Establishment and characterisation of USP29 as a novel regulator of Hypoxia Inducible Factor α Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Amelie Schober December 2016 i ABSTRACT The hypoxia signalling pathway controls the adaptation of cells to decreased oxygen availability. When oxygen becomes limiting, the central transcription factor of the pathway, hypoxia-inducible factor (HIF), is activated and induces the expression of a set of genes, which cause a glycolytic switch, enhance cell survival and induce angiogenesis. While those are necessary physiological adaptive processes, they are also hallmarks of cancer and hypoxia signalling is often found activated in cancer. However, current knowledge about the regulation of the hypoxia signalling pathway is not able to satisfactorily explain the deregulation found in cancer. Therefore, the aim of this work was to discover new regulatory mechanisms that could be responsible for sustained hypoxia signalling and therefore might represent future therapeutic targets. We focused on how post-translational modifications affected the properties of the central proteins of the pathway. Using standard biochemical approaches and fluorescence lifetime imaging, we found that the ubiquitin specific protease 29 (USP29) is a deubiquitinase for HIF-1α. Moreover, USP29 is the first ubiquitin protease towards HIF-2α. USP29 de- conjugates poly-ubiquitin from HIF-α and prevents its proteasomal degradation, leading to its stabilisation even in normoxic conditions. Importantly, this action of USP29, while dependent on its catalytic activity, was not dependent on the classical prolyl-hydroxylation and pVHL- mediated ubiquitination, and therefore we propose that USP29 is a non-canonical HIF activator. As hardly any reports about USP29 were available, we next aimed to characterise this maternally imprinted protein. We found that USP29 itself was subject to poly-ubiquitin- mediated destabilisation, but was able to remove the ubiquitination itself and therefore auto- stabilise in a catalytic activity-dependent way. Furthermore, we demonstrated that USP29 formed homodimers in living cells. Finally, we found that USP29 mRNA levels correlated with disease progression and severity in prostate cancer and suggest that USP29 might become an attractive target for therapeutic targeting of hypoxia signalling in the future. ii Table of Contents TABLE OF CONTENTS Abstract .................................................................................................................................................... i Table of Contents .................................................................................................................................... ii Abbreviations ......................................................................................................................................... vi Chapter 1: Introduction ........................................................................................................................... 1 1.1 Hypoxia signalling ........................................................................................................................ 1 1.1.1 Oxygen .................................................................................................................................. 1 1.1.2 Discovery of the hypoxia signalling pathway ....................................................................... 1 1.1.3 Hypoxia inducible factor HIF................................................................................................ 2 1.1.4 Prolyl-hydroxylase domain containing proteins (PHDs) ...................................................... 3 1.1.5 Hypoxia signalling and cancer .............................................................................................. 4 1.2 PTMs and deubiquitinases ............................................................................................................ 6 1.2.1 PTMs in hypoxia signalling .................................................................................................. 6 1.2.2 Ubiquitination........................................................................................................................ 7 1.2.3 Deubiquitination .................................................................................................................... 9 1.2.4 USP29 (Ubiquitin-specific protease 29) ................................................................................ 9 1.2.4.1 Discovery of the Usp29 gene ............................................................................... 9 1.2.4.2 Imprinting and transcription of the Peg3 domain and the Usp29 gene .............. 10 1.2.4.3 USP29 biological functions ............................................................................... 11 1.3 Hypoxia signalling and the ubiquitin proteasome system .......................................................... 11 1.3.1 DUBs in canonical hypoxia signalling ................................................................................ 12 1.3.2 DUBs in non-canonical HIF signalling ............................................................................... 12 1.3.3 Regulation of DUBs by hypoxia ......................................................................................... 14 1.3.4 Dysregulation of hypoxia-related DUBs in cancer (excerpt) .............................................. 15 1.3.5 DUBs as druggable targets for therapy ............................................................................... 15 1.4 How to investigate signalling pathways ..................................................................................... 16 1.4.1 Fluorescence and fluorescence lifetime .............................................................................. 17 1.4.2 Förster resonance energy transfer (FRET) .......................................................................... 18 1.5 Aims of the project ..................................................................................................................... 20 Chapter 2: Materials and Methods ........................................................................................................ 21 2.1 Materials ..................................................................................................................................... 21 2.2 Methods ...................................................................................................................................... 21 2.2.1 Molecular Biology ............................................................................................................... 21 2.2.1.1 Polymerase chain reaction (PCR) ...................................................................... 21 2.2.1.2 Mutagenesis ....................................................................................................... 21 2.2.1.3 Restriction digestion .......................................................................................... 22 Table of Contents iii 2.2.1.4 Gateway® Cloning............................................................................................. 22 2.2.1.5 In-Fusion® HD Cloning .................................................................................... 23 2.2.1.6 Plasmid propagation ........................................................................................... 23 2.2.2 Cell Culture and transfections ............................................................................................. 24 2.2.3 RNAi ................................................................................................................................... 24 2.2.4 RNA extraction and quantitative PCR (qPCR) ................................................................... 25 2.2.5 Immunofluorescence ........................................................................................................... 27 2.2.6 MitoTracker staining ........................................................................................................... 27 2.2.7 Western Blot ........................................................................................................................ 27 2.2.8 Antibodies ........................................................................................................................... 28 2.2.9 Ubiquitination assay with Ni-NTA purification .................................................................. 28 2.2.10 Ubiquitination assay with GFP-trap® pulldown ............................................................... 29 2.2.11 Co-immunoprecipitation using GFP-traps® ..................................................................... 29 2.2.12 Size exclusion chromatography ......................................................................................... 30 2.2.13 Sequence alignment ........................................................................................................... 31 2.2.14 Mass spectrometry (MS) analysis ..................................................................................... 31 2.2.15 Fluorescence Imaging ........................................................................................................ 31 2.2.15.1 Acquisition of confocal fluorescence images................................................... 31 2.2.15.2 RICS measurements and data analysis ............................................................. 31