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TRANSLATIONALLY by AMPK a Dissertation CHOLESTEROL 7 ALPHA-HYDROXYLASE IS REGULATED POST- TRANSLATIONALLY BY AMPK A dissertation submitted to Kent State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy By Mauris E.C. Nnamani May 2009 Dissertation written by Mauris E. C. Nnamani B.S, Kent State University, 2006 Ph.D., Kent State University, 2009 Approved by Diane Stroup Advisor Gail Fraizer Members, Doctoral Dissertation Committee S. Vijayaraghavan Arne Gericke Jennifer Marcinkiewicz Accepted by Robert Dorman , Director, School of Biomedical Science John Stalvey , Dean, Collage of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………..vi ACKNOWLEDGMENTS……………………………………………………..viii CHAPTER I: INTRODUCTION……………………………………….…........1 a. Bile Acid Synthesis…………………………………………….……….2 i. Importance of Bile Acid Synthesis Pathway………………….….....2 ii. Bile Acid Transport..…………………………………...…...………...3 iii. Bile Acid Synthesis Pathway………………………………………...…4 iv. Classical Bile Acid Synthesis Pathway…..……………………..…..8 Cholesterol 7 -hydroxylase (CYP7A1)……..........………….....8 Transcriptional Regulation of Cholesterol 7 -hydroxylase by Bile Acid-activated FXR…………………………….....…10 CYP7A1 Transcriptional Repression by SHP-dependant Mechanism…………………………………………………...10 CYP7A1 Transcriptional Repression by SHP-independent Mechanism……………………………………..…………….…….11 CYP7A1 Transcriptional Repression by Activated Cellular Kinase…….…………………………...…………………….……12 v. Alternative/ Acidic Bile Acid Synthesis Pathway…………......…….12 Sterol 27-hydroxylase (CYP27A1)……………….…………….12 Sterol 12 -hydroxylase (CYP8B1)………………….….....……14 b. Cholesterol…………..………………………………………………...18 i. Effects of Excess Cholesterol………………………………..………….19 ii. Importance of Cholesterol……………………………………..…..…..20 c. Cholesterol Synthesis……………...…………………………………..21 d. Regulation of Cholesterol Synthesis…………………………..………25 i. HMG-CoA Reductase……………………………………………....……25 ii. Transcriptional Regulation of HMG-CoA Reductase…………...….25 iii. Regulation of HMG-CoA Reductase by Phosphorylation/Dephosphorylation Events……………………..27 iv. Repression of HMG-CoA Reductase by Statin Drugs………….…..27 e. Physiological Relevance and Significance of the Bile Acid Synthesis Pathway…………………………….…………………………………28 iii f. Experimental Rationale..…………………………………..…………………..30 i. Experimental Approach, Hypothesis, and Specific aims………...……..…..…33 CHAPTER II: MATERIALS AND METHODS……………………………….…….37 PART 1: Cholesterol 7 -hydroxylase Specific Antibodies………………...…...37 a. Rational for Cholesterol 7 -hydroxylase Specific Antibody Production...37 b. Antigen (C2) used for CYP7A1 Antibody Production…………………....37 c. Sub-cloning of the C2 cDNA Sequence into E. coli-expression Vector…..38 d. Expression of the C2 antigen……………………………………………...42 i. Expression of C2 with BL21 (DE3) E. coli Cell Line.............................42 ii. Expression of C2 with the Roche RTS 500 E. coli HY kit……………..46 e. Immunization for Antibody Production……………………………….......49 f. Characterization of Anti-serum from Immunized Rabbits………………...51 g. Pre-absorption of Rabbit Serum with E. coli lysate………….……………57 h. Antibody Characterization with Purified C2 Antigen…………..………...59 i. Antibody Affinity Purification…..………………………………………..62 i.C2 Affinity Column Preparation………………………………………..62 j. C2 Antibody Affinity Purification…………………………………………63 PART 2: Expression of Recombinant CYP7A1 in E. coli…………………….....68 a. History of CYP7A1 Expression…………….……………………………..68 b.Sub-cloning WT CYP7A1 cDNA into E. coli-expression Vector…..……..68 c. Characterization of Recombinant CYP7A1 Protein…………...………….72 d. Characterization of Expressed 30KD Fragment…………………………..74 PART 3: CYP7A1 Enzymatic Assay………………………………..…………..82 a. History of CYP7A1 Assay Detection Methods……………………..…….82 b. CYP7A1 Assay Detection Method………………………………………..83 c. Internal Standard for Enzyme Assay………………………………………84 d. Evaluating Assay Detection……………………………………………….91 e. CYP7A1 Enzyme Assay…………………………………………………..91 f. Enzymatic Activity of the 30KD Fragment………………………………..99 PART 4: Mutagenesis…………………………………………………………..100 a. Site-directed Mutagenesis Strategy 1……………………………..…...…104 b. Site-directed Mutagenesis Strategy 2……………………...…………..…107 c. Site-directed Mutagenesis Strategy 3……………………...…………..…110 d. Site directed Mutagenesis Strategy 4…………………..….…………..…113 e. Mutant Sub-cloning and Characterization……………………………….116 f. Mutant Vector-construct Restriction Mapping…………………………..119 iv CHAPTER III: RESULTS AND CONCLUSIONS…………………………………124 PART 1: AICAR, an AMPK Kinase Activator, does not Affect CYP7A1 mRNA Steady-state Levels………………………………...…..……124 PART 2: CYP7A1 In-vitro Reconstituted Assay with E. coli-expressed and Microsomal HepG2 Cells Treated with AMPK, PKC, and JNK Kinases……………………………………………………...…….…128 a. Rational for Kinase Selection……………………....………………....…128 i. AMPK……………………………………………………………………..….128 ii. PKC…………………………………………………………….........129 iii. JNK………………………………………………………………………....129 b. Kinase Treatment of Microsomal HepG2 Cells……..…………………..130 c. Kinase Treatment of E. coli-expressed CYP7A1 Recombinant Protein…………………………………………………………………...134 PART 3: AMPK Kinase Treated In-vitro CYP7A1 Enzymatic Assay with Mutant CYP7A1 Recombinant Protein……………………………...137 a. Rational for Mutation Design……………………………………….…....137 b. Rational for Mutation-site Selection……………………………...……...137 c. Mutation of AMPK Phosphorylation Sites………………………............141 d. CYP7A1 In-vitro Assay with T193A /T197A Double Mutant…………..141 e. CYP7A1 In-vitro Assay with T80A Mutant……………………………..145 f. CYP7A1 In-vitro Assay with S252G Mutant……………………............148 g. CYP7A1 in-vitro assay with Truncated WT CYP7A1 (1-729) Mutant…………………………………………..…...…………………..151 CHAPTER IV: DISCUSSION………………………………………………………..154 CHAPTER V: APPENDIX…………………………………………………………...172 APPENDIX A: EXPERIMENTAL PROTOCOLS………………...……….….172 APPENDIX B: ABBREVIATIONS……………………………………………191 REFERENCES………………………………………………………...……………...195 v LIST OF FIGURES Figure 1. Differences Between Primary and Secondary Bile Acids………………7 Figure 2. Cholic Acid and Chenodeoxycholic Acid……………………………..15 Figure 3. Schematic Diagram of the Bile Acid Biosynthesis Pathway……….....17 Figure 4. Cholesterol Biosynthesis………………………………………………24 Figure 5. ClustalW (1.83) Multiple Sequence Alignment of C2 and Full-length CYP7A1……………………………………………………………..41 Figure 6. Western Blot Analysis of In-vitro Expressed C2 Fragment for Antibody Production………………………………………………………………………..45 Figure 7. RTS 500-expressed C2 Antigen for Antibody Production…..………...48 Figure 8. Western Blot of C2 Samples with the First Small Bleed of Immunized Rabbits…………………………………………………………………………...54 Figure 9. Western Blot of C2 Samples with Serum After Two Months of Immunization…………………………………………………………………….56 Figure 10. Western Blot Analysis of C2 Antigen with Pre-absorbed Rabbit Serum…………………………………………………………………….58 Figure 11. Western Blot Analysis of Purified C2………………………………..61 Figure 12. Coomassie Blue Staining of Purified C2 Polyclonal Antibody……...66 Figure 13. Western Blot Analysis with Affinity Purified C2 Polyclonal Antibodies……………………………………………………………67 Figure 14. Western Blot of E. coli-expressed WT CYP7A1 Recombinant Protein……………………………………………………………..71 Figure 15. Protein Sequence Alignment of Human Cytochrome P450 7A1 (CYP7A1) and the 30KD Isolated Fragment…………………………………….76 Figure 16. Protein Alignment of the C2 Polypeptide and the 30KD Fragment….78 Figure 17. Western Blot of E. coli-expressed WT CYP7A1 and Truncated CYP7A1 Recombinant Protein…………………………………………………..81 Figure 18a. Standard Curve of 7 -hydroxycholesterol using the HPLC-MS..…87 Figure 18b. Extracted Portion of the Standard Curve for 7 -hydroxycholesterol using the HPLC-MS……………………………………………………………..88 Figure 19. D7- and 7 -hydroxycholesterol Co-injection……………………….90 Figure 20. Complete and Extracted Ion Traces of Samples from the CYP7A1 Enzyme Assay………………………………………………………………...…96 Figure 21. CYP7A1 Reconstituted In-vitro Assay Performed with Vector only and WT CYP7A1 E. coli-expressed Recombinant Proteins……………………..…..98 Figure 22. Schematic Diagram to Illustrate Site-directed Mutagenesis Strategy 1……………………………………………………………………….106 Figure 23. Schematic Diagram to Illustrate Site-directed Mutagenesis Strategy 2……………………………………………………………………....109 vi Figure 24. Schematic Diagram to Illustrate Site-directed Mutagenesis Strategy 3….....112 Figure 25. Schematic Diagram to Illustrate Mutagenesis Strategy 4 used in Generate Truncated CYP7A1 polypeptide…………………………………………......115 Figure 26. Mini Plasmid prep of Colonies from Mutants Transformed into E.coli DH5 Cell Lines...................................................................................................118 Figure 27. Restriction Mapping of Mutant Plasmid…………………………………....122 Figure 28. Contiguous Alignment of Mutant Nucleotide Sequence …………………...123 Figure 29. AICAR, AMPK Kinase Activator does not Affect CYP7A1 mRNA Steady-state Levels……………………………………………………………………..127 Figure 30. AMPK, PKC, and JNK Kinase Activity Repressed CYP7A1 Enzymatic Activity in Microsomal HepG2 Cell Fractions…………………………………………133 Figure 31. AMPK Kinase Activity Represses CYP7A1 Enzymatic Activity in E. coli-expressed Recombinant Protein………………………………………………...136 Figure 32. Tabulated Results from Synthetic Peptides Treated with Commercially Available Protein Kinases………………………………………………………………140 Figure 33. AMPK Kinase Activity Represses T193A/T197A Double Mutant CYP7A1 Enzymatic Activity in E. coli-expressed Recombinant Protein………………………...144 Figure 34. AMPK Kinase Activity Represses T80A Mutant CYP7A1
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