Molecular Mechanisms of Biogenesis of Apolipoprotein B-Containing Lipoproteins

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DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms involved in the biogenesis of nascent apoB- containing lipoprotein particles and to identify the factors and structural features of apoB that are necessary for this process. The proposed research is driven by four hypotheses: 1) The N-terminal beta-alpha1 domain of human apoB-100, i.e., the first 1000 residues of the mature protein, folds into a triangular lipovitellin-like "lipid pocket" via the formation of a hairpin-bridge that provides a mechanism for the initiation of lipoprotein assembly without the structural requirement for microsomal triglyceride transfer protein (MTP). 2) The initial lipid transfer to the lipid pocket is mediated by phospholipid transfer protein (PLTP). 3) Translation of specific motifs in the amphipathic beta1 domain is required for MTP-mediated triglyceride (TG) recruitment. 4) The betaC region of the beta-alpha1 domain, together with MTP, provides a mechanism for delivering lipids into the lipid pocket. These hypotheses will be tested by four specific aims: 1) To establish the role of the hairpin-bridge and the four salt bridges: Arg997-Glu720, Glu998-His719, Asp999-Lys718, and Arg1000-Asp717 in the initiation of particle assembly. This aim will be accomplished by site-directed mutagenesis to disrupt the putative salt bridges and characterization of the secreted particles. 2) To investigate the putative role of PLTP in the initial lipidation of nascent apoB particles. This will be accomplished by: (a) expression of carboxyl-terminally truncated forms of apoB is equal to or more than apoB:1000 in mammalian cells that lack PLTP, with and without coexpression of PLTP; (b) inactivation of PLTP in McA-RH7777 cells expressing truncated forms of apoB using siRNA; (c) coexpression of PLTP and truncated forms of apoB in PLTP-deficient mouse hepatocytes. 3) To identify the domains in apoB that confer a high requirement for MTP and bulk lipid addition to the nascent particle. This will be accomplished as described for Specific Aim 2 using cells that lack MTP and MTP-deficient mouse hepatocytes. We propose to map the effects on the stoichiometry of lipid component of the particles of sequential inclusion of the N-terminal region of apoB-100 and to identify the sequences in the beta1 domain that confer stability in the particle beyond apoB:1000. 4). To test the hypothesis that the betaC region (beta barrel) of beta-alpha1 domain serves as a channel for lipid transfer into the lipid pocket. This will be accomplished by site- directed mutagenesis of residues critical for the structural competence of the beta barrel and binding to MTP.