The goal of this proposal is to understand the structural features of apoB that are necessary and sufficient for the assembly of apoB-containing lipoproteins. The proposed research is driven by two hypotheses: 1) The N-terminal 20-27 percent of apoB forms an intermediate structure, homologous to lipovitellin, that contains a lipid pocket required to initiate the assembly of apoB-containing lipoprotein particles. 2) The domain of apoB homologous to lipovitellin is missing a portion of the full set of lipovitellin-like structural elements necessary for the lipid pocket formation; these missing structural elements are present in microsomal triglyceride transfer protein (MTP). The binding of MTP to the nascent apoB is necessary for structural complementation to form a complete and functional lipid pocket. These hypotheses will be tested by the three specific aims: 1) To determine the regions of the N-terminal domain of apoB that are required for the assembly of apoB-containing lipoproteins.
This aim will be accomplished by construction and expression of a library of C-terminally truncated forms of apoB and analysis of particles secreted by transfected mammalian cells in culture. We propose to map the effects on apoB expression and assembly into lipoprotein particles of sequential inclusion of the N-terminal regions of apoB, focussing on those domains structurally homologous to the key structural domains of the lipovitellin lipid pocket. 2) To develop an all atom molecular model of the lipid pocket for apoB-containing lipoprotein particle assembly in the absence and in combination with MTP. To accomplish this, lipovitellin structural homologue domains of apoB and MTP, identified by us previously using sequence similarity analysis and determination of domains rich in amphipathic motifs, will be mapped onto the structural coordinates of the lamprey lipovitellin PDB file. 3) To understand the contribution of small motifs of apoB and MTP in the assembly of apoB-containing lipoprotein particles.
This aim will be accomplished by testing the effects of site-directed mutagenesis of the N-terminal domain of apoB and the hypothesized complementary domains of MTP on the assembly of apoB-containing particles in mammalian cells in culture. The results of specific aims 1 and 2 will determine the specific sites and types of mutations selected.
|Dashti, Nassrin; Gandhi, Medha; Liu, Xiaofen et al. (2002) The N-terminal 1000 residues of apolipoprotein B associate with microsomal triglyceride transfer protein to create a lipid transfer pocket required for lipoprotein assembly. Biochemistry 41:6978-87|
|Segrest, J P; Jones, M K; De Loof, H et al. (2001) Structure of apolipoprotein B-100 in low density lipoproteins. J Lipid Res 42:1346-67|