Apoprotein B100 (hereafter referred to as apoB) is necessary for the assembly and secretion of very low density lipoproteins from the liver. Although apoB is a secretory protein, it is not efficiently translocated across the endoplasmic reticulum (ER) membrane. Instead, it temporarily assumes a transmembrane topology with one or more domains exposed to the cytosol. It is in this position that nascent apoB is predisposed to rapid degradation. Completion of translocation, which depends on newly synthesized lipid, protects apoB from degradation and targets it for secretion. The authors believe that the complicated itinerary of apoB, from translation and translocation to lipoprotein assembly and secretion, requires the involvement of chaperon molecules, including cytosolic heat shock protein 70 (HSP 70), microsomal triglyceride transfer protein - protein disulfide isomerase (MTP-PDI), and calnexin. They will conduct studies to test three hypotheses related to these chaperons. Hypothesis 1. HSP 70 plays a critical role in targeting nascent apoB for secretion. These investigators will study the interaction between HSP 70 and nascent apoB under baseline conditions, after perturbations that alter translocation and/or degradation of nascent apoB, and after alterations of HSP 70 levels in the cell. Hypothesis 2. MTP-PDI plays an essential role in apoB secretion by facilitating the completion of apoB translocation across the ER membrane. The interaction of MTP-PDI with apoB is regulated by newly synthesized lipids, and requires specific sequences in apoB. They will determine if MTP-PDI binding to apoB requires newly synthesized triglyceride, or other lipids, and if MTP-PDI binding of apoB is concomitant with, or sequential, to HSP 70 binding. They will investigate the molecular basis of MTP-PDI binding to apoB using apoB truncations, constructs, and species made by site directed mutagenesis. Hypothesis 3. Calnexin plays a critical role in maintaining apoB translocation competence during the time that the secretory protein is in a transmembrane position. Calnexin binding to apoB will be studied under baseline conditions and after altering apoB translocation and secretion. Binding of apoB to Hsp 70 and calnexin, and to calnexin and MTP-PDI will be characterized. Finally, the investigators will use the truncations, constructs, and mutants of apoB described above to study the interaction of apoB with calnexin. These studies may assist scientists in designing approaches to reduce lipoprotein secretion in patients with hyperlipidemia.
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