REGULATION OF VITAMIN E AND THE TOCOPHEROL TRANSFER PROTEIN By LYNN M. ULATOWSKI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Danny Manor Department of Nutrition CASE WESTERN RESERVE UNIVERSITY May 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ________________________Lynn M. Ulatowski_______________ candidate for the ___________Doctor of Philosophy____degree *. (signed) _____Colleen Croniger____________________________ (chair of the committee) _____Danny Manor________________________________ _____Thomas Kelley_______________________________ _____Ruth Siegel__________________________________ _____Laura Nagy__________________________________ ________________________________________________ (date ) March 12, 2012 *We also certify that written approval has been obtained for any proprietary material contained therein. i Dedication I dedicate this thesis to my wonderful daughter Lindsey and my mother Joyce. I know my mom’s example shaped my ability to raise such an extraordinary daughter. Lindsey, you are my inspiration and I love you to infinity. I share the success of earning a PhD with my family and Jeff, for I am convinced without their support and love it would not have been possible. ii Table of Contents Table of Contents .............................................................................................. iii List of Tables ..................................................................................................... vi List of Figures ................................................................................................... vii Acknowledgements ............................................................................................ 1 The Regulation of Tocopherol Transfer Protein .............................................. 4 Abstract ............................................................................................................... 4 Chapter 1 ............................................................................................................. 6 Literature Review................................................................................................ 6 1.1 Vitamin E ....................................................................................................... 6 1.1.1 Vitamin E Family.............................................................................................................. 6 1.1.2. Biosynthesis of vitamin E in plants .............................................................................. 8 1.1.3. Vitamin E absorption and intracellular transport ..................................................... 11 1.1.4. Vitamin E Selectivity .................................................................................................... 18 1.1.5. Vitamin E turnover .................................................................................................... 20 1.1.6. Antioxidant activity ....................................................................................................... 21 1.1.7. Vitamin E and Fertility ................................................................................................ 24 1.1.8. Non-antioxidant activity of vitamin E ......................................................................... 25 1.2 Vitamin E binding proteins ........................................................................ 26 1.2.1. Tocopherol Transfer Protein (TTP) identification and structure .......................... 26 1.2.2. TTP Function and mutations (AVED) ........................................................................ 29 1.2.3. Regulation of TtpA/ttpA transcript and protein ......................................................... 32 1.2.4. Other Vitamin E binding proteins: heart TBP, SPF, Sec14 ................................. 33 1.2.5. TTP and vitamin E in the central nervous system ................................................ 34 Statement of purpose ....................................................................................... 36 Chapter 2 ........................................................................................................... 37 Altered vitamin E Status in Niemann-Pick type C Disease ........................... 37 Abstract ..................................................................................................................................... 38 Introduction ............................................................................................................................... 39 Materials and Methods ........................................................................................................... 41 iii Results ...................................................................................................................................... 44 Discussion ................................................................................................................................ 52 Chapter 2 Figures ................................................................................................................... 56 Chapter 3 ........................................................................................................... 63 Transcriptional Regulation of the Alpha Tocopherol Transfer Protein in Hepatocytes ...................................................................................................... 63 Abstract: .................................................................................................................................... 63 Introduction ............................................................................................................................... 64 Materials and Methods ........................................................................................................... 66 Results ...................................................................................................................................... 70 Discussion ................................................................................................................................ 76 Chapter 3 Figures ................................................................................................................... 83 Chapter 4 ........................................................................................................... 92 Tocopherol transfer protein and vitamin E in the CNS ................................. 92 Abstract: .................................................................................................................................... 92 Introduction: ............................................................................................................................. 93 Results ...................................................................................................................................... 95 Discussion .............................................................................................................................. 103 Chapter 4 figures ................................................................................................................... 106 Materials and Methods ......................................................................................................... 113 Chapter 5 ......................................................................................................... 118 Vitamin E levels in mouse models of human disease ................................. 118 Abstract: .................................................................................................................................. 118 Introduction ............................................................................................................................. 119 Description of human disease and mouse models .......................................................... 121 _Toc318047841 Materials and Methods ......................................................................................................... 126 Results .................................................................................................................................... 129 Discussion .............................................................................................................................. 138 Chapter 6 ......................................................................................................... 145 Overall Summary and Future Directions ...................................................... 145 6.1 Overall Summary ............................................................................................................ 145 iv 6.2 Regulation of TTP/TtpA ................................................................................................. 147 6.2.1 Single Nucleotide Polymorphisms effect on TtpA regulation ............................ 149 6.3 Vitamin E status is altered in Niemann-Pick type C disease ................................... 151 6.3.1 Regulation of NPC1 and NPC2 levels ................................................................. 153 6.4 Localization of TTP and tocopherol in the CNS ......................................................... 154 6.5 Levels of vitamin E in animal models of human disease .......................................... 157 6.6. Concluding Remarks (in a nutshell) .....................................................