Participation of De Novo Sphingolipid Biosynthesis in the Regulation of Autophagy in Response to Diverse Agents

PARTICIPATION OF DE NOVO SPHINGOLIPID BIOSYNTHESIS IN THE REGULATION OF AUTOPHAGY IN RESPONSE TO DIVERSE AGENTS A dissertation Presented to The Academic Faculty by Kacee Hall Sims In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Chemistry & Biochemistry Georgia Institute of Technology December 2011 PARTICIPATION OF DE NOVO SPHINGOLIPID BIOSYNTHESIS IN THE REGULATION OF AUTOPHAGY IN RESPONSE TO DIVERSE AGENTS Approved by: Dr. Alfred H. Merrill, Jr., Advisor Dr. Adegboyega K. Oyelere Schools of Biology and Chemistry & School of Chemistry & Biochemistry Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Donald Doyle Dr. M. Cameron Sullards School of Chemistry & Biochemistry School of Chemisry & Biochemistry Georgia Institute of Technology Georgia Institute of Technology Dr. Christine Payne School of Chemistry & Biochemistry Georgia Institute of Technology Date Approved: October 24, 2011 See now the power of truth; the same experiment which at first glance seemed to show one thing, when more carefully examined, assures us of the contrary. -- Galileo Galilei (1565-1642) To my wonderful mother and husband. For believing in me and teaching me that even the largest task can be accomplished if it is done one step at a time. Every time I think of you, I give thanks to my God – Philippians 1:3 ACKNOWLEDGEMENTS First and foremost, I offer my sincerest gratitude to my adviser, Dr. Al Merrill, who has supported me throughout my research with his patience and knowledge whilst giving me the room to work in my own way. His continual and convincing spirit of adventure with regard to research has made my graduate school experience both productive and stimulating. During the tough times, his joy and enthusiasm for our science was both contagious and motivational. I will always be grateful for the lessons that I have learned throughout this journey, both at and away from bench. I would also like to express my thanks to my committee members: Dr. Cameron Sullards, Dr. Donald Doyle, Dr. Christine Payne, and Dr. Yomi Oyelere. Their time, interest, helpful comments, and insightful questions have been invaluable in the completion of this work. In my daily work, I have been blessed with a group of wonderful individuals in the Merrill lab who have been a source of friendship, as well as good advice and collaboration. I am especially grateful to Elaine Wang for being an unmatched fount of knowledge and for always sharing her expertise in order to make me not only a better scientist but also a better experimentalist. I would also like to thank other members of the Merrill Lab, both past and present, who have contributed to this work in some way: Samuel Kelly, Dr. Sibali Bandyopadhyay, Dr. Holly Symolon, Dr. Jeremy Allegood, Dr. Christopher Haynes, Dr. Amin Momin, Katie Shaw, and Wenjing Zheng. It has truly been a pleasure to work with each of you. To my wonderful family and friends, thank you for all of your endless love and support. I could not have done this without you. To my mother, thank you for being my v rock, sounding board, and number one fan. You have always been a constant source of encouragement and unconditional love to me on the good days and the bad days. For this, I will be forever indebted and grateful. And to my loving, supportive, encouraging, and patient husband, Jeremy, whose faithful support during the stages of this journey is more appreciated than words can adequately express. I am so fortunate to have both of you in my life to appreciate my laughter, to dry my tears, and most of all, to help me understand and accept life’s ups and downs. To my father-in-law, Jerry, thank you for being a constant example that although there is no substitute for hard work and humility the rewards are worth it every time. I am beyond blessed to have you in my life. To my aunt, Susan, thank you for all of your prayers and continual interest in my studies. It is so nice to know that wherever life takes me, I will always have you a prayer warrior and cheerleader in you. To my amazing friends, you have each inspired me in some way, and I can’t imagine this journey without you. Finally, it would be remiss if I did not mention my sweet, loving, faithful companion, Stanley James Sims, my miniature dachshund, who brings more joy to my life than can be imagined. Thank you for allowing me to slow down and enjoy life throughout this process. Last but certainly not least, I thank my omnipresent God, for answering my prayers for the strength to plod on despite sometimes wanting to give up. Thank you so much, Dear Lord. For without You, none of this would be possible. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS v LIST OF FIGURES xi LIST OF SYMBOLS AND ABBREVIATIONS xiv SUMMARY xvi CHAPTER 1. Sphingolipids: Structure, Synthesis, and Signaling 1 1.1 Structural Diversity of Sphingolipid Species 1 1.2 Synthesis of Sphingolipid Species 3 1.2.1 De novo Biosynthetic Pathway 3 1.2.2 Turnover Pathway 4 1.2.3 Subcellular Localization of Sphingolipid Biosynthesis 6 and Turnover 1.3 Signaling: Focus on Autophagic Pathway 9 1.3.1 Autophagic Process 10 1.3.2 Methods for Monitoring Autophagy 15 1.3.3 Autophagy: Relevance to Disease 17 1.3.4 Sphingolipids and Autophagy 20 1.4 Objective(s) 23 CHAPTER 2. Kdo2-Lipid A, a TLR4 Specific Agonist, Induces de novo 25 Sphingolipid Biosynthesis, which is Essential for the Induction of Autophagy 2.1 Introduction 25 2.2 Experimental Procedures 26 2.2.1 Materials 26 2.2.2 Cells and Cell Culture 27 2.2.3 Lipid Extraction and Analysis by LC-ESI MS/MS 28 2.2.4 Stable Isotope Labeling of Sphingolipids 28 2.2.5 Confocal Immunofluorescence Microscopy 29 2.2.6 Generation of RAW264.7 Cells Stably Expressing GFP-LC3 29 vii 2.2.7 Immunohistochemical Analysis of the ER and Golgi 30 2.2.8 Immunohistochemical Analysis of Ceramide Localization 31 2.2.9 Measurement of Cell Diameter and Area 31 2.2.10 Analysis of Autophagy in CHO-LYB and CHO-LYB-LCB1 32 Cells 2.3 Results 32 2.3.1 KLA Induces Substantial Increases in the Amounts of 35 Multiple Cellular Sphingolipids 2.3.2 Correlation of Gene Expression with Metabolite Changes in 42 KLA-Treated RAW264.7 Cells 2.3.3 Analysis of de novo Sphingolipid Biosynthesis in KLA- 45 Treated RAW264.7 Cells 2.3.4 Analysis of de novo Sphingolipid Biosynthesis in RAW264.7 48 Cells by [13C] Palmitate Labeling 2.3.5 KLA Inhibits Cell Growth and Increases the Size of 51 RAW264.7 Cells 2.3.6 KLA Induces Autophagy in RAW264.7 Cells, and 54 KLA-Induced Autophagy is Dependent on de novo Sphingolipid Biosynthesis 2.3.7 Confirmation that de novo Sphingolipid Biosynthesis is 59 Required for Autophagy using CHO-LYB Cells 2.3.8 Evidence that KLA Promotes Co-Localization of Ceramide 60 with Autophagosomes 2.4 Discussion 63 CHAPTER 3. Inhibition of Dihydroceramide Desaturase and Perturbation of 67 Cellular Sphingolipid Metabolism by 4-Hydroxypalmitanilide versus 4-Hydroxyphenylretinamide (Fenretinide): Implications for Modulation of Dihydroceramide Metabolism and Cell Function by Synthetic and Natural Compounds 3.1 Introduction 67 3.2 Experimental Procedures 68 3.2.1 Materials 68 3.2.2 Cells and Cell Culture 69 3.2.3 Synthesis of C6-NBD Dihydroceramide 69 3.2.4 In situ Dihydroceramide Desaturase Assay and Lipid 70 Extraction viii 3.2.5 In vitro Dihydroceramide Desaturase Assay 70 3.2.6 HPLC Analysis of NBD-Sphingolipids 71 3.2.7 Lipid Extraction and Analysis by LC ESI-MS/MS 71 3.2.8 Stable Isotope Labeling of Sphingolipids 72 3.2.9 Viability Studies 72 3.2.10 Western Blotting 73 3.3 Results 73 3.3.1 4-HPR Increases Dihydroceramide and other Metabolites 73 with the Sphinganine Backbone in Hek293 Cells 3.3.2 Direct Evidence for 4-HPR Inhibition of DES and C6-NBD- 76 Dihydroceramide Desaturation in Hek293 Cells 3.3.3 Does Elevated de novo Sphingolipid Biosynthesis Contribute 79 to Accumulation of Dihydrosphingolipids in 4-HPR-Treated Cells? 3.3.4 The Presence of the Retiniod Group is not Essential for 81 Inhibition of DES and Dihydrosphingolipid Elevation 3.3.5 The Presence of the Phenol Group is Essential for 86 Inhibition of DES and Dihydrosphingolipid Elevation 3.3.6 4-HPR and 4-HPA Differentially Affect de novo 89 Sphingolipid Biosynthesis 3.3.7 4-HPA Uncouples Dihydroceramide Elevation and Induction 93 of Autophagy from Cytotoxicty 3.4 Discussion 96 CHAPTER 4. Palmitate Enhances Fenretinide Induced Cell Death in MCF7 Breast 99 Cancer Cells 4.1 Introduction 99 4.2 Experimental Procedures 101 4.2.1 Materials 101 4.2.2 Cells and Cell Culture 101 4.2.3 Lipid Extraction and Analysis by LC-ESI MS/MS 101 4.2.4 Confocal Immunofluorescence Microscopy 102 4.2.5 Generation of MCF7 Cells Stably Expressing GFP-LC3 102 4.2.6 Viability Analysis 103 4.2.7 Western Blotting 104 ix 4.3 Results 104 4.3.1 4-HPR Induces Autophagic Cell Death in MCF7 Breast 104 Cancer Cells 4.3.2 4-HPR Increases Dihydrosphingolipids in MCF7 Breast 107 Cancer Cells 4.3.3 4-HPR-Induced Autophagy Requires de novo Sphingolipid 112 Biosynthesis 4.3.4 Palmitate, but not Oleate, Enhances 4-HPR-Induced Cell 114 Death 4.3.5 Effects of Palmitate and Oleate on 4-HPR-Induced 116 Alterations of Sphingolipid Metabolism 4.3.6 Effect of Palmitate and Oleate on 4-HPR-Induced 118 Autophagy and Autophagic Degradation 4.4 Discussion 120 CHAPTER 5.

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