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The Role of Hedgehog Acyltransferase in Sonic Hedgehog Signalling

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The Role of Hedgehog Acyltransferase in Sonic Hedgehog Signalling The role of Hedgehog acyltransferase in Sonic hedgehog signalling Biljana Jovanović Thesis submitted for the degree of Doctor of Philosophy 29 March 2012 National Heart and Lung Institute Faculty of Medicine Imperial College London Abstract Protein acyltransferases (PATs), a group of enzymes catalysing fatty acylation of a variety of proteins, play an important role in protein sorting and signalling in the cell. In the Hedgehog (Hh) signalling pathway the PAT Hedgehog acyltransferase (Hhat) is responsible for addition of a palmitic acid to the N-terminal cysteine of Hh proteins. This modification is necessary for proper packaging and release of Hh ligands from the signalling cell and for transmitting a potent signal to the receiving cell. Hh signalling regulates cell growth and differentiation. Improper activation of the Hh signalling pathway in cancer arises either through an upregulation of ligand-dependent autocrine or paracrine signalling, or oncogenic mutations in the signal receiving cell. To date, drug therapies targeting the pathway in cancer have been directed at pathway regulators downstream of ligand binding. In this thesis, I investigate the potential of Hhat as a therapeutic target in lung cancer cell lines that express Sonic hedgehog (Shh). Studies of lung cancer cell lines A549, H209 and H82 treated with Shh pathway inhibitors showed no autocrine dependence on Shh signalling. Nonetheless, A549 cells were capable of inducing a Shh signal response in C3H10T½ cells in co-culture in a juxtacrine/paracrine fashion indicating that Shh in lung cancer cells may be involved in juxtacrine/paracrine signalling to the surrounding stroma. This effect was effectively attenuated by siRNA-mediated Hhat knock-down confirming the potential of Hhat as therapeutic target in cancer. I further provide a membrane topology model of Hhat which will aid in understanding the molecular mechanism of this enzyme. Membrane topology determination resulted in a topology model where Hhat consists of 11 transmembrane regions with the signal peptide preserved in the mature protein. Surprisingly, the suggested catalytic site, a conserved His, is localised in a transmembrane region near the cytosol away from the site of Hh palmitoylation which occurs in the lumen of the endoplasmic reticulum. Taken together, my results provide a firm basis for further investigation of the mechanism of action of Hhat and its role as a potential drug target in multiple human cancers. 3 4 Statement of originality I hereby certify that the work presented in this thesis is entirely my own unless otherwise stated. Work done by others is clearly acknowledged and referenced. The material has not previously been submitted for any other thesis or degree at this or any other university. Biljana Jovanović 29 March 2012 5 6 Acknowledgements First and foremost, I would like to thank my supervisor Prof. Tony Magee for giving me the chance to do my PhD in his group. Thank you for your guidance and for gladly sharing you vast knowledge in science. Apart from being an exceptionally kind and fair boss, you showed great trust in me and provided invaluable support when work was not going well. You are truly a scientist and a person to look up too. I would also like to thank my secondary supervisor Dr. Birgit Leitinger for helping along the way by providing objective viewpoints of ongoing experimental work and the written thesis, Dr. Ed Tate and members of his group for good support during our collaboration, and Dr. Martin Spitaler and Mark Scott for their help with imaging at the FILM imaging facility. I thank the National Heart and Lung Institute Foundation for financial support. I am further grateful to all members of the Magee lab, especially Dr. Marek Cebecauer, Dr. Antonio Konitsiotis and Anna Markiewicz, for fruitful scientific (and other) discussions and for their readiness to help with practical work and to engage into my scientific problems. Also, many thanks to Marek, Anna and Martin for countless pub sessions which added new dimensions to our conversation topics and to Anna for being a wonderful friend and a never-failing lunch companion. I would also like to thank people in my row in Sir Alexander Fleming building, especially Amy Lewis, Sara McSweeney, Antonia Solomon and Dede Lori, who helped make my PhD a joyful experience. Thanks Amy and Dede for your company in evenings and weekends making me feel normal for staying late and for explaining the wonders of English culture. Also, thanks to Amy and Jessica McCormack for fantastic Monday football sessions. My time in London was made unforgettable by friends in and outside Imperial College. I would specially like to thank Marie Kirsten for being a wonderful friend and running buddy. I am deeply indebted to Frida Einarsson who provided me with the best flat I could ever ask for and to Marie and Minttu Rönn for being lovely flatmates. This experience would not have been possible without the constant support and encouragement from my family. Hvala vam mama i tata za slobodu i podršku što ste mi 7 pružili u toku ovih proteklih godina, i pouzdanju i ponosu što mi pokazujete. Bez vašeg načina odgojanja, truda i borbe, niti bi ja imala priliku da ovo uradim, niti bi bila sposobna. Takođe, hvala ti Jaco na setrinskoj podršci, što si jaka osoba i što tako ohrabruješ sviju oko sebe. And last, but certainly not least, I wish to thank Björn Norrman, my best friend and partner, for his unwavering support throughout these years abroad, for following me to England and ending up going to the other side of the world, and for spending innumerous hours at airports later on to get to where I am. 8 Table of contents Abstract ........................................................................................................................... 3 Statement of originality .................................................................................................... 5 Acknowledgements .......................................................................................................... 7 Table of contents .............................................................................................................. 9 List of abbreviations ....................................................................................................... 17 List of figures .................................................................................................................. 21 List of tables ................................................................................................................... 25 1 Introduction ............................................................................................................ 27 1.1 Hh signalling .............................................................................................................. 28 1.1.1 Hh in development and homeostasis ................................................................ 28 1.1.2 Hh signalling in disease ...................................................................................... 30 1.1.3 The Hh signalling pathway ................................................................................. 30 1.1.3.1 Hh expression and processing .................................................................... 30 1.1.3.2 The role of palmitoylation and cholesteroylation of Hh proteins .............. 32 1.1.3.3 Hh secretion and gradient formation ......................................................... 34 1.1.3.4 Hh signal transduction ................................................................................ 37 1.1.3.5 Hh signal target genes ................................................................................ 39 1.1.4 Hh signalling in cancer ....................................................................................... 40 1.1.4.1 Modes of Hh signalling in cancer ................................................................ 40 1.1.4.2 Oncogenic mutations in the Hh pathway ................................................... 42 1.1.4.3 Overexpression of Hh ligands ..................................................................... 43 1.1.4.4 The cancer stem cell ................................................................................... 44 1.1.5 Hh signalling in lung cancer ............................................................................... 44 9 1.1.5.1 Lung cancer incidence and aetiology ......................................................... 44 1.1.5.2 Hh signalling in lung cancer ........................................................................ 45 1.1.6 Small-molecule inhibitors of Hh signalling ........................................................ 47 1.2 Protein palmitoylation .............................................................................................. 50 1.2.1 DHHC protein family .......................................................................................... 51 1.2.2 MBOAT protein family ....................................................................................... 54 1.2.3 Methods for detection of palmitoylation .......................................................... 57 1.2.4 Palmitoylation inhibitors .................................................................................... 59 1.3 Hypothesis and aims ................................................................................................. 62 2 Materials and methods ........................................................................................... 65 2.1
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