Identification of Novel Ligands of Wdr47, Using Yeast Two-Hybrid Analysis
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IDENTIFICATION OF NOVEL LIGANDS OF WDR47, USING YEAST TWO-HYBRID ANALYSIS L. McGillewie Thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Medical Sciences (Medical Biochemistry) at Stellenbosch University. Promoter: Dr Craig Kinnear Co-promoter: Prof Johanna C. Moolman-Smook December 2009 DECLARATION By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the owner of the copyright thereof (unless to the extent explicitly otherwise stated) and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date………14 October 2009………………………. Copyright © 2009 Stellenbosch University All rights reserved ii ABSTRACT The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortex. Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders. iii OPSOMMING The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortx. Reelin is a large multidomain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, ie the Lis homology and the C-terminal Lis homology domain (CTLH) was used as ‘bait’ in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders. iv TABLE OF CONTENTS INDEX PAGE ACKNOWLEDGEMENTS vi LIST OF ABBREVIATIONS vii LIST OF FIGURES xi LIST OF TABLES xvi CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: MATERIALS AND METHODS 38 CHAPTER THREE: RESULTS 61 CHAPTER FOUR: DISCUSSION 84 APPENDIX I 103 APPENDIX II 111 APPENDIX III 114 APPENDIX IV 117 APPENDIX V 118 REFERENCES 122 v ACKNOWLEDGEMENTS I would like to express my sincere gratitude to the following people who have helped me reach for the stars in the past few years: My mentor, Dr Craig Kinnear, firstly thank you for your patience. You have always believed in me and stood by me through the good, the bad and the ugly. You have been an inspiration, not only as a brilliant scientist but as a person who I truly admire. In the last few years, you became my family away from home. Thank you for everything, I am honoured to have been your student. Professor JC Moolman-Smook, for all the guidance both in my scientific career and in my personal growth, and for always encouraging me to strive for nothing but the best. Mr Ben Loos, Department of Physiology (University of Stellenbosch), for the technical assistance and patience at the fluorescence microscope throughout the co-localisation assays. To everyone in the MAGIC lab, you have not only been colleagues but many of you have become friends I will cherish forever. Thank you for all the laughs, especially to Chrizette ‘Jimmy’ Uys. The NRF for giving me the funding and financial support to complete this project. My amazing family, I could not have wished for more. Dad (David McGillewie) thank you for giving me the opportunity to spread my wings and for always being there and supporting me (in every way), and for always being so understanding you truly are an inspiration... I love you pops. Mom (Helena McGillewie), you are my guardian angel, my pillar of strength, you have always believed in me against all the odds. Thank you for always giving me nothing but the very best, and for always loving me the way only a mother can... I love you. To my brother, Danetjie, you are my best friend, thank you for always being there no matter what, words cannot describe how much I love you. Lastly, ouma Joey, I hope I have made you proud... each day that goes by you are in my thoughts, I miss you so very much! Jaco Rossouw, you have been by my side every step of the way no matter how difficult things got. Thank you for your endless support and love, you really are my one in a million. Love you always... Lastly, and most importantly dear God: you have given me everything, the opportunity to become the best I can be, you have given me the most wonderful family... without you, I would not be where I am today. Thank you. vi LIST OF ABBREVIATIONS 3D Three-dimensional 5’-UTR Five prime untranslated region aa Amino acid AD Activation domain ADE2 Phosphoribosylaminoimidazole carboxylase gene Ade Adenine Amp Ampicillin ApoER2 Apolipoprotein E receptor 2 APS Ammonium persulphate ASD Autism spectrum disorders ATP Adenosine triphosphate BD Binding domain BLAST Basic local alignment search tool BLASTN Basic local alignment search tool (nucleotide) BLASTP Basic local alignment search tool (protein) bp Base pair BRET Bioluminescence resonance energy transfer cDNA Complementary DNA Cdk5 Cyclin dependant kinase 5 Cfu Colony forming units CGE Caudal ganglionic eminence CIAP Calf intestinal alkaline phosphatase CNR Cadherin-related neuronal receptors CP Cortical plate CR Cajal-Retzius cells CS Cockayne syndrome CTLH C-terminal to the Lis homology domain Cul5 Cullin 5 Cul7 Cullin 7 dATP Deoxy-adenosine triphospate dCTP Deoxy-cytidine triphosphate ddH2O Double distilled water dGTP Deoxy-guanosine triphosphate Dab1 Disabled-1 DCX Doublecortin DMSO Dimethyl sulphoxide vii DNA Deoxyribonucleic acid dNTP Deoxy-nucleotide triphosphate DTT 1,4-Dithiothreitol dTTP Deoxy-thymidine triphosphate E.coli Escherichia coli EDTA Ethylene-diamine-tetra-acetic acid EGF Epidermal growth factor FCD Focal cortical dysplasia GABA Gamma-aminobutyric acid GAD67 Glutamate decarboxylase 67 GE Ganglionic eminence GFP Green fluorescent protein GH Glycine-histidine GSK3β Glycogen synthase kinase 3 beta Guk1 Guanylate kinase 1 HCl Hydrochloric acid HIS3 Imidazoleglycerolphosphate dehydratase gene His Histidine HRM Heterozygous reeler mouse IZ Intermediate zone kb Kilo bases kDa Kilo Dalton