This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Systems Biology Analyses of Hematopoietic Cells Milewicz, Hanna Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. 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Systems Biology Analyses of Hematopoietic Cells Hanna Jadwiga Milewicz A THESIS PRESENTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY KING’S COLLEGE LONDON 2012 Declaration I hereby declare that I alone composed this thesis and that the work is my own, except where stated otherwise. Hanna Milewicz September 2012 2 Acknowledgements Thank you to my primary supervisor Dr. Shaun Thomas for giving me the opportunity to work in your laboratory and for guiding me so well throughout this time. I am extremely grateful and indebted for the expertise, inspiration and for introducing me to the philosophy of ‘Systems Biology’. I also would like to thank Prof. Farzin Farzaneh for his ideas and guidance of my work and Prof. GJ Mufti for giving me the opportunity to work in the Department of Haematological Medicine. I would like to acknowledge our collaborators without whom this work would not be possible to complete. I would never have learnt so much without the opportunity to work within other laboratories: Prof. Tony N, Dr. Leo Carlin and all lab members for giving me the opportunity to learn advanced imaging techniques. Prof. Marcotte, Dr. Daniel Boutz and lab members for the training in proteomics and mass spectrometry. Dr.Dariusz Ladon for the help in cytogenetics. Dr. Paul Lavender and Dr. Audrey Kelly for the help in isolating chromatin. To Dr. Steve Orr, thanks for his advice and especially for the help in analysing proteomics data. To all my fellow colleagues in the Department of Haematological Medicine, thanks for being so supportive during my stay at the Institute. I would like to thank Leukaemia Lymphoma Research for funding my PhD studies. To my family, thank you for all the support and encouragement. 3 A mosaic consists of thousands of little stones. Some are blue, some are green, some are yellow, some are gold. When we bring our faces close to the mosaic, we can admire the beauty of each stone. But as we step back from it, we can see that all these little stones reveal to us a beautiful picture, telling a story none of these stones can tell by itself." -- Henri Nouwen 4 List of abbreviations ACN Acetonitrile APC antigen presenting cells APEX absolute protein expression BCR B cell receptor BSA bovine serum albumin C/NM chromatin & nuclear matrix CBP Calmodulin binding peptide Cdc42 cell division cycle 42 CFP cyan fluorescent protein ChIP chromatin immunoprecipitation CLL chronic lymphocytic leukaemia CMTMR 5-(and-6)-(((4-chloromethyl)benzoyl)amino)- tetramethylrhodamine Col Colcemid CRIB Cdc42/Rac-interactive binding CTL Cytotoxic T lymphocytes Cycs cytochrome C DetOut DetergentOut removal kit DH DBl homology DHR2 Dock-homology region-2 DSB double strand breaks DTT Dithiothreitol ESI electrospray ionisation F free F-actin filamentous actin FASP Filter-Aided Sample Preparation FC- combined Fold change combined FITC Fluorescein isothiocyanate FLIM Fluorescence Lifetime Imaging Microscopy FRET Förster Resonance Energy Transfer FSC forward scatter 5 GAP GTPase-activating protein GDI guanine nucleotide dissociation inhibitor GDP guanosine diphosphate GEF guanine exchange factor GEF guanine nucleotide exchange factor GFP green fluorescent protein GrB Granzyme B hnRNP heterogeneous nuclear ribonucleoprotein HPRD human protein reference database Hsp heat-shock protein HTS high-throughput-screening I-FISH Interphase FISH IAA Iodoacetamide IFN interferon IS immunological synapse iTRAQ isobaric Tags for Relative and Absolute Quantification LB Luria-Bertoni LC liquid chromatography LCMS/MS liquid chromatography tandem mass spectrometry LTQ LTQ-Orbitrap mass spectrometer LUMIER luminescence-based mammalian interactome mapping m/z mass to charge ratio mAb monoclonal antibody MALDI matrix assisted laser desorption ionisation Mat2A Methionine adenosyltransferase 2 A MHC major histocompatibility complex M.nase micrococcal nuclease MPM Multiphoton microscopy MS mass spectrometry MTOC microtubule organizing center Nap1L nucleosome assembly protein 1-like NChIP native chromatin immunoprecipitation NF κB nuclear factor kappa B NK cell natural killer cell 6 NKIS activating NK cell immunological synapse OMIM McKusick's Online Mendelian Inheritance in Man P pellet p-γH2AX phospho-γH2AX PAK1 p21-activayted kinase 1 PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline PH pleckstrin-homology PI Propodium Iodide PI3K1 p85 α subunit of phosphatidylinositol 3-kinase PMA Phorbol 12-myristate 13-acetate PMSF Phenylmethanesulfonylfluoride PP6 protein phosphatase 6 PRC pre-replication complex r distance between two fluorophores R0 Förster distance RFP red fluorescent protein Rpa3 replication protein 3 RPMI Rosswell Park Memorial Institute medium RT Reverse Transcriptase SD Standard deviation SILAC Stable Isotope Labeling with Amino acids in cell Culture SMAC supramolecular activation cluster SSC side scatter τ fluorescence lifetime TAP tandem affinity purification TCR T cell receptor TCSPC time-correlated single photon counting TFE Trifluorethanol Velos Velos-Orbitrap mass spectrometer w XL with formaldehyde crosslinking w/o XL without formaldehyde crosslinking Y2H yeast-two-hybrid 7 Abstract The focus of my thesis is to apply systems biology approaches to obtain a better understanding of complex cellular systems. In particular, my work concentrates on the function of haematological cells. The first part of the thesis applies a novel, predictive strategy to identify new regulators of a signalling pathway in human immune cells. Cdc42 is a membrane associated GTPase that is an important regulator of cytoskeleton rearrangements and is required for natural killer (NK) cell activation. Previous work had shown that Cdc42 activity oscillates during NK immune surveillance after an initial increase, suggesting that Cdc42 activity is regulated in NK cells by a number of signalling molecules. The aim of the project is to predict proteins required for Cdc42 activity in NK cells and test the predictions. Using a bioinformatics methodology, 13 different proteins were predicted to interact with Cdc42 and form feedback loops. To determine whether any of the predicted proteins were required for Cdc42 activity, NK cells were transfected with a Cdc42 biosensor, each of the predicted targets was downregulated with siRNA and Cdc42 activity was quantified by FRET/FLIM microscopy in the presence of target cells. The screen identified AKT1 and the p85 α subunit of PI3K as novel regulators of Cdc42 activity. Depletion of each of these targets also results in an impaired cellular cytotoxic response. This proof of principle study demonstrates the power of a predictive approach in the NK immune surveillance context to identify novel regulators of Cdc42. The second part of my thesis is based on using a predictive methodology, called the ‘Phenolog approach’ to predict novel proteins involved