Apo-B Domains and Lipoprotein Structure and Assembly

Boston University Medical Campus, Boston MA

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Abstract

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