The long-term goal of the project is further to define the structure- function relationships of apoB, the major protein of LDL. The original approach was to identify natural mutants of apoB and over the first 4 years of this grant 7 new truncations of apoB associated with hypobetalipoproteinemia were identified and characterized. We now propose to expand our studies in the kindreds identified by us and by others, and to identify new truncations. Also, to study the production of truncated apoBs at the cellular-molecular levels, HepG2 cells lines """"""""heterozygous for apoB-100/apoB truncations will be produced via mutagenesis by """"""""in & out"""""""" homologous recombination. In probands and kindreds, identified by screening low cholesterol populations, molecular genetic defects of apoB, and apoB haplotypes are ascertained. To assess any relationships between the lengths of apoB truncations and their metabolic behaviors, in vivo kinetic protocols are performed using I.V. infusions of stable isotopically labelled amino acids. Kinetic parameters are calculated using multicompartmental models. ApoB concentrations of heterozygotes studied to date are only about 5th percentile rather than the 50th percentile expected. To determine the kinetic basis for this, the kinetics of apoB-100 in heterozygotes and in normolipidemic controls are compared. Tissue loci of catabolism of mutant LDLs are identified in in vivo kinetic studies performed in rabbits using dual label protocols. ApoB-100 LDL and apoB- truncated LDL are labelled with 125l- or 131l-dilactitol tyramine, a protein residualizing label, injected intravenously, and traced in plasma, and into tissues. Little is known about the lipid transporting functions of lipoproteins containing apoB-truncations. To assess their structures, compositions, and metabolic behaviors as lipolytic substrates for LPL, apoB-100- and apoB truncation-containing lipoproteins are separated from plasma and their sizes, lipid and apoprotein compositions quantified and their LPL catabolized lipolysis rates assessed. Hepatic production of mutant and wildtype apoBs is studied in """"""""heterozygous"""""""" HepG2 cell lines. The steady state levels and metabolism of mutant and wildtype mRNAs are assessed as well as the synthesis, secretion and degradation of the proteins. Finally, advantage is taken of the repertoire of apoB truncations to identify binding regions on apoB in intact lipoproteins where heparin and apo(a) may bind. These broad based studies are expected further to define the relationships between apoB structure and its production, intravascular fate and tissue sites of and cellular catabolism.
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