Signalling Functions of Polyubiquitin Chains and Ubiquitin- binding Domains Shengkai Zhao University College London and Cancer Research UK London Research Institute PhD Supervisor: Dr. Helle D Ulrich A thesis submitted for the degree of Doctor of Philosophy University College London September 2010 Declaration I, Shengkai Zhao, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The ubiquitin signalling system has been shown to regulate many important biological events, ranging from DNA repair to the immune response. Different polyubiquitin chains linked by various linkages have been identified in vivo, and can be recognised by proteins containing ubiquitin-binding domains that act as downstream effectors. However, functions for many of them are not well understood. I have studied the function of K63-linked and linear polyubiquitin chains on a common substrate. The other branch of my study was to investigate the role of ubiquitin binding for a novel ubiquitin-interacting protein, SPC25. K63-linked and linear polyubiquitin chains have a similar topology, but whether they convey a similar signal in vivo remains unclear. I have used the eukaryotic replication clamp PCNA, a natural substrate of K63-linked polyubiquitylation, as a model substrate to directly compare the consequences of modification by different types of polyubiquitin chains. I have shown that K63-polyubiquitylated PCNA is not subject to proteasomal degradation. In contrast, linear, non-cleavable ubiquitin chains do not promote DNA damage tolerance, but function as general degradation signals. I found that a linear tetraubiquitin chain is sufficient to afford proteasomal targeting through the Cdc48-Npl4-Ufd1 complex without further modification. In the second part of my thesis, I describe the identification of SPC25, a subunit of the Ndc80 complex, as a novel ubiquitin-binding protein, using tetra-ubiquitin chains as baits in a genome-wide two-hybrid screen. I have shown that the C-terminal region of SPC25 interacts with ubiquitin in vivo and in vitro. This region does not exhibit significant similarity with any known ubiquitin-binding domains. Further genetic evidence suggests that this ubiquitin-binding domain contributes to the stability of the kinetochore complex. 3 Acknowledgement First and foremost I would like to thank my supervisor Helle Ulrich. It has been a great experience for being her student. She taught me the way science should be. She guided me all the way through my projects, from the big questions to the little technical details. I appreciate her continuous support on my projects and allowing me to pursue many interesting ideas. She was always available for answering my questions and giving countless brilliant suggestions. She was also very patient with my unprofessional English, carefully correcting my reports, meeting abstracts, posters and the PhD thesis, thanks, Helle. I also would like to thank my thesis committee Jesper Svejstrup and Simon Boulton for their constructive suggestions, generous support with lab reagents and continuous encouragement during the progress of the projects. Thanks give to all current and past Ulrich lab members for creating a friendly and organised working environment. Without all of you, I would not have such enjoyable lab experience in the past four years. Special thanks go to Irene, Jo and Addie for teaching me many techniques and sharing their valuable scientific experiences; Thank you Nicola for your constructive ideas and helpful discussions; Thank you Yasu for the good time while we were waiting for 84. Thank you Andrea, Diana, Oliver, Olivier, Laure, Liz, Magda and other people who have worked in D36. I can not forget my friends in Clare Hall, specially Ling, Feng, Namita and Ozan, the admin office, mass spectrometry and other Cancer Research UK-LRI services for their kind help during my PhD. Special thanks go to Sally and Erin for organising various student events and making the life of an international student much easier. Of course, I want to thank Cancer Research UK for funding the research and providing generous studentship. Last but not least, I would like to thank my mother Xiaomin Wang and my father Jianying Zhao for their endless love and continuous encouragement on my study since I was a child. Sorry for being far away from both of you in the past few years. I want to thank my girl friend Luo, for her love, being with me, giving me support on daily life, bringing me a lot of laugh during the time of thesis writing and telling me that life is more than just science. 4 Table of Contents Abstract ..................................................................................................................................... 3 Acknowledgement ................................................................................................................. 4 Table of Contents .................................................................................................................... 5 Table of figures .................................................................................................................... 12 List of tables .......................................................................................................................... 14 Abbreviations ....................................................................................................................... 15 Chapter 1. Introduction ................................................................................................ 20 1.1 Ubiquitylation .................................................................................................................. 20 1.1.1 Ubiquitin ...................................................................................................................................... 20 1.1.2 Biochemistry of Ubiquitylation .......................................................................................... 21 1.1.3 Ubiquitin Conjugation Enzymes......................................................................................... 22 1.1.4 Ubiquitin Deconjugation Enzymes .................................................................................... 24 1.2 Ubiquitin Signals ............................................................................................................. 24 1.2.1 Monoubiquitylation ................................................................................................................ 25 1.2.2 K48-linked Polyubiquitin Chains ....................................................................................... 26 1.2.3 K63-linked Polyubiquitin Chains ....................................................................................... 27 1.2.4 K11-linked Ubiquitin Chains ............................................................................................... 28 1.2.5 Other Lysine-linked Ubiquitin Chains ............................................................................. 30 5 1.2.6 Linear Ubiquitin Chains ......................................................................................................... 30 1.3 Ubiquitin-binding Domains ......................................................................................... 31 1.3.1 Ubiquitin Recognition by Different Types of UBDs .................................................... 32 1.3.2 Linkage-Specific Recognition of Polyubiquitin Chains by UBDs .......................... 34 1.4 The Proteasome-dependent Degradation Pathway ........................................... 36 1.4.1 The 26S Proteasome ............................................................................................................... 36 1.4.2 The N-end Rule and the UFD Pathway ............................................................................ 37 1.5 Proteolysis-Independent Functions of Ubiquitin Signalling ........................... 40 1.5.1 NF-κB Pathway.......................................................................................................................... 41 1.5.2 Apoptosis ..................................................................................................................................... 43 1.5.3 Genome Stability ...................................................................................................................... 43 1.6 The Aims of the Thesis .................................................................................................. 45 Chapter 2. Materials and Methods ............................................................................ 46 2.1 Strains ................................................................................................................................. 46 2.1.1 Yeast Strains ............................................................................................................................... 46 2.1.2 E.coli strains ............................................................................................................................... 46 2.2 Plasmids ............................................................................................................................. 46 2.2.1 List of Plasmids ......................................................................................................................... 46 2.2.2 Construction of Linear Fusions of Ubiquitin to PCNA ............................................... 46 2.2.3