We have recently identified two potential new steps in the lipoprotein assembly pathway-the interaction of apoB with a protein (designated """"""""protein X"""""""") in the endoplasmic reticulum and the involvement of protein disulfide isomerase (PDI) in apoB secretion. This grant proposes to test the hypothesis that apoB interacts with protein X and with PDI within the ER and that these interactions affect the proportion of apoB that is secreted from hepatocytes. We shall ask if protein X plays a role in apoB secretion in mouse hepatocytes. We have discovered that co-expression of a domain of protein X with apoB dramatically increases the apoB secretion rate in baculovirus-infected insect cells, implying a protein-protein interaction within the secretory pathway. In addition, we have detected a physical interaction between apoB and protein X within these cells. We shall extend these studies with the following experiments in primary mouse hepatocytes. We shall measure the apoB secretion rate from primary mouse hepatocytes of normal and protein X knockout mice. We shall study the effect of several types of protein X on apoB secretion. We shall investigate the mechanism by which protein disulfide isomerase (PDI) is involved in apoB disulfide bond formation and apoB secretion. We have found that co-expression of an enzymatically inactive mutant form of PDI inhibits apoB secretion. The inhibition is reversible by oleate supplementation at high physiological concentration (1 mM). We shall co-express PDI active site mutants with apoB48 in order to discover which of PDI's enzymatic activities plays a role in apoB secretion. We shall directly assess the formation of disulfide bonds in nascent apoB in order to test the hypothesis that triglyceride transfer to the secretory pathway affects disulfide bond formation. We shall try to isolate a PDI-apoB mixed disulfide intermediate by expressing a mutant form of PDI lacking redox activity but possessing shufflase activity. We shall disrupt the redox potential of cells expressing apoB48 with several novel dithiol reagents with reduction potentials close to that of the ER lumen, to study the potential role of disulfide bond formation in lipid acquisition by newly-synthesized apoB.
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