certain point mutations and failure to form disulfide bonds target it to degradation. Limited proteolysis of 617 and B20.5 indicates that apoB homology to LV is approximate but not exact. Structural determination of isolated proteolytic and expressed domains using CD, NMR and X-ray diffraction will define B20.5 domains at the molecular level. The assembly with lipids as driven by the primary sequence of apoB is studied in MTP-deficient C127 cells transfected with C-terminally truncated forms. The role of molecular chaperones in folding, lipidation and in quality control is studied both in C127 and hepatoma-derived cells. The region from B20.5 to B29 recruits surface molecules, mainly PL to the nascent particle. As the peptide lengthens from B20.5 to B29 the number of surface molecules increases from approximately 5 to about 70 per particle. The sequence between B32 and B41 recruits almost 200 TAG (about 1 TAG/2.3 aa) which form nascent particles with a cryo-EM discernable core which is not seen in B32.5. Biophysical analysis shows that B37 and B41 peptides must interact directly with the core. This region contains 24 amphipathic beta strands (ApS) 12-15 aa long. Consensus 12 aa ApS and 27 aa p sheet peptides bind to oil/water (O/W) interfaces, are elastic and are not displaced at high pressure. ApoB also contains 2 major regions a2 and a3 of AaH. Consensus AaH peptides bind to O/W and air/water interfaces, but are ejected at a critical pressure into the aqueous phase. Native apoB also binds to the TAG/W interface and cannot be fully displaced by high pressure, but parts of the apoB come off when compressed and snap back on rapidly when the surfaces are again expanded. Thus, we suggest the flexible parts of apoB are AaH and the non-exchangeable part is AbetaS. This project dissects the complex pathway by which "bad cholesterol" (apoB associated cholesterol, VLDL, LDL) is made and secreted by the liver. The long term goal is to find procedures or drugs to reduce bad cholesterol, heart attack and stroke.