Assembly of ApoB-Containing Lipoproteins

Wake Forest University Health Sciences, Winston-Salem NC

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Abstract

Elevated plasma concentrations of apolipoprotein B (apoB)-containing lipoproteins is a positive risk factor for the development of premature coronary artery disease. The goals of the proposed research are to define (1) the mechanism of initial lipid acquisition by apoB and (2) the role of intracellular trafficking in apoB particle expansion and the modulation of this process in the intestine by apoAIV. Three specific aims are proposed. Aim I: Identify phylogenetic precursors of apoB and define their MTP-dependence, mechanisms of forma- tion, and relationships to human VLDL and chylomicron (CM) assembly. The mechanism of initial lipid ac- quisition by apoB evolved from the assembly of primitive lipoproteins, such as vitellogenin and insect apol- ipophorin. The evolution of this mechanism will be explored by comparing compositional properties and MTP-dependence of primitive apoproteins to the behavior of apoB 19.5, the smallest form of apoB capable of forming a precursor lipoprotein particle. The different roles of MTP in apoB assembly will be assessed by comparing characteristics of insect MTP, which is responsible only for f'trst-step precursor particle formation, to human MTP, which participates in both first and second-step assembly and may have evolved additional functions to achieve these reactions. Aim II: Identify the structural basis for initial lipid acquisition by apoB. Removal of 22 amino acids from apoB 19.5 abolishes lipoprotein formation. In the studies proposed here, we will conduct a head-to-head comparison of the structure, conformation, and interfacial properties of two forms of apoB that terminate on either side of this initiation boundary. The apoB truncation mutants will be investigated by a variety of spectroscopic and surface chemistry techniques to distinguish between two alter- native models of apoB assembly, one involving formation of a globular lipid binding cavity and the other in- volving formation of a domain with strong interfacial properties that perturbs membrane surfaces and facili- tates lipid desorption and nascent particle formation. Aim III: Identify the intracellular site(s) of apoB particle assembly and elucidate the role of apoAIV as a dedicated cofactor that alters the intracellular trafficking of apoB and affects particle expansion. Controversy exists regarding the relative role of the endoplasmic reticu- lum (ER) and Golgi in apoB particle expansion. The relative contribution of each compartment will me es- tablished by modifying apoB48 with the KDEL ER retention/recycling signal, and assessing its impact on apoB particle expansion. The function of apoAIV will be explored by defining how it interacts with apoB in the secretor), pathway and the resulting impact on apoB-containing particle expansion and lipid absorption.