Hussain , Mushtaq (2013) Phylogenomic and structure-function relationship studies of proteins involved in EBV associated oncogenesis. PhD thesis. http://theses.gla.ac.uk/5357/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten:Theses http://theses.gla.ac.uk/ [email protected] Phylogenomic and Structure-Function Relationship Studies of Proteins Involved in EBV Associated Oncogenesis by Mushtaq Hussain The research presented in this thesis was sponsored by Dow University of Health Sciences, Karachi, Pakistan and Higher Education Commission, Pakistan and is author’s original work unless otherwise stated. Mushtaq Hussain Mushtaq Hussain, 2013 2 In the memory of my Paternal Grand Father and Maternal Grand Mother Wali Bhai Dadu Bhai Hawan Ji Bai Chand Junagadh Wala (Late) Ibrahim (Late) Mushtaq Hussain, 2013 3 Abstract This study covers the investigation of evolutionary and structure-function relationship aspects of several cancer related proteins. One part of the study deals with the investigation of a critical protein of Epstein-Barr Virus (EBV) the Nuclear Antigen 1 (EBNA1), and its interactions with different host proteins. One of these host proteins is a member of a large gene family, encoding ubiquitin specific proteases (USP), known as USP7. The second section of the thesis deals with the molecular evolution of the USP gene family. Another set of cellular proteins deregulated during EBV associated oncogenesis are members of the glycoside hydrolase (GH18) family. Their phylogenetic relationships and protein structures were investigated in the third section of this thesis. EBNA1 is the only EBV protein that consistently expressed in all latent forms of the EBV infections. The protein is involved in the genome maintenance and a substantial body of evidence suggests that it has a role in EBV associated oncogenesis. In this study, full length molecular models of the EBNA1 protein were generated using the programmes, I- TASSER, MOE and Modeller. The best models were selected on the basis of plausibility in structural and thermodynamical parameters and from this models of EBNA1 homologues of primates lymphocryptoviruses (LCVs) were generated. The C-terminal DNA binding and homodimerisation domain was predicted to be structurally similar between different LCV EBNA1 homologues, indicative of functional conservation. The central glycine alanine repeat (GAr) domain was predicted to be primarily composed of α helices, while almost all of the protein interaction region was found to be unstructured, irrespective of the prediction approach used and sequence origin. Predicted USP7 and Casein kinase 2 (CK2) binding sites and GAr were observed in the EBNA1 homologues of Old World primate LCVs, but not in the marmoset homologue suggesting the co-evolution of both these sites. Dimer conformations of the EBNA1 monomer models were constructed using SymmDock, where the C-terminal tail was predicted to wrap around the proline rich loop of another monomer, possibly contributing to dimer stability. This feature could be exploited in therapeutic design, hence an inhibitor peptide was designed and a preliminary evaluation was conducted to explore its ability to inhibit EBNA1 function in cell survival. The peptide array libraries of EBNA1 were used to investigate the binding regions and critical contact points between EBNA1 and partner proteins. Human EBP2 and USP7 proteins were expressed in bacteria and probed on the EBNA1 array. The data confirm the previously known binding region for EBNA1-EBP2 and EBNA1-USP7 interactions. In addition further information was gained regarding the critical contact residues and the potential role of phosphorylation of serine residues of EBNA1 in its binding with EBP2 and USP7. Mushtaq Hussain, 2013 4 The human genome encodes nearly 100 USPs which contribute to regulate the turnover of cellular proteins. These homologues are divided into 16 paralogous groups, all sharing a characteristic peptidase C19 domain. Evolutionary relationships between these homologues were explored by datamining and the phylogenetic reconstruction of peptidase C19 domain sequences. The data reveal an ancient relationship between the genes, with expansion occurring throughout the course of evolution, but particularly at the base of the vertebrates, at the time of the two whole genome duplications. A comparison between the phylogenetic architecture and protein interaction networks suggests the parallel emergence of many molecular pathways and the associated USPs. The GH18 gene family includes chitinases and related non catalytic proteins. Most mammals encode at least three chitinases (CHIT1, CHIA/AMCase and CTBS), as well as several homologues encoding catalytically inactive chitinase-like proteins or chilectins. Phylogenomic analysis shows that the family has undergone extensive expansion, initiating with a duplication event at the root of the vertebrate tree, resulting in the origin of the ancestors of CHIT1 and CHIA. Two further duplications of ancestral CHIA predate the divergence of bony fishes, one leading to a newly identified paralogous group (we have termed CHIO). In tetrapods, additional CHIA duplications predate and postdate the amphibian/mammalian split and relics of some exist as pseudogenes in the human genome. Homology modelling of structurally unresolved GH18 homologues in mouse and human was conducted using Modeller and I-TASSER. All resolved and predicted structures share a TIM barrel (β/α)8 and α+β domain. A central ligand binding cavity was also found in all GH18 homologues. The variation in size and shape of different paralogous proteins, indicate the difference in their ligands specificity and in turn potential functions. Mushtaq Hussain, 2013 5 Acknowledgements The present work would not be possible without the invaluable support of many individuals. Firstly, I would like to express my deepest gratitude to my supervisor Dr Joanna Beatrice Wilson for her perpetual extension of ideas, feedbacks and freedom I was given during my studies. She has all the attributes to sculpt a heavily dependent student like me into an independent researcher. Her immediate recognition and understanding of my interests and strengths allows me to switch from a immunology based project to a bioinformatics based research. Her artistic skills, as evident from her paintings and interior decoration of her house and garden, came very handy in beautifying many of the figures in this thesis and associated publications. It would be impossible to imagine the shape of the present thesis without her extensive help in editing, proofreading and more importantly filtering out the scientific errors. In short, it will be my honour to take my experience with her, as a role model for supervision in the impending future. Her thanks could not be complete without mentioning the regular supply of homemade chilly pickle (from home grown chillies), lemon tea (when I catch a cold) and coffee during my late stays in the lab. And of course, trips to Loch Lomond and “A play A pie and A pint” at Oran Mor with her are some of the most cherishable memories of mine. Finally, my experience with her as a demonstrator provided me an opportunity to learn skills to engage students especially with reference to critical thinking. My sincere thanks are due to Prof. Dr. George Baillie, Ruth MacLeod and Ryan Cameron for manufacturing the peptide arrays and providing the expression vectors. I am also grateful to Dr. Mark Bailey for his ever present help in conducting the phylogenetic studies. I always found his joyful and constructive criticism immensely helpful in undertaking phylogenomic studies. Dr. Derek Gatherer is a real help in constructing the molecular models of EBNA1 and in inferring the results pragmatically. I would like to extend my thanks to Donald Campbell, who is beyond any doubt the most resourceful person when it comes to lab issues. His constant supply of documentaries, movies and music made my weekends informative and entertaining. Without him I would not be able to understand dynamics of World War II and the events revolving around it. The friendship with him is the treasure which I will always relish. I am very thankful to all the lab members of Lab412 especially Jin (for generating some of the array results) and Xiao Gao. This acknowledgement would not be complete without mentioning, my lab colleague Saeed Al-Ghamdi (dubbed The King; Ya Marhaba) from the Kingdom of Saudia Arabia. His generous and brotherly help is one of the biggest reason I was able to complete this study. My trip to Loch Tay, Rosslyn Chapel and many other places in UK are only Mushtaq Hussain, 2013 6 become possible because of him. In short, wherever I need, I had found him amongst the first to support. I cannot miss mentioning my dearest friend Dr. Nusrat Jabeen for being always remain in touch. Keeping me informed about the folks in Pakistan and independently sorting out many of the formalities for me in Pakistan. To me, the friendship with Nusrat is an example of unconditional sincerity. My thanks is also due to the Higher Education Commission, Govt. of Pakistan and Dow University of Health Sciences, Pakistan for sponsoring the higher studies of mine and many other of my country fellows despite severe economical constraints. Being first time abroad, it is not easy for me and for my family, I had left behind in Pakistan. I am indebted to my family members, Muhammad Hussain (Dad), Mumtaz Hussain (Mom), Dr. Saba Hussain (Sis) and Zohaib Hussain (Bro) for taking care of my wife and kids and enduring my absence from the home.