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University of California, San Diego UNIVERSITY OF CALIFORNIA, SAN DIEGO The Mechanism of TAT-Mediated Cellular Transduction: Role of Glycans and Rab GTPases A dissertation in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Jacob Morris Gump Committee in charge: Professor Steven F. Dowdy, Chair Professor Jeffrey Esko Professor James Feramisco Professor Yitzhak Tor Professor Benjamin Yu 2009 Copyright © Jacob Morris Gump, 2009 All rights reserved. The Dissertation of Jacob Morris Gump is approved, and is acceptable in quality and form for publication on microfilm and electronically: ____________________________________________________________________ ____________________________________________________________________ ____________________________________________________________________ ____________________________________________________________________ ____________________________________________________________________ Chair UNIVERSITY OF CALIFORNIA, SAN DIEGO 2009 iii DEDICATION I dedicate this dissertation to my wife Meagan and my son Eli. Without your constant love and support, this would not have been possible. I love you both; you are the greatest joys of my life. iv EPIGRAPH "Science is a wonderful thing if one does not have to earn one's living at it." — Albert Einstein v TABLE OF CONTENTS SIGNATURE PAGE ………..……………………………………………………………….. III DEDICATION ………………..……………………………………………………………… IV EPIGRAPH ………………….……………………………………………………………… V TABLE OF CONTENTS …………………………………………………………………….. VI LIST OF ABBREVIATIONS ………………………………………………………………….. VIII LIST OF FIGURES …………………………………………………………………………. XI ACKNOWLEDGEMENTS …………………………………………………………………... XIII VITA ………………………………………………………………………………………. XV ABSTRACT OF THE DISSERTATION ….………………………………………….... ………XVIII CHAPTER 1: INTRODUCTION ……………………………………………………………… 1 ABSTRACT …..…………………………………………………………………… 1 HISTORY …..…………………………………………………….……………… 2 GLYCANS …………………….………………………………..……..……..…… 5 MACROPINOCYTOSIS ...…….…………………………………..……..………… 7 UPTAKE AND TRAFFICKING BY RAB GTPASES …….….……….……………… 11 BIOPHYSICS OF MEMBRANE TRANSLOCATION …..…….……….……………… 13 REFERENCES ..………………………………………………………………….. 16 CHAPTER 2: TAT TRANSDUCTION: THE MOLECULAR MECHANISM AND THERAPEUTIC PROSPECTS……………………………....……………….…………………..… 29 ABSTRACT …..…………………………………………………………………… 29 GENERAL INTRODUCTION TO MACROMOLECULAR THERAPEUTICS …………… 30 MECHANISM OF TAT-MEDIATED TRANSDUCTION INTO CELLS ………………… 32 THERAPEUTIC PROSPECTS …………………………………………..………… 41 CONCLUDING REMARKS ………………………………………………….…….. 45 GLOSSARY ………………………………………………………………………. 47 REFERENCES ……………………………………………………………………. 48 vi CHAPTER 3: REVISED ROLE FOR GLYCANS IN TAT-MEDIATED TRANSDUCTION…..…. 58 ABSTRACT…..………………………………………………………………….. 58 INTRODUCTION …………………………………………………………………. 59 EXPERIMENTAL PROCEDURES …………………………………………………. 62 RESULTS ………………………………………………………………………. 67 DISCUSSION …………………………………………………………………... 71 FIGURES ……………………………………………………………………….. 75 REFERENCES ………………………………………………………………….. 81 CHAPTER 4: TAT PROTEIN AND PEPTIDE TRANSDUCTION OCCURS BY ACTIVATING RAC1-DEPENDENT MEMBRANE RUFFLING AND RAB5 & RAB34-MEDIATED MACROPINOCYTOSIS .……………………………….…………………………. 86 ABSTRACT…..………………………………………………………………….. 86 INTRODUCTION ………………………………………………..……………….. 87 EXPERIMENTAL PROCEDURES ………………………………………………… 90 RESULTS ……………………………………………………………………… 96 DISCUSSION …………………………………………………………………….. 99 FIGURES ……………………………………………………………………….. 103 REFERENCES …………………………………………………………………. 108 CHAPTER 5: DISCUSSION ……………………………………………………………….. 114 REFERENCES …………………………………………………………………. 121 vii LIST OF ABBREVIATIONS Arf6 - Actin Remodeling Small GTPase ATP - Adenosine Triphosphate Bcl-XL - B-cell Lymphoma Extra-Large Protein CCD - Charge-Coupled Device CDC42 - Member of the Rho Family of Small GTPases CHO - Chinese Hamster Ovary Derived Cell Line CMV - Cytomegalovirus Promoter CPP - Cell Penetrating Peptide Cre - Cyclization Recombination Protein DNA - Deoxyribonucleic Acid EDTA - Ethylenediaminetetraacetic Acid EGFP - Enhanced Green Fluorescent Protein FACS - Fluorescent-Activated Cell Sorting FITC - Fluorescein Isothiocyanate FGF2 - Fibroblast Growth Factor 2 (Basic FGF) FSC - Forward Scatter GAG - Glycosaminoglycan GDNF - Glial Cell Line-Derived Neurotrophic Factor GTP - Guanosine Triphosphate GTPase - GTP Hydrolysing Enzyme HIV - Human Immunodeficiency Virus HPLC - High Performance Liquid Chromatography HS - Heparan Sulfate viii Hsp90 - Heat Shock Protein 90 kDa HSPG - Heparan Sulfate Proteoglycan kDa - Kilodalton loxP - Locus of X-Over P1 Recombination Site LTR - Long Terminal Repeat MPG - PTD from N-methylpurine DNA glycosylase Protein p53 - Tumor Protein 53 kDa Transcription Factor PAK1 - p21-Activated Kinase PBS - Phosphate Buffered Saline PDGF - Platelet-Derived Growth Factor PG - Proteoglycan PI3K - Phosphatidylinositide-3 Kinase PKC - Protein Kinase C PLC-γ - γ Phospholipase C Enzyme PMA - Phorbol 12-myristate 13-acetate PTD - Protein Transduction Domain R8 - Octaarginine Peptide (RRRRRRRR) Rab - Small GTPases with Roles in Endo & Exocytosis Rac - Subfamily of Rho GTPases Ras - Canonical Small GTPase RNA - Ribonucleic Acid RNAi - RNA Interference SA - Sialic Acid siRNA - Small Interfering RNA SOD - Superoxide Dismutase ix SPPS - Solid Phase Peptide Synthesis Src - Tyrosine Kinase (from Sarcoma) SSC - Side Scatter Tat - HIV Transactivator of Transcription Protein TAT - Transduction Domain from HIV Tat Protein TMR - Tetramethylrhodamine WGA - Wheat Germ Agglutinin x LIST OF FIGURES Chapter 3 Figure 3.1 TAT peptide cell association and cell surface binding in glycan- deficient cells ………………. ………………………………………….. 75 Figure 3.2 TAT fusion protein cell association and cell surface binding in glycan- deficient cells ………………. ………………………………………….. 76 Figure 3.3 TAT-Cre transduction occurs in the absence of glycosaminoglycans and sialic acids in CHO parental and glycan-deficient cell lines.…. 77 Figure 3.4 Macropinocytotic inhibitors and TAT-Cre transduction in glycan- deficient cells .………………………………………………………….. 78 Figure 3.5 TAT-induced macropinocytotic fluid-phase uptake is intact in glycan- deficient cells .…………………………………………………………... 79 Figure 3.6 Enzymatic depletion of heparan sulfate and sialic acids does not impair TAT transduction in parental or glycan-deficient cells despite efficient removal of glycans.…………………………………………………….. 80 Chapter 4 Figure 4.1 TAT PTD activates Rac1 and TAT transduction is Rac1-dependent ……………..…………………..………………………..……………….. 103 Figure 4.2 TAT PTD induces Rac1-dependent membrane ruffling and macropinocytosis …………………………………………………….. 104 Figure 4.3 GFP-Rab34 localizes to TAT-induced membrane ruffles and Rab34 Q111L expression alters TAT transduction and macropinocytosis.. 105 xi Figure 4.4 TAT PTD traffics through Rab5 endosomes and TAT transduction is altered in cells expressing Rab5 Q79L …...........................……….. 106 Figure 4.5 Rab5 Q79L expression impairs TAT induction of macropinocytosis with no effect on transferrin uptake …………………………….………….. 107 xii ACKNOWLEDGEMENTS First, I would like to thank my thesis advisor, Dr. Steven F. Dowdy for the opportunity to join his dynamic and consistently interesting lab. I thank Steve for the many scientific and life lessons learned in his lab. Among these are the value of positive thinking especially with respect to your project and the value of “just doing” an experiment you are positive won’t work. I also thank Steve for his generous support, especially in hard times, I am greatly indebted. I would also like to thank the current and former members of the Dowdy lab. Especially all the β side members who have helped, supported, pretended to listen and drank beer with me when appropriate. In particular, I thank Bryan Meade and Gary Shapiro for thoughtful arguments and discussions on music, the existence of free will, Bush, lab gossip and occasionally our work. Their scientific and personal insights will remain with me forever. Other β’s of note who helped guide my scientific maturation are Aki, Hiro (not tight pants), Scott, Donger and Britta. Thanks to all. Thanks to Gina and Leanne in the BMS office who help take the BS out of BMS. I have enjoyed the fun times we have had together (especially at the retreat!). Their help from beginning to end has been invaluable. Thanks to Baja, our weekend sojourns to her beautiful and rugged coast brought fiery adventure and renewed vigor. I thank Meagan, my wife and the love of my life, for the sacrifices she made to support my graduate career. I know it was not easy for her to postpone her dreams so that I could be miserable for 6 years. In return, I have given her my first born son. A special thanks to my parents and my sister for their love and support, they made me who I am. xiii The text of Chapter 2 is a reprint of the material as it appears in Trends in Molecular Medicine, 2007, Vol 13, No 10, 443-448. Jacob M. Gump and Steven F. Dowdy. The dissertation author was the primary author of this article. Chapter 3, in part has been submitted for publication of the material. Jacob M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material. Chapter 4, in part is currently being prepared for submission for publication of the material. Jacob M. Gump and Steven F. Dowdy. The dissertation author was the primary investigator and author of this material. xiv VITA EDUCATION: 2002-2009 Ph.D., Biomedical Sciences Program Department of Cellular and Molecular Medicine, University of California San Diego, School of Medicine, La Jolla, CA. Graduate Advisor: Professor
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