PROTEIN INTERACTIONS AT THE INNER NUCLEAR MEMBRANE By QIAN LIU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Qian Liu 2 To my parents, for their unconditional love and support throughout the years 3 ACKNOWLEDGMENTS First and foremost, I would like to thank my mentor, Dr. Brian Burke, who has provided me with an unforgettable graduate experience. I have come to respect Brian’s brilliance as a scientist as well as his knowledge and understanding of things beyond. He also carries with him a great sense of humor, which makes my graduate life much enjoyable. I especially appreciate the immense patience he has exercised with me through the years and the support he has offered as an advisor. I would like to thank Kyle, for whom I have gained a great deal of respect and appreciation. He has been a remarkable teacher, leader and motivator in my graduate study. Moreover, he taught me a lot about knowledge of life and American culture. It is always fun to talk with him and I have learned so much. I would also like to thank my committee members (Steve Sugrue, John Aris and Jorg Bungert) for offering their expertise, suggestions and support through my graduate experience. I would especially like to acknowledge to Dr. Sugrue, who has served as an exceptional chair to our department and challenged each of us through his vision and leadership. I would like to thank the Sugrue, LuValle and Sarosi labs for constant exchange and use of equipment and reagents as well as many hours of valuable discussion. I am grateful for all the support and guidance I have received from everyone in the open lab, especially Daein (for being a good friend and coworker), Janet (for teaching me American culture and pushing my limit for better), Cuc (for being a considerate and inspiring friend who offers generous help), Caitlin (for constant encouragement and support), Connie (for offering me help scientifically and socially), Mo (for being a good friend in and out of the lab), Gus (for his wonderful friendship), Johnny (for his help and constant vocal entertainment in the lab), Deby (for always being helpful in my 4 research), Lora, Jeff, Dustin Yong, Julie, Todd (for his computer assistance), Lynda (for always keeping things in order), Kim, Mary, PJ and also Susan Gardner. Final thanks go to my close friends and family. I thank Rui, for her undying support and friendship from the other other end of the earth. I thank Qiong, for always being there for me, listening and giving me suggestions. I thank Shangli, Xiaolei, Zhiqun, for their amazing friendship. To Qing, Jian, Wei, Yao, Xin, Ye, for making my life warmer and more fun. Most importantly, I cannot express gratitude enough for my parents, for always being there. I could never have come this far without them. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 LIST OF ABBREVIATIONS ........................................................................................................10 ABSTRACT ...................................................................................................................................14 CHAPTER 1 INTRODUCTION ..................................................................................................................17 Introduction .............................................................................................................................17 The Nuclear Envelope ............................................................................................................17 The Nuclear Pore Complexes .................................................................................................19 The Nuclear Lamina ...............................................................................................................20 Laminopathies .........................................................................................................................23 Hutchinson-Gilford Progeria Syndrome (HGPS) ...................................................................27 Inner Nuclear Membrane Proteins ..........................................................................................31 Nesprin Targeting and SUN Domain Proteins .......................................................................32 2 FUNCTIONAL ASSOCIATION OF SUN1 WITH NUCLEAR PORE COMPLEXES .......43 Abstract ...................................................................................................................................43 Introduction .............................................................................................................................43 Results .....................................................................................................................................47 Discussion ...............................................................................................................................56 Materials and Methods ...........................................................................................................61 3 DYNAMICS OF LAMINA PROCESSING: IMPLICATIONS OF LOPINAVIR AND FTI-277 IN THE TREATMENT OF HIV AND PROGERIA ...............................................77 Abstract ...................................................................................................................................77 Introduction .............................................................................................................................77 Results .....................................................................................................................................80 Discussion ...............................................................................................................................85 Materials and Methods ...........................................................................................................88 6 4 CONCLUSION .......................................................................................................................98 Overview of Findings .............................................................................................................98 Significance ..........................................................................................................................100 Sun1 Can Be Involved in Nuclear Pore Membrane (NPM) Curvature, de Novo NPC Assembly and Defining Distinct Regions of Nuclear Periphery ...............................100 The Mechanisms of Nuclear Lamina Assembly Has Implications in the Clinical Use of both HIV Protease Inhibitors and Farnesyltransferase Inhibitors ..................101 Future Work ..........................................................................................................................102 Functional Investigation of Sun1 ...................................................................................102 Withdrawl of Lop and FTI-277 on Animal Models ......................................................103 LIST OF REFERENCES .............................................................................................................105 BIOGRAPHICAL SKETCH .......................................................................................................125 7 LIST OF TABLES Table page 1-1 Properties of representative inner nuclear membrane proteins ..........................................42 8 LIST OF FIGURES Figure page 1-1 Overview of nuclear envelope organization in a eukaryotic interphase cell. ....................39 1-2 LaA Processing.. ................................................................................................................40 1-3 Model for the LINC complex. ...........................................................................................41 2-1 Mammalian SUN protein family.. .....................................................................................67 2-2 Sun1 and Sun2 are segregated within the plane of the NE. ...............................................68 2-3 Sun1, but not Sun2, is closely associated with NPCs as revealed by immuno-electron microscopy.. .......................................................................................................................69 2-4 Sun1 contains a single transmembrane domain.. ...............................................................70 2-5 The Sun1 nucleoplasmic domain has overlapping NE localization motifs. ......................72 2-6 Sun1 forms homotypic oligomers in vivo.. ........................................................................73 2-7 Sun1 association with NPCs requires both the nucleoplasmic and lumenal domains. ......74 2-8 Perturbation of Sun1 affects NPC distribution.. ................................................................75 2-9 Sun1 topology and interactions.. ........................................................................................76 3-1 HIV PIs block LaA maturation, which is reversed rapidly following PI removal. ...........91 3-2 Aberrant cellular phenotypes are recovered