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Characterization of the E3 Ubiquitin Ligase Siah2 As an Anti-Cancer Target

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Characterization of the E3 Ubiquitin Ligase Siah2 As an Anti-Cancer Target CHARACTERIZATION OF THE E3 UBIQUITIN LIGASE SIAH2 AS AN ANTI-CANCER TARGET ANUPRIYA GOPALSAMY M.Sc (Bioinformatics), University of Madras A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013 For my parents... DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Anupriya Gopalsamy 21 May 2014 i ACKNOWLEDGEMENTS First and foremost, I thank GOD for blessing me with privilege and much-needed strength to pursue this program. It is only with His grace that I have come so far in this journey of learning. I express my supreme gratitude to Prof. Kunchithapadam Swaminathan, Department of Biological sciences for kindly agreeing to be my supervisor when my former supervisor left. I feel profound privilege to thank Prof. Swaminathan for believing in me and took me under his wings to embark on the same project with different prospective. I am thankful to him for patiently tolerating my mistakes and politely pointing them out to me and, in the process, moulding me into a better researcher. I will be deeply indebted to him for all his support over the years. I express my heartfelt gratitude and thanks to Prof. Thilo Hagen, Department of Biochemistry for agreeing to be a collaborator and for hosting me in his lab, providing with all facilities and guidance. Prof. Hagen taught me several experimental techniques and also taught me how to think as a researcher. He also expertly showed me how to design experiments and communicate results. I want to thank all the wonderful people in his lab for their valuable discussions on my project. I extend my special thanks to Christine, Yee Liu and Shin Yee for voluntarily providing timely help in my experiments. I also express my appreciation and thanks to Dr. Lijun, Department of obstetrics and Gynaecology, who allowed me to share their lab facilities to carry out certain experiments. I thank Zhang Zhi Wei and Seok Eng for providing me the needful in their lab. I am grateful to my former supervisor Dr. Annamalai Loganath for his support and guidance for the first year of my candidature. I would like to extend my sincere thanks to my other supervisors Prof. Arijit Biswas and Prof. Yap Seng Chong of the department of Obstetrics and Gynaecology, who have given me financial, academic and technical support to pursue my research smoothly. ii A word of praise will not be sufficient to acknowledge Lilly, Liha and Apple for their timely help to solve all the administrative works. I am thankful to the all my present and former SBL4 labmates Roopa, Deepthi, Umar, Shani, Pavithra, Madhuri, Divya, Raghu, Jobi, Pankaj, Fengxia, Jikun, and Kanmani for their help as and when required. My Special thanks to Thomas, undergraduate student for his contribution to this work. I also would like to thank Prof. Sivaraman and his lab members (SBL5) for their help and valuable discussions on my project. A few people were not only my lab mates but also happened to be my closest friends during the course of my research. I would like to say “Thank you” to Roopa, (my close friend, senior and labmate) for always being there for me and shared most of the best moments in Singapore. She always provided a source of help in research, advice on life and living, and support during hard times. I would also take this opportunity to thank Lakshmi Akka (former colleague) for providing a great company and unforgettable friendship throughout the stay at NUS. I would like to thank Lalitha (my close friend and rooomate) for all the good times and also for listening to and letting me vent out all my thoughts, worries and frustrations. I want to thank my housemates Ambica, Deepthi, Divya, Lavanya, Lalitha and Roopa for all the fun, memorable times in Singapore and for their kind help and cooperation during this dissertation. I thank NUS for having given me the opportunity to pursue my Ph.D. with a research scholarship. Finally and most importantly, my indebtedness to my parents for their constant support for the choices I made and their sacrifice are beyond expression. I dedicate this thesis for them as a humble tribute to their love and blessings that has brought me to where I am now. iii TABLE OF CONTENTS DECLARATION ............................................................................................... i ACKNOWLEDGEMENTS ............................................................................ ii TABLE OF CONTENTS ............................................................................... iv SUMMARY ................................................................................................... viii LIST OF TABLES ........................................................................................... x LIST OF FIGURES ........................................................................................ xi LIST OF PUBLICATIONS ......................................................................... xiii LIST OF SYMBOLS AND ABBREVIATIONS ........................................ xiv CHAPTER 1. INTRODUCTION ................................................................... 1 1.1 Biological background ......................................................................... 1 1.1.1 The importance of regulated protein degradation .............................. 1 1.1.2 Ubiquitination and protein degradation ............................................. 1 1.1.3 E3 ubiquitin ligases ............................................................................ 4 1.1.4 The Siah family of proteins ................................................................ 5 1.1.5 Siah Structure ..................................................................................... 6 1.1.6 Diverse role and substrates of Siah .................................................... 8 1.1.7 Siah1 and cancer .............................................................................. 10 1.1.8 Role of Siah2 in cancer .................................................................... 10 1.1.8.1 Hypoxia signaling connected to Siah2 in cancer ..................... 12 1.1.8.2 RAS signaling connected to Siah2 in cancer ........................... 14 1.1.9 E3 ubiquitin ligases as a cancer target ............................................. 15 1.1.10 Protein-Protein Interactions as drug targets ..................................... 16 1.2 Drug Development ............................................................................. 17 1.2.1 Introduction to drug discovery ......................................................... 17 1.2.2 Stages in drug development and the role of in silico approaches .... 17 1.2.3 Computer-aided drug design (CADD) ............................................. 20 1.2.3.1 Homology modeling ................................................................ 21 1.2.3.2 Molecular dynamics simulation ............................................... 23 iv 1.2.3.3 Structure based drug design (SBDD) ....................................... 24 1.2.3.4 Active site identification ....................................................... 26 1.2.3.5 Virtual screening .................................................................. 26 1.2.3.6 Docking ................................................................................ 28 1.2.3.7 Scoring methods ................................................................... 30 1.2.3.8 Adsorption, distribution, metabolism and excretion (ADME) . .............................................................................................. 32 1.2.4 Successes and limitations of SBDD ................................................. 33 1.3 Protein crystallization and data collection ...................................... 35 Objectives ........................................................................................................ 36 CHAPTER 2. MATERIALS AND METHODS .......................................... 37 2.1 Homology modeling ........................................................................ 37 2.2 Validation of the modeled structure ................................................. 37 2.3 Protein simulation ............................................................................ 38 2.4 Docking ............................................................................................ 38 2.5 Virtual screening .............................................................................. 39 2.6 Cell lines and cell culture ................................................................. 39 2.7 Molecular cloning ............................................................................ 40 2.7.1 Human plasmid constructs ........................................................... 40 2.7.2 Bacterial expression plasmid constructs ...................................... 40 2.8 Transient transfection ....................................................................... 41 2.8.1 Plasmid transfection ..................................................................... 41 2.8.2 Small-interfering (siRNA) transfection ....................................... 41 2.9 Protein isolation and concentration determination........................... 42 2.10 SDS-PAGE .....................................................................................
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