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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL056593-01A2
Application #
2602842
Study Section
Metabolism Study Section (MET)
Project Start
1998-05-01
Project End
2002-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Blasiole, Daniel A; Oler, Angie T; Attie, Alan D (2008) Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB. J Biol Chem 283:11374-81
Attie, Alan D; Flowers, Matthew T; Flowers, Jessica B et al. (2007) Stearoyl-CoA desaturase deficiency, hypercholesterolaemia, cholestasis and diabetes. Novartis Found Symp 286:47-53;discussion 54-7, 162-3, 1
Blasiole, Daniel A; Davis, Roger A; Attie, Alan D (2007) The physiological and molecular regulation of lipoprotein assembly and secretion. Mol Biosyst 3:608-19
Flowers, Jessica B; Rabaglia, Mary E; Schueler, Kathryn L et al. (2007) Loss of stearoyl-CoA desaturase-1 improves insulin sensitivity in lean mice but worsens diabetes in leptin-deficient obese mice. Diabetes 56:1228-39
Attie, Alan D; Flowers, Matthew T; Flowers, Jessica B et al. (2007) Stearoyl-CoA desaturase deficiency, hypercholesterolemia, cholestasis, and diabetes. Nutr Rev 65:S35-8
Nassoury, Nasha; Blasiole, Daniel A; Tebon Oler, Angie et al. (2007) The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR. Traffic 8:718-32
Yi, Nengjun; Shriner, Daniel; Banerjee, Samprit et al. (2007) An efficient Bayesian model selection approach for interacting quantitative trait loci models with many effects. Genetics 176:1865-77
Flowers, Matthew T; Groen, Albert K; Oler, Angie Tebon et al. (2006) Cholestasis and hypercholesterolemia in SCD1-deficient mice fed a low-fat, high-carbohydrate diet. J Lipid Res 47:2668-80
Lan, Hong; Chen, Meng; Flowers, Jessica B et al. (2006) Combined expression trait correlations and expression quantitative trait locus mapping. PLoS Genet 2:e6
Benjannet, Suzanne; Rhainds, David; Essalmani, Rachid et al. (2004) NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol. J Biol Chem 279:48865-75

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