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Molecular and Cellular Studies of Drosophila Neuroserpin Spn4a And Molecular and Cellular Studies of Drosophila Neuroserpin Spn4A and its Polymer-Forming Mutants by Christine Chieh-Lin Lai B.Sc. University of British Columbia, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology & Immunology) The University of British Columbia (Vancouver) August 2010 © Christine Chieh-Lin Lai, 2010 1 Abstract Serpins (Serine Protease Inhibitors) are expressed by most organisms and perform a variety of functions. Most serpins inhibit proteases by undergoing a unique conformational change. They are clinically relevant in two ways. First, introduction of single amino acid point mutations transforms the serpins’ labile conformations into pathogenic, inactive polymers causing “serpinopathies”. In particular, human neuroserpin is a brain-specific serpin that, when mutated, causes a debilitating early onset dementia through unknown cellular pathways. Second, serpins are currently under investigation as therapeutic inhibitors of proprotein convertases (PCs). PCs are associated with some bacterial and viral infections as well as cancer. However, no comprehensive investigation into the cellular effects of PC inhibitor expression in mammalian cells has been performed. This thesis details the use of the Drosophila serpin, Spn4A, to address the cellular pathways mediated by serpin polymers or PC inhibition. Spn4A is a neuron-specific, secretory pathway serpin that inhibits Drosophila or human PCs. We hypothesized that Spn4A mutants, encoding homologous disease-causing mutations in human neuroserpin, would form pathogenic polymers and represent an ideal candidate for generating a cell-based and transgenic Drosophila serpinopathy model. Further, we hypothesized that we could evaluate the cellular response to PC inhibition and polymer accumulation by transcriptome profiling of H4 human neuroglioma cells expressing Spn4A wild-type and mutants. We established an expression system using Spn4A and its mutants in H4s. Subsequently, we used microarray analysis to simultaneously address how serpin polymers may induce cytotoxicity as well as the effects of proprotein processing inhibition in neuroglioma cells. We demonstrated that Spn4A mutants formed polymers, were retained in the endoplasmic reticulum, and lacked inhibitory function, but induced few changes on the transcriptome (under 20 genes differentially regulated). To this end, we have developed transgenic Drosophila overexpressing Spn4A variants to further investigate the biological impact of Spn4A mutants. Next, we analyzed the response to the PC inhibitor, Spn4A, and found marked changes in genes related to malignancy. Our genome-wide gene expression studies have provided novel insights into cellular changes in response to polymeric or PC-inhibiting serpins, and establish the foundation for future functional studies. ii Preface Ethics certificates for transgenic Drosophila experiments were obtained by Dr. Carl Hashimoto at Yale University. All transgenic fly experiments were performed by Dr. Hashimoto. Phylogenetic analysis of Spn4A was performed and Fig. 2.2 was generated by Dr. Victoria Svinti (Dr. François Jean’s lab). Catherine Verreault (Dr. Jean’s lab) cloned four Spn4A variants (Spn4A-S S49P, S52R, H338R, and G392E). Adenoviruses Ad. Emp, Ad. Spn4A WT-S, Ad. RFP Spn4A WT-S, and Ad. RFP Spn4A WT-R were generated previously by Dr. Jean’s lab. I prepared RNA for microarray experiments, Dr. Anne Haegart (Vancouver Prostate Center Microarray Facility, Vancouver, Canada) performed the microarray experiment and Dr. Robert Bell (Vancouver Prostate Center Microarray Facility, Vancouver, Canada) performed the statistical analysis and provided me with fold changes and p-values. I performed all the other experiments and prepared the figures. iii Table of Contents Abstract ........................................................................................................................................... ii Preface ............................................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ xi List of Abbreviations ................................................................................................................... xiv Acknowledgements .................................................................................................................... xviii Dedication .................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................... 1 1.1 Serpins.................................................................................................................................. 1 1.2 Serpin structure and inhibitory mechanism ......................................................................... 1 1.3 Human serpins ..................................................................................................................... 2 1.3.1 Function and localization.............................................................................................. 2 1.3.2 Serpinopathies .............................................................................................................. 2 1.4 Drosophila Spn4A ............................................................................................................... 3 1.5 Project overview .................................................................................................................. 4 Chapter 2: Development and transcriptome profiling of a cell model expressing Spn4A polymer- forming mutants .............................................................................................................................. 8 2.1 Introduction .......................................................................................................................... 8 2.1.1 Serpinopathies and neurodegenerative diseases ........................................................... 8 2.1.2 FENIB ........................................................................................................................... 8 2.1.2.1 Human neuroserpin ................................................................................................... 8 2.1.2.2 Human pathology of FENIB ..................................................................................... 9 2.1.2.3 Biochemical studies of hNS mutants ........................................................................ 9 2.1.2.4 Cellular studies of hNS mutants ............................................................................. 10 2.1.2.5 Models of FENIB in lower organisms .................................................................... 11 iv 2.1.2.6 Further questions and Spn4A .................................................................................. 12 2.1.3 Hypothesis, aims, and experimental approach ........................................................... 13 2.2 Methods.............................................................................................................................. 14 2.2.1 Phylogenetic analysis ................................................................................................. 14 2.2.2 Cell lines ..................................................................................................................... 14 2.2.3 Plasmid constructs ...................................................................................................... 14 2.2.4 Recombinant adenovirus constructs and production .................................................. 15 2.2.5 Transfection and infection conditions ........................................................................ 16 2.2.6 SDS-PAGE, native PAGE, and western blot ............................................................. 16 2.2.7 Immunofluorescence .................................................................................................. 18 2.2.8 Furin complex formation assay .................................................................................. 18 2.2.9 MTS cell viability assay ............................................................................................. 19 2.2.10 Flow cytometry ........................................................................................................... 19 2.2.11 RNA extraction and cDNA synthesis ......................................................................... 19 2.2.12 Microarray and data analysis ...................................................................................... 20 2.2.13 Real-time PCR ............................................................................................................ 21 2.3 Results ................................................................................................................................ 22 2.3.1 Phylogenetic analysis of Spn4A ................................................................................
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