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Regulation of Inositol Biosynthesis and Cellular Consequences of Inositol Depletion: Implications for the Echm Anism of Action of Valproate Rania M

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Regulation of Inositol Biosynthesis and Cellular Consequences of Inositol Depletion: Implications for the Echm Anism of Action of Valproate Rania M Wayne State University Wayne State University Dissertations 1-1-2014 Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The echM anism Of Action Of Valproate Rania M. Deranieh Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Biology Commons, Cell Biology Commons, and the Molecular Biology Commons Recommended Citation Deranieh, Rania M., "Regulation Of Inositol Biosynthesis And Cellular Consequences Of Inositol Depletion: Implications For The Mechanism Of Action Of Valproate" (2014). Wayne State University Dissertations. Paper 1045. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. REGULATION OF INOSITOL BIOSYNTHESIS AND CELLULAR CONSEQUENCES OF INOSITOL DEPLETION: IMPLICATIONS FOR THE MECHANISM OF ACTION OF VALPROATE by RANIA M. DERANIEH DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2014 MAJOR: BIOLOGICAL SCIENCES Approved by: _____________________________________ Advisor Date _________________________________________ _________________________________________ _________________________________________ _________________________________________ DEDICATION In memory of my beloved parents ii ACKNOWLEDGEMENTS My everlasting gratitude and appreciation to Dr. Miriam L. Greenberg, my mentor, advisor, teacher, and friend. Thank you, Miriam, for your unwavering support and encouragement, for each and everything you have taught me, and for each and every advice you have given me. You are a gem! I am lucky, and proud to be your student. Special thanks to my committee members, Dr. David Njus, Dr. Karen Beningo, Dr. Arun Anantharam, and Dr. Krishna Rao Maddipati. I thank you all for your continuous support and advice. You are a great committee! I thank the American- Jordanian Fulbright Commission for the Fulbright Scholarship, the Biological Sciences Department for funding, and the Graduate School for the University Graduate Research Fellowship. I thank our collaborators, Dr. Patricia Kane for her wonderful collaboration and many discussions, Dr. Marie Migaud, and Dr. Robin Williams. My thanks to Dr. Golenberg who constantly stood by me through various stages of ups and downs. I would also like to thank Dr. Phil Cunningham for all that he taught me during my first few years in his lab. Also, thank you to my lab-mates, past and present. I am grateful to friends and family for always being there, even from a distance. Shifra, Miriam, and Rebecca, thank you for being my family here and for all your warmth, support, and fun times. Aunt Ikram, you are an angel. Ali, you have been my solid rock and you have kept me sane! Rana, Muna, Laila, Hania, Dima, Steffi, Sophie, Micha, Peggy, Brad, Mechthild, Airlie, and Ricky, thank you all for your friendship and continuous support... I am blessed to have you all... Thank you! iii TABLE OF CONTENTS Dedication ............................................................................................................... ii Acknowledgements ................................................................................................ iii List of Tables ............................................................................................................ vi List of Figures ......................................................................................................... vii CHAPTER 1 - INTRODUCTION ................................................................................... 1 CHAPTER 2 - PHOSPHORYLATION OF MYO-INOSITOL PHOSPHATE 3-SYNTHASE: A NOVEL REGULATORY MECHANISM OF INOSITOL BIOSYNTHESIS Introduction ...................................................................................... 21 Materials and Methods ................................................................... 25 Results ............................................................................................. 35 Discussion ....................................................................................... 51 CHAPTER 3 - INOSITOL DEPLETION PERTURBS THE VACUOLAR-ATPase: A NOVEL MECHANISM OF ACTON OF VALPROATE Introduction ..................................................................................... 57 Materials and Methods .................................................................. 61 Results ............................................................................................ 66 Discussion ...................................................................................... 84 CHAPTER 4 - VALPROATE DECREASES ENDOCYTOSIS IN YEAST AND MAMMALIAN CELLS Introduction .................................................................................... 92 Materials and Methods .................................................................. 97 Results ........................................................................................... 103 iv Discussion ........................................................................................ 118 CHAPTER 5 – FUTURE DIRECTIONS ....................................................................... 125 References ............................................................................................................ 138 Abstract ................................................................................................................ 181 Autobiographical Statement ................................................................................ 184 v LIST OF TABLES Table 2.1 Strains used in Chapter 2 ....................................................................... 32 Table 2.2 Plasmids used in Chapter 2 .................................................................... 33 Table 2.3 Primers used for site-directed mutagenesis of yeast and human MIPS...34 Table 3.1 Strains used in Chapter 3 ........................................................................ 65 Table 4.1 Yeast strains and plasmids used in Chapter 4 ....................................... 102 Table 4.2 Genes leading to increased sensitivity to VPA when overexpressed .... 104 vi LIST OF FIGURES Figure 1.1 The inositol biosynthetic pathway ..........................................................4 Figure 2.1. MIPS catalyzes the rate-limiting step in inositol biosynthesis .............. 22 Figure 2.2 The tagged MIPS is functional and rescues growth of ino1Δ in inositol- deficient media ..................................................................... 36 Figure 2.3 MIPS is a phosphoprotein ..................................................................... 38 Figure 2.4 Domains and phosphosites of MIPS ...................................................... 39 Figure 2.5 MIPS is phosphorylated at 5 serine and threonine residues ................. 41 Figure 2.6 Effect of site mutations of yeast MIPS on growth and enzyme activity .................................................................................................... 44 Figure 2.7 Effect of dephosphorylation on MIPS activity ........................................ 47 Figure 2.8 Effect of site mutations of human MIPS on growth and enzyme activity .................................................................................................... 48 Figure 2.9 The double mutation (DM) confers a growth advantage, increases MIPS activity, and partially rescues sensitivity to VPA ........................... 50 Figure 3.1 The vacuolar ATPase ................................................................................. 60 Figure 3.2 Vacuolar defects in VPA-treated cells are rescued by supplementation with inositol .............................................................................................. 67 Figure 3.3 VPA inhibits V-ATPase activity and reduces coupling in WT cells ..............69 Figure 3.4 Inositol depletion inhibits V-ATPase activity and reduces coupling ..........71 Figure 3.5 VPA does not cause a vma- phenotype..................................................... 73 Figure 3.6 Inositol rescues sensitivity of V-ATPase V0 deletion mutants to VPA.........74 Figure 3.7 Cytosolic pH is not altered by VPA treatment........................................... 75 Figure 3.8 VPA alters the distribution of vacuolar PI3,5P2......................................... 78 vii Figure 3.9 VPA enhances vacuolar fusion................................................................ 79 Figure 3.10 Synthesis of PI3,5P2 is perturbed in VPA-treated cells. A, Levels of PI3,5P2 .................................................................................................... 81 Figure 3.11 Levels of PI3P are not altered by VPA..................................................... 83 Figure 3.12 Model: Inositol depletion perturbs the vacuolar V-ATPase.................... 91 Figure 4.1 VPA treatment phenocopies overexpression of AKL1........................... 106 Figure 4.2 Endocytosis is decreased in VPA-treated cells....................................... 107 Figure 4.3 VPA alters the trafficking of PI4P........................................................... 109 Figure 4.4 VPA causes cell wall defects and increases thermosensitivity............... 111 Figure 4.5 VPA perturbs the cell wall integrity pathway......................................... 113
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