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Cholesterol 7 alpha-hydroxylase is Regulated Post-translationally by AMPK

Nnamani, Mauris E.C
http://rave.ohiolink.edu/etdc/view?acc_num=kent1239735961

Abstract Details

2009, PHD, Kent State University, College of Arts and Sciences / School of Biomedical Sciences.
Type II diabetes and dyslipidemia are common metabolic disorders resulting in ahigher risk of cardiovascular disease, larger bile acid pools, and excretion rates; suggesting an association between abnormal bile acid and cholesterol biosynthesis with type II diabetes and cardiovascular disease. The rate at which cholesterol is converted to bile acids is determined by the activity of the liver enzyme, cholesterol 7 alpha-hydroxylase. AMP-activated protein kinase (AMP), has long been recognized as regulating cholesterol synthesis by controlling 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the second and rate-limiting step of cholesterol synthesis. In addition, AMPK is significant for its roles in energy metabolism. 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is an activator of AMPK. Previous work in the Stroup lab have shown that AICAR treatment changes the rate at which the human hepatoma cell line (HepG2) synthesizes bile acids without significantly changing message RNA levels, as measured with real-time PCR. Also recombinant human cholesterol 7-alpha-hydroxylase can be covalently modified by commercially available AMPK in kinase assays. To test the hypothesis that cholesterol 7 alpha-hydroxylase activity can be regulated posttranslationally by phosphorylation, catalytically active cholesterol 7 alpha-hydroxylase was expressed in E. coli, purified and reacted with purified AMPK, cAMP-dependant protein kinase (PKA), and protein kinase C (PKC). Cholesterol 7 alpha-hydroxylase activity was determined by measuring the conversion of cholesterol into 7 alpha-hydroxycholesterol by recombinant CYP7A1 using LC-MS. The product was measured without chemical derivation with an orthogonal atmospheric pressure chemical ionization source (APCI) detector, using a method developed for this study. The effects of the kinases on activity of the recombinant enzyme were compared to that of extracts prepared from HepG2 cells. The activities measured from the kinase-treated extracts were significantly different than controls, and were specific to the kinases used. Site-directed mutagenesis was performed to determine phosphorylation sites responsible for the effects noticed by kinase activity. AMPK repressed E. coli-expressed recombinant CYP7A1 enzymatic activity by phosphorylating S252. The finding that the activity of cholesterol 7 alpha-hydroxylase can be regulated post-translationally by AMPK provides a possible mechanism of coordinating cholesterol disposal with cholesterol synthesis
Diane Stroup, PhD (Advisor)
Gail Fraizer, PhD (Committee Member)
Arne Gericke, PhD (Committee Member)
Marcinkiewicz Jenniefr, PhD (Committee Member)
Vijayaraghavan Srinivasan, PhD (Committee Member)
Twieg Robert, PhD (Other)
205 p.
Biochemistry; Biomedical Research
cholesterol 7-alpha hydroxylase; AMPK

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Citations

  • Nnamani, M. E.C. (2009). Cholesterol 7 alpha-hydroxylase is Regulated Post-translationally by AMPK [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1239735961

    APA Style (7th edition)

  • Nnamani, Mauris. Cholesterol 7 alpha-hydroxylase is Regulated Post-translationally by AMPK. 2009. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1239735961.

    MLA Style (8th edition)

  • Nnamani, Mauris. "Cholesterol 7 alpha-hydroxylase is Regulated Post-translationally by AMPK." Doctoral dissertation, Kent State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=kent1239735961

    Chicago Manual of Style (17th edition)

kent1239735961
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© 2009, all rights reserved.
This open access ETD is published by Kent State University and OhioLINK.