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A Chemical Proteomic Approach to Investigate Rab Prenylation in Living Systems

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A Chemical Proteomic Approach to Investigate Rab Prenylation in Living Systems A chemical proteomic approach to investigate Rab prenylation in living systems By Alexandra Fay Helen Berry A thesis submitted to Imperial College London in candidature for the degree of Doctor of Philosophy of Imperial College. Department of Chemistry Imperial College London Exhibition Road London SW7 2AZ August 2012 Declaration of Originality I, Alexandra Fay Helen Berry, hereby declare that this thesis, and all the work presented in it, is my own and that it has been generated by me as the result of my own original research, unless otherwise stated. 2 Abstract Protein prenylation is an important post-translational modification that occurs in all eukaryotes; defects in the prenylation machinery can lead to toxicity or pathogenesis. Prenylation is the modification of a protein with a farnesyl or geranylgeranyl isoprenoid, and it facilitates protein- membrane and protein-protein interactions. Proteins of the Ras superfamily of small GTPases are almost all prenylated and of these the Rab family of proteins forms the largest group. Rab proteins are geranylgeranylated with up to two geranylgeranyl groups by the enzyme Rab geranylgeranyltransferase (RGGT). Prenylation of Rabs allows them to locate to the correct intracellular membranes and carry out their roles in vesicle trafficking. Traditional methods for probing prenylation involve the use of tritiated geranylgeranyl pyrophosphate which is hazardous, has lengthy detection times, and is insufficiently sensitive. The work described in this thesis developed systems for labelling Rabs and other geranylgeranylated proteins using a technique known as tagging-by-substrate, enabling rapid analysis of defective Rab prenylation in cells and tissues. An azide analogue of the geranylgeranyl pyrophosphate substrate of RGGT (AzGGpp) was applied for in vitro prenylation of Rabs by recombinant enzyme. Alternatively, geranylgeranylated proteins (including Rabs) were labelled with AzGG via metabolic labelling of live cells with AzGGOH. Once Rabs were tagged with an azide moiety they could be labelled via a bioorthogonal ligation reaction with a trifunctional alkyne probe. The probe contained a fluorophore for in-gel fluorescence analysis and a biotin affinity label for affinity purification of labelled proteins. The conditions for protein tagging, labelling, affinity purification and LC-MS/MS analysis were optimised significantly during this work. Affinity purified proteins were identified and in some cases quantified using LC-MS/MS techniques, with iTRAQ labelling for quantification. Rab prenylation was probed in cell culture, in cells treated with the drug Mevastatin and in tissue from mouse models with defects in the prenylation machinery. 3 Acknowledgments I would like to thank everyone who has helped me over the course of my PhD. In particular I would like to give heartfelt thanks to my supervisor Ed Tate for his support, encouragement and input to my project and for giving me the opportunity to work in his group. Thank you also to the members of the Tate group and the Leatherbarrow group for being so friendly and making it such a good place to work. In the Tate group especial thanks to Will Heal for lots of helpful advice, initial capture reagent and for proof-reading and further thanks to Megan Wright for capture reagent, useful discussions and sharing of experiences with click chemistry. I would also like to thank my second supervisor Miguel Seabra for letting me work in his group and to the members of the Seabra group for being so helpful and friendly. In the Seabra group, particular thanks go to Raj Singh for the bone marrow, Elham Ostad-Saffari for the fibroblast cells and Marie Kirsten for recombinant protein and help with lab techniques. I would like to sincerely thank Tanya Tolmachova for the mouse samples, helpful advice and for proof reading. Last but not least of the Seabra group thank you to Abul Tarafder for being a great teacher at the start of my project and for helpful advice and discussions and for proof reading even though he had moved to Portugal, I really appreciate it. I am grateful to Andrew Bottrill at the University of Leicester for carrying out the trypsin digests and mass spectrometry for the proteomics experiments described in this thesis. For financial support I am grateful to the Institute of Chemical Biology who funded me through the EPSRC and provided lots of opportunities over the past few years. Outside of the lab, thanks to Richard, Sarah and Lucy for making 537 do delightful and to the reflections team of Lucy, Jemima, Arwen, John, Chirag and Rob for trips to the Holland Club and further afield. I would like to thank my parents for being so supportive, especially my Mum for not asking me too often if I had nearly finished. Finally, I want to thank Owen for all his support and for putting up with me through lab work and writing up, I doubt I could have finished without him. 4 Publications and Presentations Arising from this Thesis Publications Berry, A. F.; Heal, W. P.; Tarafder, A. K.; Tolmachova, T.; Baron, R. A.; Seabra, M. C.; Tate, E. W., Rapid Multilabel Detection of Geranylgeranylated Proteins by Using Bioorthogonal Ligation Chemistry. ChemBioChem 2010, 11, 771-773 Oral Presentations A. F. H. Berry et al., ‘A chemical proteomics approach to investigating Rab prenylation’, Postgraduate research symposium, Imperial College London, UK. (2011). A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, DTC conference, University of Warwick, UK. (2010) Poster presentations A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, Chemistry of the Cell, University of Oxford, UK. (2010) A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, Department of Chemistry Postgraduate Symposium, Imperial College London, UK. (2010). Winner of best poster prize (Chemical Biology) A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, 42nd IUPAC conference, Glasgow, UK. (2009) A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, FASEB Protein Lipidation and membrane domains conference, Vermont, USA. (2009) A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, DTC Conference, Imperial College, London, UK. (2009). Winner of best poster prize A. F. H. Berry et al., ‘Investigating Rab prenylation in Choroideremia: A chemical proteomics approach’, Biological and Medicinal Chemistry Symposium for Postgraduates, Chemistry Department of the University of Cambridge, UK. (2008) 5 Contents Abstract ................................................................................................................................................... 3 Acknowledgments ................................................................................................................................... 4 Publications and Presentations Arising from this Thesis ........................................................................ 5 Contents .................................................................................................................................................. 6 Abbreviations ........................................................................................................................................ 11 Chapter 1 Introduction ...................................................................................................................... 14 1.1 Ras superfamily of proteins .................................................................................................. 14 1.1.1 Rab proteins ................................................................................................................... 15 1.2 Protein prenylation ............................................................................................................... 23 1.2.1 Protein Lipidation ........................................................................................................... 23 1.2.2 Protein Prenylation ........................................................................................................ 25 1.2.3 Protein Prenyltransferases ............................................................................................. 26 1.2.4 RGGT and REP ................................................................................................................ 31 1.2.5 REP and RabGDI ............................................................................................................. 33 1.2.6 Post-prenylation modifications ...................................................................................... 35 1.2.7 Rab Prenylation and Disease .......................................................................................... 36 1.3 Chemical proteomics ............................................................................................................ 42 1.3.1 Bioorthogonal Ligation ................................................................................................... 42 1.3.2 Tagging-by-substrate ..................................................................................................... 45 1.3.3 Methods used to detect protein prenylation ................................................................ 47 1.4 Mass spectrometry-based proteomics ................................................................................
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