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Molecular and cellular studies of Drosophila neuroserpin Spn4A and its polymer-forming mutants Lai, Christine Chieh-Lin 2010

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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 polymerforming 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 ................................................................................. 22  2.3.2  Development of Spn4A variants................................................................................. 22  2.3.3  Polymer formation studies of Spn4A variants ............................................................ 23  2.3.4  Spn4A mutants do not have inhibitory activities in vitro and in cellulo .................... 24  2.3.5  Cellular characterization of recombinant adenoviruses expressing Spn4A WT and mutants… .................................................................................................................................. 25  2.3.6  Microarray strategy..................................................................................................... 26  2.3.7  Spn4A WT and mutant overexpression induces ER stress genes .............................. 27  2.3.8  Retained and secreted non-inhibitory and polymeric Spn4A mutants affect few genes in the H4 transcriptome ............................................................................................................. 28  2.4  Discussion .......................................................................................................................... 29  2.4.1  Molecular and cellular comparisons of Drosophila Spn4A and human neuroserpin mutants… .................................................................................................................................. 29  2.4.2  Cellular model development ....................................................................................... 31  2.4.3  Transcriptome profiling of retained and secreted Spn4A mutants ............................. 32  2.4.4  Conclusions and future directions .............................................................................. 33  Chapter 3: Transcriptome profiling of human neuroglioma cells expressing the proprotein convertase inhibitor, Spn4A.......................................................................................................... 64  3.1  Introduction ........................................................................................................................ 64  3.1.1  Proprotein convertases ................................................................................................ 64  3.1.1.1  Structural features ................................................................................................... 64  3.1.1.2  Activation and subcellular localization ................................................................... 65  v     3.1.1.3  Substrate specificity and physiological substrates .................................................. 65  3.1.2  Furin as a therapeutic target........................................................................................ 66  3.1.2.1  Bacterial and viral infections requiring furin-like enzymes ................................... 66  3.1.2.2  Cancers associated with increased furin-like activity ............................................. 67  3.1.3  Furin-targeted inhibitors ............................................................................................. 68  3.1.3.1  Small molecule inhibitors ....................................................................................... 69  3.1.3.2  Prodomain-based inhibitors .................................................................................... 69  3.1.3.3  Peptide-based inhibitors .......................................................................................... 69  3.1.3.4  Protein-based inhibitors .......................................................................................... 70  3.1.4  Project rationale, hypothesis, and experimental approach ......................................... 71  3.2   Methods.............................................................................................................................. 73   3.3  Results and discussion ....................................................................................................... 74  3.3.1  RFP Spn4A inhibitory activities ................................................................................. 74  3.3.2  Experimental procedures and microarray strategy ..................................................... 74  3.3.3  Ontological, pathway, and network analysis of genes regulated by Spn4A in the secretory pathway ...................................................................................................................... 75  3.3.4  Regulation of specific genes implicated in cell proliferation ..................................... 76  3.3.5  Regulation of specific genes implicated in cell movement ........................................ 77  3.3.6  Regulation of specific genes implicated in angiogenesis ........................................... 79  3.3.7  Regulation of specific genes implicated in stress and apoptotic responses ................ 80  3.3.8  Ontological, pathway, and network analysis of genes regulated by RFP-tagged Spn4A in the secretory pathway................................................................................................ 81  3.3.9  Ontological, pathway, and network analysis of genes regulated by RFP-tagged Spn4A retained in the ER .......................................................................................................... 82  3.3.10  Real-time PCR validation ........................................................................................... 83  3.4   Conclusions and future directions ...................................................................................... 84   Chapter 4: Conclusion................................................................................................................. 108  4.1   Implications for cellular and animal models of serpinopathies ....................................... 108   4.2   Implications for the use of PCs as therapeutic targets ..................................................... 109   4.3   Closing remarks ............................................................................................................... 110   References ................................................................................................................................... 113  Appendix 1: Spn4A hinge mutant inhibitory studies .................................................................. 133  Appendix 2: Supplemental methods and materials ..................................................................... 141  vi     Appendix 3: Chapter 2 supplemental figures and tables ............................................................ 144  Appendix 4: Chapter 3 supplemental figures and tables ............................................................ 148  Appendix 5: Gene lists and annotations...................................................................................... 171     vii     List of Tables Table 2.1. Serpin rate of polymer formation correlates with severity of disease. ........................ 56  Table 2.2. Serpin clades A to I, prototypical serpins belonging to the clades, and their amino acid sequence identity to Spn4A. ......................................................................................................... 57  Table 2.3. Functional annotation clusters for genes upregulated by secreted or retained RFP Spn4A WT and mutants expression.............................................................................................. 58  Table 2.4. ER stress genes, gene descriptions, and fold changes for WT-R, S52R-R, H338R-R, WT-S, and H338R-S. .................................................................................................................... 61  Table 2.5. Genes differentially regulated in response to polymeric serpins. ................................ 62  Table 2.6. Functional annotations for genes differentially regulated by polymeric serpins expression. .................................................................................................................................... 63  Table 3.1. DAVID GO functional annotation clusters for genes downregulated by Spn4A WT-S expression. .................................................................................................................................. 101  Table 3.2. DAVID GO functional annotation clusters for genes upregulated by Spn4A WT-S expression. .................................................................................................................................. 105  Table 3.3. Top 10 networks generated from genes differentially regulated by Spn4A WT-S expression as identified by Ingenuity Pathway Analysis. ........................................................... 106  Table 3.4. Top 10 downregulated or upregulated genes associated with cell movement as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S expression. .................................................................................................................................. 107  Supplemental Table A2.1. Primer names and sequences used for cloning. ............................... 141  Supplemental Table A2.2. Titers of Adenovirus constructs. ...................................................... 142  Supplemental Table A2.3. Primers and probes for qPCR. ......................................................... 143  Supplemental Table A3.1. Functional annotation clusters for genes downregulated by secreted or retained RFP Spn4A WT and mutants expression...................................................................... 147   viii     Supplemental Table A4.1. Fold changes of genes discussed in Figure 3.8. ............................... 151  Supplemental Table A4.2. DAVID GO functional annotation clusters for genes downregulated by RFP Spn4A WT-S expression ............................................................................................... 153  Supplemental Table A4.3. DAVID GO functional annotation clusters for genes upregulated by RFP Spn4A WT-S expression. ................................................................................................... 155  Supplemental Table A4.4. Top 10 networks generated from genes differentially regulated by RFP Spn4A WT-S expression as identified by Ingenuity Pathway Analysis. ............................ 156  Supplemental Table A4.5. DAVID GO functional annotation clusters of unique and downregulated genes in response to Spn4A WT-S compared to RFP Spn4A WT-S. ................ 157  Supplemental Table A4.6. DAVID GO functional annotation clusters of unique and upregulated genes in response to Spn4A WT-S compared to RFP Spn4A WT-S ......................................... 159  Supplemental Table A4.7. DAVID GO functional annotation cluster of unique and downregulated genes in response to RFP Spn4A WT-S compared to Spn4A WT-S. ................ 160  Supplemental Table A4.8. DAVID GO functional annotation clusters of shared and downregulated genes in response to RFP Spn4A WT-S and Spn4A WT-S. .............................. 161  Supplemental Table A4.9. DAVID GO functional annotation clusters of shared and upregulated genes in response to RFP Spn4A WT-S and Spn4A WT-S........................................................ 163  Supplemental Table A4.10. DAVID GO functional annotation clusters of genes downregulated by RFP Spn4A WT-R expression. .............................................................................................. 164  Supplemental Table A4.11. DAVID GO functional annotation clusters of genes upregulated genes by RFP Spn4A WT-R expression. .................................................................................... 165  Supplementary Table A4.12. Top 10 networks generated from genes differentially regulated by RFP Spn4A WT-R expression as identified by Ingenuity Pathway Analysis. ........................... 166  Supplemental Table A4.13. DAVID GO functional annotation clusters of unique and downregulated genes in response to RFP Spn4A WT-S compared to RFP Spn4A WT-R. ....... 167  Supplemental Table A4.14. DAVID GO functional annotation clusters of unique and upregulated genes in response to RFP Spn4A WT-S compared to RFP Spn4A WT-R. ................................ 168  ix     Supplemental Table A4.15. DAVID GO functional annotation clusters of unique and downregulated genes in response to RFP Spn4A WT-R compared to RFP Spn4A WT-S. ....... 169  Supplemental Table A4.16. DAVID GO functional annotation clusters of shared and downregulated genes in response to RFP Spn4A WT-R and RFP Spn4A WT-S. ..................... 170  Supplemental Table A5.1. Lists of genes downregulated and upregulated by secreted or retained RFP Spn4A WT and mutants...................................................................................................... 171  Supplemental Table A5.2. List of genes downregulated and upregulated by Spn4A WT-S that are associated with DAVID GO annotations. ................................................................................... 173  Supplemental Table A5.3. Genes associated with cell growth and proliferation as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S. ....................... 176  Supplemental Table A5.4. Genes associated with cell movement as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S. ........................................ 181  Supplemental Table A5.5. Genes associated with significant canonical pathways identified by Ingenuity in response to Spn4A WT-S. ...................................................................................... 185   x     List of Figures Figure 1.1. Serpin structures and mechanisms................................................................................ 5  Figure 1.2. Serpin polymer formation............................................................................................. 6  Figure 1.3. Flow chart of themes, questions addressed, and experimental approaches in this thesis project. ............................................................................................................................................ 7  Figure 2.1. Unfolded protein response signaling pathways. ......................................................... 35  Figure 2.2. Serpin variants used in this study. .............................................................................. 36  Figure 2.3. Phylogenetic relationship of Drosophila Spn4A to one archetypal member of each serpin clade (A-I) that contains human serpins............................................................................. 37  Figure 2.4. Sequence alignment between human nueroserpin and Spn4A. .................................. 38  Figure 2.5. Polymer formation studies of human nueroserpin and Spn4A WT and mutants and quantification of intracellular and extracellular levels of Spn4A WT and mutants. .................... 39  Figure 2.6. Localization and staining patterns of human neuroserpin and Spn4A WT and mutants. ......................................................................................................................................... 41  Figure 2.7. Heat and SDS-stable complex formation assay between furin and Spn4A WT and mutants. ......................................................................................................................................... 47  Figure 2.8. Qualitative and quantitative measurements of Spn4A expression levels as delivered by recombinant adenoviruses and cell viability measurements of virus-induced toxicity. .......... 48  Figure 2.9. Strategy for microarray analysis of H4 neuroglioma cells infected with recombinant adenovirus. .................................................................................................................................... 52  Figure 2.10. The mRNA levels of three ER stress genes in response to RFP Spn4A WT-R, S52RR, or H338R-R determined by qPCR. .......................................................................................... 53  Figure 2.11. Distribution of genes differentially regulated in response to polymeric Spn4A mutants. ......................................................................................................................................... 54   xi     Figure 2.12. The mRNA levels of small proline-rich protein 2D in response to RFP Spn4A WTR, S52R-R, or H338R-R determined by qPCR............................................................................. 55  Figure 3.1. Structural features of PCs. .......................................................................................... 87  Figure 3.2. Activation of furin is spatially regulated. ................................................................... 88  Figure 3.3. Cellular localization of the PCs. ................................................................................. 89  Figure 3.4. Spn4A and furin EI complex formation assay. .......................................................... 90  Figure 3.5. Top 10 most significant cellular and molecular functions for genes differentially regulated by Spn4A WT-S expression identified by Ingenuity Pathway Analysis. ..................... 91  Figure 3.6. The most significant canonical pathways deduced from genes differentially regulated by Spn4A WT-S expression.......................................................................................................... 92  Figure 3.7. The top two most significant networks of genes regulated by Spn4A WT-S expression. .................................................................................................................................... 93  Figure 3.8. Points of the cell cycle where genes are differentially regulated in response to Spn4A-S expression. ..................................................................................................................... 96  Figure 3.9. Summary of DAVID gene ontology analysis for each list of regulated genes unique to or shared between WT-S and RFP WT-S. ................................................................................ 97  Figure 3.10. Summary of DAVID gene ontology analysis for each list of regulated genes unique to or shared between RFP WT-S and RFP WT-R. ....................................................................... 98  Figure 3.11. Comparison of the number of genes annotated with cell growth and proliferation or cell movement that are regulated in response to WT-S, RFP WT-S, and RFP WT-R. ................ 99  Figure 3.12. The mRNA levels of three cell cycle genes and IL8 in response to RFP WT-R determined by microarray and qPCR. ......................................................................................... 100  Figure 4.1. Flow chart of key findings and future directions in the thesis projects. ................... 111  Figure 4.2. PCs as anti-cancer treatment targets. ........................................................................ 112  Supplemental Figure A1.1. Sequence alignment of the hinge and reactive site loop region of Drosophila Spn4A and human maspin. ...................................................................................... 135  xii     Supplemental Figure A1.2. Serpin hinge mutant constructs....................................................... 136  Supplemental Figure A1.3. In vitro EI complex formation assay and immunofluorescence of Spn4A hinge mutants. ................................................................................................................. 137  Supplemental Figure A1.4. In cellulo EI complex formation assay with Spn4A hinge mutants. ..................................................................................................................................................... 139  Supplemental Figure A1.5. In cellulo EI complex formation assay with adenovirus expressing Spn4A hinge mutants. ................................................................................................................. 140  Supplemental Figure A3.1. Enlarged figures of RFP Spn4A WT-S and H338R-S. .................. 144  Supplemental Figure A3.2. Example flow cytometry graphs used to calculate percentage of cells expressing RFP Spn4A. .............................................................................................................. 145  Supplemental Figure A3.3. Immunofluorescent images of transgenic Drosophila pupae expressing RFP Spn4A WT-R, RFP Spn4A T328D-R, and RFP Spn4A H338R-R. ................. 146  Supplemental Figure A4.1. Top 10 most significant cellular and molecular functions for genes differentially regulated by RFP Spn4A WT-S expression identified by Ingenuity Pathway Analysis....................................................................................................................................... 148  Supplemental Figure A4.2. The most significant canonical pathways deduced from differentially regulated genes in response to RFP Spn4A WT-S. .................................................................... 149  Supplemental Figure A4.3. Top 10 most significant cellular and molecular functions for genes differentially regulated by RFP Spn4A WT-R expression identified by Ingenuity Pathway Analysis....................................................................................................................................... 150   xiii     List of Abbreviations Ad AhR AJAP1 ALPP ATF6 BCL2 BiP BMF C5ORF13 CCN CD24 CD36 CDC25A CDK CDKN1C cDNA CFSE CNS CT DAVID DCN DDIT3 DMEM E2F2 ECM EDTA EI eLF2α Emp EOR EPYC ER ERAD ERGIC ERRFI1 ES ETH ETV5 F FC FENIB  Adenovirus Aryl hydrocarbon receptor Adherens junctions associated protein 1 Alkaline phosphatase, placental (Regan isozyme) Activating transcription factor 6 B cell lymphoma 2 Binding immunoglobulin protein BCL2 modifying factor Chromosome 5 open reading frame 13, also known as P311 Cyclin CD24 molecule CD36 molecule (thrombospondin receptor) Cell division cycle 25 Cyclin-depndent kinase Cyclin-dependent kinase inhibitor 1C Complementary deoxyribonucleic acid Carboxyfluorescein succinimidyl ester Central nervous system Threshold cycle Database for Annotation, Visualization and Integrated Discovery Decorin DNA-damage-inducible transcript 3 Dulbecco’s modified eagle medium E2F transcription factor 2 Extracellular matrix Ethylenediaminetetraacetic acid Enzyme-inhibitor Eukaryotic translation initiation factor 2 alpha Empty ER overload response Epiphycan Endoplasminc reticulum ER associated degradation ER-Golgi Intermediate Compartment ERBB receptor feedback inhibitor 1 Enrichment score Ecdysis triggering hormone Ets variant 5 FLAG polypeptide tag Fold change Familial Encephalopathy with Neuroserpin Inclusion Bodies xiv     FGF FLIP FRAP G1 G2 GADD gB GDF15 GUCY1A3 HA hCMV HDEL HEK293 HERPUD1 HF HGG HIV-1 hNS Hsp47 HSPA5 IGF IGFR IL8 IPA IRE1 ITGA KDEL Ki KRT81 LPC M MCM MEF MGP ML MMP MOI mRNA MT1-MMP MTS MYC  Fibroblast growth factor Fluorescence loss in photobleaching Fluorescence recovery after photobleaching Gap1 Gap2 Guanidinylated aryl 2,5-dideoxystreptamine derivatives Glycoprotein B Growth differentiation factor 15 Guanylate cyclase 1, soluble, alpha 3 Hemagglutinin Human cytomegalovirus His-Asp-Glu-Leu Human embryonic kidney 293 cells Homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 1 His and FLAG polypeptide tag High grade gliomas Human immunodeficiency virus 1 Human neuroserpin Heat shock protein 47 kDa Heat shock 70 kDa protein 5 Insulin growth factor Insulin growth factor receptor Interleukin-8 Ingenuity Pathway Analysis Inositol requiring enzyme 1 Integrin alpha Lys-Asp-Glu-Leu Inhibition constant of the enzyme-inhibitor complex Keratin 81 Lymphoma proprotein convertase Mitosis Mini chromosome maintenance Mouse embryonic fibroblast Matrix gla protein Maximum likelihood Matrix metalloproteases Multiplicity of infection Messenger ribonucleic acid Membrane type 1 matrix metalloprotease 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium inner salt v-Myc myelocytomatosis viral oncogene homolog xv     Nek2 NFκB NI OLR1 ORC PA PAGE PAI-1 PAS PBS PBS-S PC PCR PCSK-9 PDB PDGF PEA PERK PI8 PLAG-1 PLAT PLAUR PPAP2A qPCR RCL RFP RGS1 ROS RSL S S100A4 SCID SDS SEM Serpin SKI-1 SMC SOCS2 SP Spn4A SPP1 SPRR2D STS  Never in mitosis gene A-related kinase 2 Nuclear factor kappa-light-chain-enhancer of activated B cells Non-infected cells Oxidized low density lipoprotein (lectin-like) receptor 1 Origin of replication complex Protective antigen Polyacrylamide gel electrophoresis Plasminogen activator inhibitor 1 Periodic acid/Schiff reagent Phosphate buffered saline PBS with 0.05% saponin Proprotein convertase Polymerase chain reaction Proprotein convertase subtilisin kexin-9 Protein data bank Platelet-derived growth factor Pseudomonas exotoxin A PKR-like endoplasmic reticulum kinase Proteinase inhibitor 8 Pleiomorphic-adenoma gene 1 Tissue type plasminogen activator Urokinase plasminogen activator receptor Phosphatidic acid phosphatase type 2A Quantitative real-time polymerase chain reaction Reactive center loop Red fluorescent protein Regulator of G-protein signaling 1 Reactive oxygen species Reactive site loop Synthesis S100 calcium binding protein A4 Severe combined immunodeficiency Sodium dodecyl sulfate Standard error of the mean Serine protease inhibitor Subtilisin/kexin-like isozyme-1 Structural maintenance of chromosome proteins Suppressor of cytokine signaling 2 Signal peptide Serpin 4A Secreted phosphoprotein 1 Small proline-rich protein 2D Staurosporine xvi     TGFA TGFβ TGFβR TGN TNF tPA uPA UPR VEGF WAG WT XBP1  Transforming growth factor alpha Transforming growth factor beta Transforming growth factor beta receptor Trans-Golgi network Tumor necrosis factor Tissue-type plasminogen activator Urokinase-type plasminogen activator Unfolded protein response Vascular endothelial growth factor Whelan and Goldman Wild type X-box binding protein 1       xvii     Acknowledgements I would like to acknowledge numerous people who have supported me through my Masters at UBC. I thank my supervisor, Dr. François Jean, for the opportunity to learn so much about serpins, guidance on how to present and write scientifically, and many discussions that left me with more questions than answers. Also, I thank my committee members, Dr. Michael Gold and Dr. Steven Hallam, for their time, advice on my projects, and encouragements. I would like to thank Dr. Carl Hashimoto for his gift of the Drosophila Spn4A and human neuroserpin cDNA and collaboration to make transgenic flies. I am grateful to the members of the Jean lab who are always available for questions, laughs, and chocolate. Especially Martine Boutin, who ordered reagents on short notice and lent me a hand when I was too ambitious with experiments. I also thank Catherine Verreault, a past co-op student, who cloned the first four serpinopathy-linked variants to start the project. Further, Dr. Victoria Svinti helped with the phylogenetic analysis of Spn4A. Lastly, I thank Steve McArthur, a Master student, who critically read my thesis. I thank my family and friends for all their support; especially my dad, who rushed to fix my computer when it crashed while writing my thesis, and my mom, who brought me food when I was too tired to cook. I am thankful for my older brother, Ben, who helped me through my undergraduate and graduate years with advice on science and life. Also, I thank Mark for his emotional and technical support. The microarray analysis would not have been possible without his scripts. Last, I am grateful for the Graduate Entrance Scholarship from the UBC Microbiology and Immunology Department and Master’s Award from the Canadian Institute of Health Research (CIHR CGM-98317). This work is supported by a CIHR grant (III-94585) to Dr. François Jean.    xviii     Dedication For my parents, brother, and puppeh.  xix     Chapter 1: Introduction 1.1  Serpins Serpins (Serine Protease Inhibitors) comprise a protein superfamily found in organisms  ranging from humans and plants to viruses and prokaryotes (1-4), as identified by conserved sequence motifs (5). In humans, 36 serpins have been identified to date (2). These serpins can localize extracellularly or intracellularly and most inhibit serine proteases. A few others also inhibit cysteine proteases, or lack inhibitory activity altogether (2). Phylogenetic analyses categorize serpins into 16 clades labeled A to P, with 10 additional unclassified serpins (5). Generally, human serpins reside in clades A to I, while clades J to P group according to nonvertebrate species (5).  1.2  Serpin structure and inhibitory mechanism Serpins are identified by a total of approximately 50 highly conserved amino acid  residues and an ~30% amino acid sequence homology (5) to the prototypical serpin, α1antitrypsin, an inhibitor of neutrophil elastase (6). Despite low amino acid sequence homology, all serpins share a conserved tertiary structure (6). Most serpins have three β-sheets and nine αhelices like α1-antitrypsin (Fig. 1.1A) (7). The reactive site loop (RSL) or reactive center loop (RCL) lies approximately 20 to 25 amino acids above the serpin core and contains a peptide sequence to bait target proteases (6). The native structure exists in a metastable or “stressed” state (8). Once a protease recognizes the pseudosubstrate peptide in the RCL, the P1-P1’ bond is cleaved and an acyl ester bond forms between the protease and the serpin (6). Like a mousetrap, the serpin undergoes a conformation change that involves the end-to-end translocation of the protease by incorporation of the clipped RSL into the serpin’s β-sheet A (9) (Fig. 1.1C). The conformational transition is accompanied by an increase in serpin stability, bringing it into the “relaxed” state (8). In the new conformation, the protease active site is distorted, preventing the deacylation step and the reaction from running to completion (9). Thus, serpins are suicide inhibitors, trapping the protease in an irreversible and heat- and SDS (sodium dodecyl sulfate)stable covalent complex. Aberrations such as mutations within the RSL limit its insertion rate into the β-sheet, resulting in serpin cleavage instead (10) (Fig. 1.1D). For example, the P14 position (14th residue on the amino terminal side of the cleavage site), usually a serine or threonine, acts like a hinge 1     for RSL insertion (11). Mutations at P14 to charged or bulky amino acids force the serpin into the substrate pathway, producing active protease and non-inhibitory, cleaved serpin (11;12).  1.3  Human serpins  1.3.1  Function and localization In humans, the majority of the serpins are inhibitory. Some are secreted into the  circulating plasma to control extracellular proteolytic cascades such as inflammation (α1antitrypsin inhibition of elastase (13)), the complement pathway (C1 inhibitor and C1 esterase (14)), blood clotting (antithrombin and thrombin (15)), or tissue remodeling (neuroserpin and tissue plasminogen activator (16)). Other serpins localize to the cytoplasm to control inappropriate protease activities (cytoplasmic antiproteinase 9 inhibition of granzyme B (17;18)). The small number of non-inhibitory serpins localize extracellularly or intracellularly. Non-inhibiting serpins may function as hormone precursors like angiotensinogen, which regulates blood pressure (19), or hormone transporters, such as thyroxine-binding globulin (20). Intracellularly, well studied and non-inhibitory serpins include the chaperone Hsp47 (heat shock protein 47 kDa) (21) and the tumor suppressor maspin (22). Non-inhibitory serpins may have regions beside the RSL that are involved in biological activities (6). 1.3.2  Serpinopathies Serpin diseases can arise from mutations affecting the inhibitory mechanism or serpin  stability. In the latter, point mutations cause serpins to fold into disease-causing conformations, known as pathogenic polymers. The new conformational transition involves domain swapping of one RSL and β-sheet strand from one serpin to the β-sheet of another serpin (23;24) (Fig. 1.2). The inactive long-chain polymers are known to cause several diseases, collectively known as “serpinopathies” (25;26). Serpin polymers cause gain and/or loss of function diseases. The best studied example is α1-antitrypsin deficiency. α1-Antitrypsin is synthesized in liver cells and secreted into the plasma. It maintains the integrity of the lung tissue by inhibiting neutrophil elastase. The most common and clinically significant mutation of α1-antitrypsin, called the Z allele, leads to polymer formation and subsequent retention of the serpin within hepatocytes. Inclusion body formation manifests physiologically as hepatitis, cirrhosis, and hepatocellular carcinoma (27;28). Further, 2     decreased serum levels and inhibitory activity expose lung tissue to uncontrolled elastase proteolysis, leading to emphysema. Pathogenic polymers also underlie the hereditary dementia, Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Central nervous system neurons secrete neuroserpin, which controls synaptic plasticity by inhibiting tissue plasminogen activator (29). Several point mutations identified in neuroserpin result in neuronal inclusion body formation. Clinically, the inclusion bodies are associated with dementia, which manifests as early as childhood (30) with mortality in early adulthood (31). The loss of extracellular neuroserpin and regulation of synaptic contact formation may also cause epilepsy (30).  1.4  Drosophila Spn4A Fewer serpins are well characterized in other organisms compared to humans.  Nonetheless, the study of serpins in model organisms has broadened our knowledge of their functional diversity and cellular localization. One of the best examples comes from the Spn4 (Serpin 4) gene in Drosophila. Spn4 can be alternatively spliced into eight protein isoforms (AH) using four different RSL sequences. Isoforms A-D differ from E-H by the presence of an Nterminal signal peptide (SP) (32), targeting proteins to the secretory pathway. Spn4A contains an ER retention motif at the C-terminus (32), and unlike Spn4B-D, is not secreted to the extracellular space (33). Without the N-terminal targeting sequence, Spn4E-H are not secreted (33). In vitro, Spn4A can inhibit Drosophila and human furin as well as Drosophila PC2 (proprotein convertase 2) (33-36), serine endoproteases important for activation of proproteins, such as hormones, in the secretory pathway. In Drosophila, Spn4A localizes to the central nervous system. Its overexpression leads to lethal larval molting defects (35) resembling the Drosophila PC2 (37) and ETH (ecdysis triggering hormone) (38) loss of function phenotype. This was the first demonstration of an endogenous serpin co-localizing and inhibiting a PC. Further evidence of the novel function was recently confirmed in a Branchiostoma lanceolatum serpin, B1-Spn1, which has an N-terminal signal peptide and ER retrieval signal and inhibits PCs (39). The PC inhibitory activity of Spn4A is of therapeutic interest. PCs control the maturation of proteins such as growth factors and extracellular matrix proteins important for tumor 3     malignancy as well as the activation of viral proteins and bacterial toxins (40). The ability of Spn4A to inhibit PC activity under human pathological conditions has been demonstrated by our lab (unpublished data). Previous work showed that Spn4A blocked PC-mediated processing of viral coat proteins from HIV-1 (human immunodeficiency virus I) by Vesna Posarac (Jean lab, Master’s student, 2008), hCMV (human cytomegalovirus) by Martine Boutin (Jean lab, technician), and H5N1, H7N3, and H7N7 influenza viruses by Dr. Sandra Diederich (Jean lab, postdoctoral researcher). Therefore, Spn4A may be an effective treatment for various PCmediated pathologies including infectious diseases and cancer.  1.5  Project overview The ability of polymer-forming serpin mutants to cause disease and the development of  serpin PC inhibitors as cancer therapeutics are two clinically significant research fields. However, many questions remain regarding the pathogenesis of polymers (discussed in Chapter 2) and the potential of PC-targeted therapeutics (discussed in Chapter 3). Drosophila Spn4A offers a unique vantage point in studying the pathobiology and therapeutic application of serpins. As a neuron specific serpin, Spn4A mutants encoding homologous FENIB-causing substitutions may be molecular tools for studying the cellular pathways associated with FENIB in cell culture and transgenic Drosophila models. Further, Spn4A has pharmacological significance as a potent inhibitor of PCs. We addressed these two different roles. The overall objective of this thesis project was to use Spn4A variants to study the cellular responses to serpin polymers and furin inhibition (Fig. 1.3). Accordingly, we describe the development and transcriptome profile of a cellular model expressing Spn4A polymer formingmutants (Chapter 2). We show that Spn4A mutants, with amino acid substitutions equivalent to those found in FENIB, formed polymers in human H4 neuroglioma cells. Gene expression profiling revealed that fewer than 20 genes were differentially regulated in response to Spn4A polymers. Further, to evaluate Spn4A as a therapeutic agent, we investigated the transcriptome profile of neuroglioma cells expressing Spn4A and found marked changes in cell proliferation and movement (Chapter 3). Consequently, we hypothesize mechanisms through which PC inhibition may block tumorigenesis and malignancy. Finally, we developed a non-inhibitory hinge mutant of Spn4A as a negative control to demonstrate functional specificity (Appendix 1).  4     Figure 1.1. Serpin structures and mechanisms. A) The structure of native SERPINA1 (α1antitrypsin, Protein Data Bank (PDB) code IQLP (41)) is shown. The A sheet is in red, the B sheet in green, the C sheet in yellow, helices in blue, and the reactive center loop (RCL) in magenta. The dashed magenta line indicates the path of RCL insertion. The breach and shutter region are as labeled. B) The multicoloured trypsin protease docks on the RCL of α1-antitrypsin (PDB code IOPH (42)). C) The protease cleaves the peptide bond, releasing the serpin’s Cterminal tail (not shown), and is translocated to the other end of the serpin, which traps the protease in an irreversible serpin-enzyme complex (PDB code IWZX (9)). D) Alternatively, serpins follow the substrate pathway, producing cleaved α1-antitrypsin (PDB code 7API (43)) and active protease. Reprinted with permission from BioMed Central: Genome Biology, Law RA, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, Rosado CJ, Langendorf CG, Bird PI, and Whisstock JC, An Overview of the Serpin Superfamily (2), © 2006.  5     Figure 1.2. Serpin polymer formation. A) Unfolded serpins fold to a short-lived intermediate. B) In the native protein conformation, the β-strand s4A folds into β-sheet A. C) Mutations promote the insertion of one serpin’s domain into another. Monomer 2 donates the RCL (reactive centre loop) and s5A into a neighbouring monomer. A third monomer could donate its β-strands to the empty space in monomer 2, allowing polymer growth. Reprinted by permission from Macmillan Publishers Ltd: Nature, Whisstock JC and Bottomley SP, Structural biology: Serpins’ mystery solved (24), © 2008.  6     Figure 1.3. Flow chart of themes, questions addressed, and experimental approaches in this thesis project.     7     Chapter 2: Development and transcriptome profiling of a cell model expressing Spn4A polymer-forming mutants 2.1  Introduction  2.1.1  Serpinopathies and neurodegenerative diseases Serpins (Serine protease inhibitors) are a superfamily of proteins that typically function  in the control of proteolytic cascades (6). However, mutations in serpins can cause aberrant protein conformational transitions, resulting in inactive serpin polymers retained inside the cell. Disease is attributed to a toxic gain of function due to intracellular protein inclusion body formations and/or a loss of extracellular inhibitory function (25;26). Polymeric α1-antitrypsin, C1 inhibitor, antithrombin, α1-antichymotrypsin, and neuroserpin lead to liver cirrhosis and emphysema, angioedema, thrombosis, emphysema, and dementia and epilepsy, respectively. Together, they are known as the serpinopathies (25;26). The most recently described serpinopathy is caused by mutations to neuroserpin, an inhibitor of tissue-type plasminogen activator (tPA) (29;44), that result in the formation of neuronal inclusion bodies. Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), described in six families to date, is a disorder characterized by early-onset dementia, seizures, and/or tremors (45). Although affecting only a handful of families worldwide, FENIB shares common disease hallmarks – conformational instability, oligomerization, and inclusion body deposition – with other neurodegenerative syndromes such as Alzheimer’s, Parkinson’s, and Huntington’s diseases as well as the prion encephalopathy. Unlike other disorders, the causative protein and its function, mechanism, and native and disease-causing structures are clear in FENIB (29;46;47). Thus, models of FENIB are likely to provide insight into the pathogenesis of other neurodegenerative diseases (25;26;48). 2.1.2  FENIB  2.1.2.1 Human neuroserpin Human neuroserpin (hNS) is expressed primarily in embryonic and adult neurons of the central nervous system (CNS), with low levels in the pancreas, heart, kidney, and testis (29). In vitro, neuroserpin has inhibitory activity toward tPA and to a lesser extent, urokinase-type plasminogen activator (uPA) and plasmin (29;44). Detection of an enzyme-bound serpin 8     complex from cultured cells expressing both neuroserpin and tPA suggest that the complex also occurs in vivo (49). In the brain, neuroserpin and tPA are both secreted from growth cones at the tips of axons especially during neurite outgrowth, synaptogenesis, and synaptic plasticity (50). Further, neuroserpin is neuroprotective in blocking tPA-dependent seizures (51) and stroke (52;53). 2.1.2.2 Human pathology of FENIB First described in 1999, FENIB is an inherited, autosomal dominant disorder caused by mutations to hNS (46). It presents with myoclonus (muscle twitching) and pre-senile dementia, affecting individuals as early as eight years of age to as late as 50 years of age (45;46). FENIB patients have problems with shortened attention span and oral fluency in the early stages (54). Global cortical atrophy is found in more severe cases (54). Neuroserpin inclusion bodies, named Collins bodies, are histologically unique (periodic acid/Schiff reagent (PAS)-positive, but diastase-resistant), can be 1-50 μm in diameter (46;55), and deposit in the cerebral cortex and substantia nigra (located in the midbrain) (46). Isolated Collins bodies were shown to be composed exclusively of neuroserpin by amino-acid sequencing (46). Subsequent genetic sequencing of affected individuals revealed point mutations resulting in the following substitutions S49P, S52R, H338R, G392E, or G392R (30;31;46;56). 2.1.2.3 Biochemical studies of hNS mutants To understand how single amino acid point mutations may lead to disease, neuroserpin mutants were cloned and expressed in Escherichia coli (57-59) or in mammalian cells (60;61). Polymers are identified by ladder formations on non-denaturing polyacrylamide gel electrophoresis (PAGE) (57-60) and punctate inclusions within the endoplasmic reticulum (ER) by confocal microscopy (60;61). Decreased levels of extracellular mutant serpins are found in the cultured media, demonstrating protein retention (61). Strikingly, the molecular instability of neuroserpin, assessed biochemically by polymer formation rate, is directly related to the amount of inclusion body formed in human brain histology samples and to disease severity (Table 2.1) (31). Further, neuroserpin mutants are poor inhibitors of tPA (the S49P mutant had a 100 fold difference in inhibitory constant compared to the wild type [WT] and the S52R mutant was not inhibitory at all) (57-59), which may intensify epileptic episodes and other neurological symptoms (30;45).  9     2.1.2.4 Cellular studies of hNS mutants The cellular response to polymer accumulation is currently under investigation. Since the ER is the site of polymer retention, pathways within the ER may mediate toxic effects. The unfolded protein response (UPR), ER overload response (EOR), ERAD (ER associated degradation), or autophagy are pathways activated by cells to cope with increased intracellular protein load. Excessive signaling from these pathways, or lack thereof from ERAD, may lead to apoptosis (62;63). The UPR includes the PERK (PKR-like endoplasmic reticulum kinase), IRE1 (inositol requiring enzyme 1), and ATF6 (activating transcription factor 6) signaling pathways in response to increased ER protein load or pathological stresses, such as the presence of misfolded proteins (Fig. 2.1). The ER molecular chaperone, BiP (binding immunoglobulin protein), normally binds to these three transmembrane signal transducers and is released when misfolded protein accumulates (64). Freed IRE1 then dimerizes, which activates its endonuclease activity leading to alternative splicing of the XBP1 (X-box binding protein 1) mRNA (messenger ribonucleic acid), which produces an active transcription factor (65;66). Release of ATF6 leads to its translocation to the Golgi, where it is proteolytically processed to an active transcription factor (67). Last, PERK dimerizes and phosphorylates eLF2α (eukaryotic translation initiation factor 2 alpha) to attenuate general mRNA translation while promoting the translation of select mRNAs such as ATF4 (68;69). These three transcription factors (Xbp1, ATF6, and ATF4) regulate stressresponse genes, ultimately leading to protein translation attenuation, increased chaperone expression, and protein degradation through ERAD, enabling the cell to cope and recover from stress (62). The inability to resolve the excess load results in signaling from the transcription factor CHOP, which represses transcription of the anti-apoptotic protein Bcl2 (B cell lymphoma 2) and activates several apoptotic genes (62). Alternatively, ER protein buildup may activate EOR (70;71). EOR involves the release of calcium from the ER into the cytoplasm and subsequent production of reactive oxygen species (ROS) mediates the activation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) (70;71). The existence of EOR is currently under scrutiny as NFκB can also be activated by PERK signaling (72), but others have shown UPR-independent NFκB activation (73). NFκB induces the transcription of many genes involved in pro- and anti-apoptotic pathways, inflammation, immunity, and proliferation (74). 10     Protein buildup in the cell may be cleared by activating ERAD or autophagy (75). ERAD involves targeting misfolded proteins to the ubiquitin-proteasome pathway by retro-translocation from the ER to the cytoplasm (75). Autophagy involves the degradation of large aggregates by membrane vesicles called autophagosomes that engulf the protein load and fuse with lysosomes (76). Autophagy has been linked to apoptosis, although its direct role is unclear (77). Recently, Davies et al. demonstrated that neuroserpin polymers fail to activate the UPR, but found that S52R and G392E neuroserpin mutants can activate NFκB in the rat adrenal pheochromocytoma cell line, PC12, and mouse embryonic fibroblast (MEF) cells (73). Further, Kroeger et al. demonstrated that mammalian cells employ ERAD and, in MEF cells, autophagy, to clear polymeric mutant serpins (78). 2.1.2.5 Models of FENIB in lower organisms To assess the effects of polymer accumulation in model organisms, transgenic Drosophila melanogaster expressing hNS S49P, S52R, H338R, or G392E mutants (61) and mice expressing hNS S49P, S52R, or G392E mutants (79;80) were generated. Intracellular accumulation is detected in both model organisms. Flies expressing mutant hNS suffer from locomotor dysfunction, but no differences in longevity are observed (61). Also, WT and mutant neuroserpin are cleared with equal efficiency from fly brains (78). The transgenic mice exhibit clinical features such as ataxia and tremors (79). Neuronal apoptosis, measureable by caspases and cytoplasmic cytochrome C, is not apparent (80). Dose-dependence of protein aggregate formation is demonstrated in transgenic hemizygous G392E mice, which do not form protein aggregates, and their homozygous counterparts, which do (80). Together, these results indicate that human mutant neuroserpin forms intracellular aggregates that can independently cause neurodegeneration. Further, a rat model of FENIB was generated from the overexpression of human wildtype megsin (81), a serpin targeting plasmin in vitro (82). While homozygous transgenic megsin rats suffer from renal and pancreatic lesions (83), heterozygous transgenic rats fail to show any abnormities in those organs (81). Instead, the heterozygous transgenic rats display characteristic inclusions in neurons, express ER stress and apoptotic proteins (BiP, hypoxia upregulated 1 protein, caspase 12 and 3), have significant neuronal loss, but have the same life span as control non-transgenic rats (81).  11     2.1.2.6 Further questions and Spn4A Within a decade of the discovery of FENIB, hNS mutants were identified and demonstrated to form polymers that led to disease. However, the details of the molecular and cellular events contributing to pathology are still unclear. Importantly, the signaling pathways that activate NFκB and its consequent pathways leading to cell survival, proliferation, or death (74) still needs to be elucidated. NFκB can control the transcription of over 150 genes depending on cell type and stimulus (84). Relevant genes in response to pathogenic polymers can be addressed using cDNA (complementary deoxyribonucleic acid) microarray technology. Further, the effects of the modulated cellular pathways will need to be assessed in a model organism. Drosophila is an economical and genetically amenable model organism in which we could dissect the cellular mechanisms underlying FENIB. The current transgenic Drosophila FENIB models express human neuroserpin mutants and recapitulate some clinical symptoms of neurological disease, but not life span or neuronal loss (61). Further, they could clear hNS WT or mutants with equal efficiency (78). In comparison, the serpinopathy model of α1-antitrypsin deficiency in Drosophila uses the endogenously-expressing serpin Necrotic, homologous to α1-antitrypsin (85). Transgenic Drosophila expressing polymeric Necrotic mutants were observed to have a temperature-dependent lethality phenotype. The lack of a strong toxic phenotype in the FENIB Drosophila model may be due to the levels of expression, since higher levels of mutant hNS are associated with greater disease severity (Table 2.1), or the production of foreign human proteins in transgenic models. Increased expression of polymeric mutants or the use of endogenous serpins with homologous mutations may be advantageous.  To date, only Serpin 4 (Spn4) is known to be expressed in the Drosophila CNS (35). Spn4 can be alternatively spliced to produce eight different protein isoforms, changing between four different reactive site loops (RSL) to bait different proteases, and omitting or including an N-terminal signal peptide, allowing different cellular localizations (32). Spn4A (isoform A) contains an N-terminal signal peptide and a 13 amino acid C-terminus extension, similar to hNS (86). Several studies have demonstrated that Spn4A is an inhibitor of human and Drosophila furin, as well as Drosophila PC2 (proprotein convertase 2) (33-36). Transgenic Drosophila generated from the native and neuron-localized serpin, Spn4A, may provide a good model system for studying FENIB. The ability of Spn4A mutants to form polymers like hNS would first have to be evaluated. The expression of Spn4A mutants in a mammalian cell line may be used to 12     study polymer formation. Further, this cellular expression system may be used to address the subsequent cellular responses to serpin polymers. 2.1.3  Hypothesis, aims, and experimental approach The overall goal was to develop a cell-based and transgenic Drosophila model of FENIB  to study the serpinopathy disease mechanisms, using Spn4A. We hypothesized that mutant Spn4A could form polymers, modulate cellular stress pathways, and cause disease symptoms in transgenic Drosophila, similar to mutant hNS. Specific Aim 1: Establish a cell-based system to evaluate polymer formation, retention, and non-inhibitory function of Spn4A mutants. First, we evaluated the phylogenetic relationship between Spn4A and hNS, and generated Spn4A mutants with amino acid substitutions equivalent to those found in FENIB-causing hNS residues. Next, we tested the ability of Spn4A mutants to form polymers and be retained in the ER, which are cellular signatures of hNS mutants. We used transfection and adenovirus infection systems to express Spn4A WT and mutants in an H4 mammalian neuroglioma cell line. We chose to use H4 cells, instead of macrophage-like Drosophila Schneider 2 cells, in order to investigate polymer formation and consequences in a neuronal cell context (specific aim 2). Further, tools such as antibodies and microarray chips are more readily available for human systems. Polymer formation and retention were assessed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), nondenaturating PAGE, and confocal microscopy. Lastly, the inhibitory activities toward furin were also studied. Specific Aim 2: Determine transcriptome changes in response to Spn4A mutants. Polymer accumulation may lead to the activation of stress signaling cascades, which may affect cell health and eventually lead to cell death. To inspect the whole transcriptome for genes that were regulated in response to mutant Spn4A expression, we performed a microarray experiment using the Illumina HumanHT-12 v3 Beadchip. Specific Aim 3: Generate transgenic Drosophila overexpressing Spn4A mutants. In collaboration with Dr. Carl Hashimoto (Yale University), we generated transgenic flies overexpressing Spn4A variants. I cloned the mutants and Dr. Hashimoto’s lab generated transgenic flies. The model will be useful for further studies investigating the impact of Spn4A mutants on a whole organism.  13     2.2  Methods  2.2.1  Phylogenetic analysis Serpin primary sequences were aligned using CLUSTALW2 (87) for construction of the  phylogenetic tree. Two methods for building phylogenetic trees, maximum likelihood (ML) and Bayesian methods, are preferred because these algorithms take into account how the data evolves (model of evolution), which leads to more accurate trees. The Bayesian method used for this analysis is faster than ML since it combines the ML tree search and the bootstrapping steps into one. This saves time while producing trees that are comparable in accuracy. The tree was built using Mr. Bayes v3.1.2 (88;89). The amino acid substitution rates used were those derived from the Whelan and Goldman (WAG) matrix (90). The analysis was run twice for 1 million generations with sampling frequency of 1000 and the number of chains set to 4. The trees retained are those obtained after the convergence of the two runs. This set of trees was summarized into a majority rule consensus tree, with values on the branches reflecting clade credibility. 2.2.2  Cell lines H4 neuroglioma cells, human embryonic kidney 293 cells (HEK293 or AD293) and  HEK293-C4 furin-overexpressing cells (Dr. Richard Leduc, University of Sherbrooke) were maintained in DMEM (Dulbecco’s Modified Eagle Medium) containing 10% fetal bovine serum and 100 units of penicillin and streptomycin per mL. Cells were incubated at 37°C in 5% CO2. 2.2.3  Plasmid constructs Plasmid cloning reagents include pfu polymerase (Fermentas), restriction enzymes (New  England Biolabs), Dephosphorylation and Ligation kits (Roche), and DH5α competent cells. Plasmids were purified from DH5α using QIAprep Spin Miniprep Kit (Qiagen) or Nucleobond Xtra Midi kit (Macherey-Nagel). PCR (polymerase chain reaction) fragments were purified using the QIAquick PCR Purification kit (Qiagen). Plasmids used for transfection experiments were constructed in the mammalian expression vector pcDNA3.1 plus (Invitrogen). Primers are listed in Appendix 2, Supplemental Table A2.1. His and FLAG-tagged (HF) Spn4A, with or without tagRFP (91) (red fluorescent protein), were localized to three different cellular compartments by deletion of the C-terminal 14     HDEL ER retention motif or the N-terminal SP (signal peptide) (Fig. 2.2). Martine Boutin (Jean lab) had cloned the HF Spn4A ER-retained (designated Spn4A WT-R) and secreted (HDEL deletion, designated Spn4A WT-S) variants into pGEM-T. I further sub-cloned Spn4A WT-S and WT-R into pcDNA3.1 using KpnI and NotI sites. The RFP-tagged Spn4A WT-R and WT-S variants were cloned by Martine Boutin in pcDNA3.1. For protein localization to the cytoplasm, I removed the SP. Spn4A fragments without the SP were amplified from either Spn4A WT-S (for non-RFP-tagged) or RFP Spn4A WT-R (for RFP-tagged) plasmids using KpnI or NotI flanking primers, then inserted into the empty vector pcDNA3.1 using the same sites. Mutations at S36P, S39R, H317R, and G374E were inserted using the Stratagene site directed mutagenesis method. A plasmid containing full length hNS was a gift from Dr. Hashimoto (Yale University). I cloned the hNS cDNA into pcDNA3.1 using BamHI and NotI flanking primers. A FLAG tag (F) after the SP was inserted using phosphorylated primers carrying the tag (Fig. 2.2). The primers amplified the entire plasmid, which were ligated together after PCR to form a circular vector for transformation into DH5α competent cells. Site-directed mutagenesis was used to make point mutations to hNS at S49P and H338R. All cloned constructs were sequenced to verify integrity. 2.2.4  Recombinant adenovirus constructs and production Martine Boutin had previously generated adenovirus without transgene (Ad. Empty) and  recombinant adenovirus expressing RFP Spn4A WT-S or RFP Spn4A WT-R (Ad. RFP WT-S, Ad. RFP WT-R). I cloned and produced adenovirus expressing the polymeric mutants RFP Spn4A S52-R or H338R-R following the AdEasy XL Adenoviral Vector System from Stratagene. Briefly, the two mutants were cloned into a pShuttle vector using KpnI and NotI digest sites, linearized with PmeI, and transformed into BJ5183-AD-1 competent cells (Stratagene), where in vivo recombination into the plasmid pAdEasy occurs (pAdEasy contains most of the adenovirus genome, but not genes for E1 and E3). Positive recombinants were selected based on the restriction digest pattern with PacI and amplified in DH5α competent cells. Plasmids were purified using the Nucleobond Midi kit from Clonetech, further digested with PacI, and phenol/chloroform extracted. Linearized plasmids were transfected into low-passage AD293 cells (Stratagene) using TransIT-LT1 (Mirus). AD293 cells carry the E1 gene, which is necessary for production of viral particles (Stratagene). Primary viral stocks were prepared by 15     harvesting and lysing transfected AD293 cells and used for large scale production of recombinant adenovirus, which was purified with the Adeno-X Maxi Purification kit (Clonetech). I also cloned RFP Spn4A H338R-S and H338R-C into the pShuttle vector using KpnI/NotI sites for adenovirus production by ViraQuest Inc (North Liberty, IA). I titered all viruses used in this thesis with Adeno-X Rapid Titer kit (Clonetech) and titers are reported in Appendix 2, Supplemental Table A2.2. 2.2.5  Transfection and infection conditions Two hundred thousand H4 or HEK293 cells were seeded per well the day before  transfection into 6-well plates and transfected at 70% confluency following the manufacturer’s protocol for TransIT-LT1 (Mirus). Specifically, transfections were performed using 1.5 μg DNA and 4.5 μL TransIT-LT1 transfection reagent. For immunofluorescence experiments, 40 000 H4 cells were seeded onto coverslips in 24-well plates the day before transfection and transfected with 0.3 μg DNA and 0.9 μL TransIT-LT1. Adenovirus infection experiments were performed using the same number of cells per well as the transfections. Cells were counted, resuspended in growth media to the proper concentration, immediately infected with viruses, and seeded onto 6- or 24-well plates. 2.2.6  SDS-PAGE, native PAGE, and western blot Media and cell samples were collected from both transfection and infection experiments  for western blot analysis. Samples from transfections were prepared in the absence of SDS loading buffer to allow for loading onto native PAGE. Media and cell samples from infections were immediately mixed with SDS loading buffer to inactivate viruses and were not used on native gels. After 48 hours of transfection, media and cell samples were collected. A total of 500 µL of media was collected from each well, centrifuged for 3 minutes at 3000 x g to pellet cell debris, and the top 300 µL kept. Cells were washed with PBS (phosphate buffered saline), scraped from the wells in PBS, and centrifuged for 3 minutes at 3000 x g. The supernatant was removed and cells were lysed in 100-120 μL of hypotonic buffer (20 mM Tris pH 7.4, 10 mM MgCl2, 10 mM  16     CaCl2) supplemented with fresh Complete, EDTA (ethylenediaminetetraacetic acid)-free protease inhibitor cocktail (Roche). For SDS-PAGE, 24 μL of cell lysate or media were mixed with 5 x SDS loading buffer containing fresh β-mercaptoethanol and boiled at 95°C for 10 minutes. Samples were separated on 8%, 10%, or 15% SDS-PAGE gels depending on the protein of interest. To detect polymers, 24 μL of cell lysates or media were mixed with 5 x native loading buffer lacking SDS and βmercaptoethanol, incubated for 30 minutes at 37°C, and loaded onto 8% native PAGE gels (5760). After 48 hours of infection, media and cells were harvested as described. To 120 μL of media samples, 30 μL of 5 x SDS loading buffer containing β-mercaptoethanol was added. Cells were lysed in 150-200 μL of hypotonic buffer pre-mixed with SDS loading buffer and βmercaptoethanol. Samples were immediately boiled at 95°C for 10 minutes to inactivate viruses and used for SDS-PAGE analysis. Electrophoresis was performed at 100-110 V. Protein on gels were transferred to nitrocellulose blotting membrane (Pall Corporation) using a BioRad Trans-Blot Semi-dry Transfer machine. Transfers were performed for 45 minutes at 25 V. Blots were blocked using Li-Cor Odyssey Blocking Buffer diluted 1:1 in PBS for at least one hour. Primary and secondary antibodies were diluted in Odyssey Blocking Buffer diluted 1:1 in 0.1% Tween in PBS (PBS-T). Primary antibodies used include 1:1000 mouse anti-FLAG M2 (Sigma, F1804), 1:1000 rabbit anti-FLAG (ABR Affinity BioReagents, PA1-984B), 1:1000 rabbit anti-β tubulin (Abcam, ab6046), 1:1000 mouse anti-Hsp47 (Stressgen, SPA-470), 1:500 mouse anti-His (Applied Biological Materials, G020) and 1:500 rabbit anti-caspase3 (Epitomics, 1476-1). Secondary antibodies were all diluted at 1:12000 and include IRDye 800CW donkey anti-mouse IgG (LI-COR, 926-32212), IRDye 800CW donkey anti-rabbit IgG (LI-COR, 92632213), IRDye 680CW donkey anti-mouse IgG (LI-COR, 926-32222), and IRDye 680CW donkey anti-rabbit IgG (LI-COR, 926-32223). Blots were scanned and quantified on an Odyssey Infrared Imaging System (Li-Cor Biosciences) using the Odyssey v2.0 and v3.0 software. Western blot quantification of intracellular and extracellular levels of Spn4A was performed by normalizing the band intensities of Spn4A to that of Hsp47 (loading control), as detected by SDS-PAGE. Normalized intensities are expressed as a percentage compared to the 17     WT. Statistical significance was calculated based on two-tailed Student’s t-test from three experiments performed in duplicates. 2.2.7  Immunofluorescence After 48 hours of transfection or infection, H4 cells were fixed for 30 minutes in 3.7%  formaldehyde. Cells were permeablized in 0.05% saponin in PBS (PBS-S) for 30 minutes. Primary labeling was performed using mouse anti-FLAG M2 (Sigma, F1804) or rabbit antiFLAG (ABR Affinity BioReagents, PA1-984B) at 1:100 dilution, rabbit anti-β tubulin (Abcam, ab6046), rabbit anti-calnexin (Sigma, C4731), or mouse anti-GM130 (BD, 610823) at 1:200 in PBS-S with 3% BSA for at least one hour. Secondary labeling was performed using Alexa Fluor 488 donkey anti-mouse IgG (Invitrogen, A-21202), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen, A-21206), Alexa Fluor 568 donkey anti-mouse IgG (Invitrogen, A10037), and Alexa Fluor 568 donkey anti-rabbit IgG (Invitrogen, A-10042) at 1:300 in PBS-S with 3% BSA for at least one hour. Lastly, the nuclei were stained for 25 minutes using Hoechst dye (Invitrogen) diluted in PBS to 5 µg/mL. Slides were mounted in mounting solution (2.5% DABCO in 90% glycerol, 20 mM Tris pH8.8). Fluorescence was visualized using an Olympus Fluoview FV1000 laser scanning confocal microscope. 2.2.8  Furin complex formation assay Enzyme-inhibitor complex formation of furin and Spn4A was assessed by incubating cell  lysates with recombinant furin and detecting the presence of a heat- and SDS-stable complex using SDS-PAGE. Per reaction, 100 ng of recombinant His-tagged human furin (R&D Systems, 1503-SE) was incubated with 15 μL of cell lysates in hypotonic buffer mixed with 15 μL of 2 x furin assay buffer (100 mM Hepes pH 7.5, 1 mM CaCl2, 0.5% Triton-X100) (36). The reaction proceeded for 10 minutes at 30°C and was quenched with a final concentration of 50 mM EDTA. SDS loading buffer was added to the reaction and the samples were boiled for 10 minutes at 95°C and loaded onto an SDS-PAGE for western blot analysis. Spn4A-furin complex formation was also assessed using HEK293-C4 cells that overexpress FLAG-tagged furin (92). Plasmids were transfected into HEK293-C4 cells and media were harvested after 48 hours for PAGE analysis, as described in sections 2.2.5 and 2.2.6.    18     2.2.9  MTS cell viability assay Cell viability was assessed using the CellTiter 96 AQueous One Solution Cell  Proliferation Assay (Promega) following the manufacture’s protocol. Two thousand H4 cells were infected and seeded into clear 96-well plates (Sarstedt Tissue Culture Plate Flat Bottom). After 72 hours, 20 µL of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium inner salt) solution was added to each well, the plates incubated for 45 minutes at 37°C, and absorbance readings taken at 490 nm (SpectraMax 190, Molecular Devices). 2.2.10 Flow cytometry RFP expression was quantified from infected cells using flow cytometry. Two hundred thousand H4 cells were infected and seeded into 6-well plates. After 48 hours of infection, cells were washed with PBS, trypsinized, and collected by centrifugation for 3 minutes at 3000 x g. The supernatant was removed and cells were resuspended in 900 µL of PBS. Cells were fixed by adding 100 µL of 37% formaldehyde and incubating at 37°C for 15 minutes. Cells were collected by centrifugation for 3 minutes at 3000 x g, washed in PBS, and resuspended in flow cytometry buffer (4% fetal bovine serum, 2 mM EDTA in PBS). Flow experiments were performed on a BD FACScan Flow Cytometer. A total of 30 000 events were collected per sample using CellQuest and analyzed with FlowJo v4.0.2 (TreeStar). Events with very low forward scatter, which indicates degenerated cells, were excluded from the analysis. 2.2.11 RNA extraction and cDNA synthesis RNA was extracted from infected cells for microarray experiments. H4 cells were infected and seeded into 6-well plates in duplicates. Cells were incubated for 48 hours, washed with PBS, lysed in RLT buffer containing β-mercaptoethanol, and passed through QiaShredder columns. RNA was extracted using the Qiagen RNeasy Mini kit with DNase on-column digest (Qiagen) or Qiagen RNeasy Plus Mini kit and eluted in 40 µL of RNase-free water. RNA purity was assessed with absorbance readings (A260: A280 ratios between 1.9-2.1 and A260:A230 ratios between 1.8-2.3) using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and integrity tested on agarose 2.2 M formaldehyde RNA gels.  19     Complementary DNA synthesis was performed using TaqMan Reverse Transcription Reagent (Applied Biosystems) with 200 ng of RNA and oligo dT primers in a reaction volume of 12.5 µL. To ensure RNA preparations were not contaminated with genomic DNA, end-point PCR was performed on 0.5 µL of each cDNA reaction using intron-flanking β-actin primers. 2.2.12 Microarray and data analysis Extracted RNA from three independent experiments was used on an Illumina HumanHT12 v3 Expression BeadChip. Microarray experiments were carried out by Dr. Anne Haegart and data statistical analysis by Dr. Robert Bell at the Vancouver Prostate Centre Microarray Facility (Vancouver, Canada). Briefly, RNA quality was verified using an Agilent 2100 Bioanalyzer, and 200 ng of all RNA samples were biotin-labeled with the Ambion Illumina TotalPrep RNA Amplification Kit and hybridized onto the Illumina BeadChip, following the Whole-Genome Gene Expression Direct Hybridization Assay Guide. Arrays were scanned using an Illumina iScan scanner. The quantified data was loaded into Illumina GenomeStudio, where array quality control was assessed. Raw data were imported into Agilent GeneSpring v7.3.1, where data was normalized. Fold changes (FCs) were calculated between averages of each condition and t-tests were performed between the conditions. Genes above the threshold cut-off of 1.7 fold (p<0.05), either up or downregulated, were considered significant and used for analysis. We used the Database for Annotation, Visualization and Integrated Discovery (DAVID v6.7) (93) for gene ontology and enrichment analysis. Briefly, gene lists were uploaded onto the DAVID online software, where various gene ontology terms are assigned to each gene. In a second step, genes are classified into functional annotation terms, and highly related/redundant annotation terms can be further grouped into annotation clusters. To examine the significance (p-value) of the functional annotation term, the probability of obtaining the number of genes in a term is compared to the total number of genes in that term from all the genes in the microarray, and is calculated by a modified Fisher’s exact test (93). To examine the significance (enrichment score [ES]) of the annotation clusters, the geometric mean of all the enrichment p-values of each annotation term in the cluster is calculated. Because the geometric mean is a relative score, the negative log transformation is applied onto the mean, and reported as ES. ES above two are considered significant.    20     2.2.13 Real-time PCR Quantitative real-time PCR (qPCR) was performed on an AB 7500 Fast Real-time PCR system with 7500 Software v2.0.1. Reaction components included 4 µL of 1:10 diluted cDNA, Brilliant II Fast qPCR Master Mix (Stratagene), probes from Roche Universal Probe Library, and primers from Integrated DNA Technologies in a 20 µL reaction. Roche probes and primer sets are listed in Appendix 2, Supplemental Table A2.3. Cycling conditions were 95°C for 5 minutes followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Differential gene expression was calculated by the ∆∆CT (threshold cycle) method (94) using β-actin as the reference amplicon. For all real time assays, acceptable doubling efficiencies (86% - 110%) were ensured by calculating PCR efficiency from slopes of standard curves (CT vs. serially diluted cDNA, slope between -3.1 to -3.7). FC averages were calculated from three independent experiments performed in duplicates and presented with the standard error of the mean (SEM). Statistical significance was calculated from two-tailed Student’s t-tests.    21     2.3  Results  2.3.1  Phylogenetic analysis of Spn4A Our goal was to use Drosophila Spn4A as a tool to study the serpinopathy associated  with hNS. Thus, we assessed the relationship of Drosophila Spn4A with human serpins. Serpins are classified into 16 clades (A to P), with an additional ten unclassified orphan serpins, according to their amino acid sequence and secondary structure alignments (5). In general, clades A to I contain vertebrate serpins, while clades J to P contain serpins in nonvertebrate species (5). For comparative analysis, we selected one archetypal member from each vertebrate clade (A to I) representing serpins found in humans, Triticum aestivum (bread wheat) Serpin Z1A belonging to the plant clade P (5), and Spn4A which belongs to the insect clade K (5;95). We made an amino acid alignment of these sequences and built a phylogenetic tree using Serpin Z1A as the outgroup sequence. We summarized the trees resulting from a Bayesian tree search into a majority rule consensus tree to explore the relationship between Spn4A and other serpins. Fig. 2.3 showed that hNS and PAI-1 (plasminogen activator inhibitor 1) were highly related, while Spn4A was most closely related to both groups. In terms of sequence characteristics, Spn4A shared a higher amino acid alignment score with hNS compared to PAI-1 (Table 2.2). Further, Spn4A contains a 13 amino acid C-terminal targeting sequence, which is shared only between Spn4A, hNS, pancpin, and Hsp47 among more than 500 serpins tested (86). Spn4A is expressed specifically in Drosophila neurons and like hNS, is directed to the secretory pathway (35). The overexpression of either Spn4A or hNS produces similar molting defects in Drosophila (35), despite their divergent protein functions. Further, the hNS amino acids related to disease are conserved in Spn4A (Fig. 2.4). Therefore, we predicted that Spn4A could be used as a tool to study serpinopathy associated with FENIB. 2.3.2  Development of Spn4A variants In Drosophila, Spn4A possesses an N-terminal signal peptide and a C-terminal HDEL  motif (Spn4A-R or WT-R), which suggests it may cycle between the ER and early Golgi compartments (34;36). Since hNS is a secreted serpin whose mutants are partially retained in the ER as polymers (61), we used a secreted Spn4A variant (Spn4A-S or WT-S) that lacked the HDEL signal (Fig. 2.2) to study the trafficking and possible intracellular polymer retention of Spn4A. To test if the polymer formation was an ER-specific phenomenon, we engineered a 22     cytoplasmic-localized Spn4A by deletion of the signal peptide, designated Spn4A-C or WT-C (Fig. 2.2). Notably, Spn4E is a naturally occurring splice variant of Spn4 that contains the HDEL signal but not the N-terminal signal peptide, like Spn4A-C. All three constructs have been tagged with RFP (91), a monomeric red fluorescent protein, to facilitate immunofluorescence and in vivo experiments. The hNS constructs were cloned as positive controls for establishing methods. Four mutations at sites S36P, S39R, H317R, and G374E (equivalent to human S49P, S52R, H338R, and G392E) were introduced to each WT construct (Fig. 2.2). We will use the hNS template for Spn4A amino acid numbering from here on to avoid confusion. We have cloned one negative control mutant, S49A, in order to verify that mutations linked to FENIB, and not any point mutation, formed serpinopathy specific polymers. 2.3.3  Polymer formation studies of Spn4A variants Mutant polymeric hNS are retained in the ER as previously demonstrated through native  PAGE and confocal imaging analysis (60). Following the same polymer detection techniques, we confirmed that Spn4A mutants also formed polymers and localized to the ER in human H4 neuroglioma cells. First, we demonstrated that Spn4A mutants formed polymers similar to hNS mutants by native PAGE analysis. FLAG-tagged hNS WT as well as S49P and H338R mutants were cloned and transfected into H4 cells to validate our system. H4 cells were harvested and cultured media collected for both non-denaturating and SDS-PAGE analysis. Polymers were detected as ladders on a native PAGE only when hNS mutants were expressed (Fig. 2.5A). Also, hNS mutants had higher levels of protein in cell lysates and less in media samples compared to the WT, as seen on the SDS-PAGE (Fig. 2.5A). Accordingly, we performed the same experiments with Spn4A WTS and mutants. We demonstrated that Spn4A mutants, with the exception of S49A, could also form polymers (Fig. 2.5B). We used SDS-PAGE analysis to standardize protein expression to Hsp47 (heat shock protein 47 kDa) and to quantify intracellular and extracellular protein amounts. Spn4A variants, with the exception of S49A, were retained intracellularly to a significant extent and had an approximately 60% reduction extracellularly compared to the WT protein (Fig. 2.5C). Most likely, the mutants were secreted at a lower level or were more prone to extracellular proteolysis due to greater protein destabilization. Further, we tested RFP-tagged Spn4A-S, with similar 23     results to the non-tagged (Fig. 2.5D). We noted that Spn4A-R (retained) mutants, RFP-tagged and untagged, exhibited the same ladder formation in native PAGE analysis (data not shown). Additionally, cytoplasmic Spn4A mutants, both RFP-tagged and untagged, were capable of forming polymers as detected by native PAGE, unlike the WT-C (Fig. 2.5E). Spn4A-S had an approximately 4 kDa increase in molecular weight compared to Spn4A-C, presumably due to its glycosylation in the secretory pathway (Fig. 2.5B and E). We then fixed transfected H4 cells and used cellular markers to visualize the localization and staining patterns of both WT and mutant hNS and Spn4A. The hNS mutants exhibit a specific punctate characteristic in transfected monkey kidney fibroblast COS-7 cells (60;61) and in stably transfected rat pheochromocytoma PC12 cells (61). The punctate phenotype of hNS mutants was reproduced in H4 cells (Fig. 2.6A). We further demonstrated similar staining patterns in Spn4A H338R-S but not WT-S (Fig. 2.6B and Appendix 3, Supplemental Fig. A3.1). Spn4A WT-R localized to the ER, but not the Golgi (Fig. 2.6C and D) and Spn4A WT-C stained the cytoplasm (Fig. 2.6E), while their mutant counterparts exhibited distinct dotted features (Fig. 2.6C to E). We noted that some cells expressing cytoplasmic mutants exhibited the dispersed cytoplasmic staining pattern like the WT (data not shown). Immunofluorescence experiments of H4 cells transfected with RFP Spn4A constructs were performed first with the non-RFP-tagged constructs to ensure similar staining patterns (data not shown for non-RFP-tagged constructs). 2.3.4  Spn4A mutants do not have inhibitory activities in vitro and in cellulo WT Spn4A is an inhibitor of human furin and Drosophila PC2 (36). It forms complexes  with furin with a Ki (inhibitory constant of the enzyme-inhibitor complex) of 13 pM and a 1:1 stoichiometry of inhibition (36). To assess the inhibitory activities of polymeric mutants, we performed complex formation assays using recombinant His-tagged furin or HEK293-C4 cells that overexpress FLAG-tagged furin (92). Inhibition is achieved through a covalent complex between Spn4A and furin (Fig. 2.7A) that is heat- and SDS-stable, and therefore detectable by SDS-PAGE and western analysis. Incubation of furin with cell lysates transfected with Spn4A WT-R resulted in the detection of a high molecular weight band, but not with those expressing Spn4A H338R-R (Fig. 2.7B). Both WT-R and mutant H338R-R resulted in some cleaved product, suggesting that although Spn4A H338R-R does not have inhibitory activity against furin, its reactive center loop may be accessible to cleavage. 24     We also tested all of the Spn4A mutants for inhibitory activities in HEK293-C4 cells. Transfected cells were analyzed for polymer formation by native PAGE and complex formation by SDS-PAGE (Fig. 2.7C). We found that Spn4A mutants were able to form polymers in HEK293 cells as indicated by the ladder formation in native PAGE (Fig. 2.7C). Free furin was detected as doublet bands intracellularly and singlet bands extracellularly on SDS-PAGE, as previously reported (92). Enzyme-inhibitor (EI) complexes were observed intracellularly and extracellularly as high molecular weight bands in cells transfected with Spn4A WT-S, negative control mutant Spn4A S49A-S, and RFP Spn4A WT-S (Fig. 2.7C and D). Cells expressing serpinopathy-linked mutants did not form complexes intracellularly or extracellularly with furin, except those transfected with Spn4A S49P-S (Fig. 2.7C and D). A high molecular weight band was detected in the media samples of cells expressing Spn4A S49P-S, but not intracellularly. However, the amount of extracellular free furin was visibly greater in the media sample from cells transfected with S49P compared to the WT and S49A negative control mutant (Fig. 2.7C), which may bring into question the identity of the high molecular weight band (discussed below). 2.3.5  Cellular characterization of recombinant adenoviruses expressing Spn4A WT and mutants Next, we established an infection system using recombinant adenoviruses in order to  achieve higher and dose-dependent expression levels of Spn4A. We reasoned that very high expression levels of Spn4A (higher than can be achieved by transfection) might exacerbate the cellular effects of polymers. In addition, the adenovirus expression system might allow us to delineate how different doses of polymer formation affect cells by infecting with different multiplicity of infection (MOI). We constructed recombinant adenoviruses containing two polymeric mutants that were ER-retained and one mutant that was secreted. Since the ER is the site of polymer localization, we reasoned that cellular stress signals would be amplified if all polymers were retained. On the other hand, if Golgi or extracellularly-localized polymers are affecting the cell, then a comparison of polymers that are retained to those that are trafficked through the secretory pathway could elucidate those specific effects. We chose to make recombinant adenovirus expressing S52R or H338R retained mutants (Ad. RFP S52R-R and Ad. RFP H338R-R) because they formed the most polymers indicated on native PAGE analysis. In their hNS counterpart, the H338R mutant causes an earlier onset of disease than S52R (45). We have also generated 25     secreted Ad. RFP H338R-S. We used adenoviruses without transgene (Ad. Emp [Empty]) as a negative control, Ad. RFP WT‐R for comparison with the S52R‐R and H338R‐R mutants, and Ad. RFP WT-S for comparison with H338R-S. Ad. Emp, Ad. RFP WT-R, and Ad. RFP WT-S were previously generated by the Jean lab. We used several methods to quantify the levels of expression at various MOIs. First, we infected H4 cells at MOI 1 and 5 and used western blot analysis to ensure a dose response with increasing MOI (Fig. 2.8A). Second, for cellular response analysis, we titrated for the MOI sufficient to express Spn4A in all cells using flow cytometry (Fig. 2.8B). We found that infections at MOI 50 were sufficient for 100% of cells to contain retained Spn4A-R and at least 95% of cells to contain secreted Spn4A-S. Third, the dose response was further examined using confocal experiments (Fig. 2.8C). Cells infected with MOI 1 compared to those at MOI 50 expressed less Spn4A. Fourth, consistent with flow cytometry data, cells infected at MOI 50 showed that all cells expressed Spn4A (Fig. 2.8D). Further, staining patterns of WT and polymer-forming Spn4A in adenovirus infected cells (Fig. 2.8D) were the same as those from transfection experiments (Fig. 2.6). Last, MTS cell viability assays ensured that transgene expression of the retained Spn4A-R (Fig. 2.8E) and secreted Spn4A-S (data not shown) by adenovirus was not cytotoxic in H4s. 2.3.6  Microarray strategy We showed that Spn4A mutants had the same cellular signatures as hNS polymers in  mammalian cells and predicted that Spn4A polymers would modulate the same cellular stress pathways as hNS. In order to address the cellular response to serpin polymers, we analyzed the transcriptome of H4 cells infected with Spn4A polymeric mutants. H4 cells were infected with Ad. Emp, RFP WT-R, RFP S52R-R, RFP H338R-R, RFP WT-S, or RFP H338R-S. RNA was extracted and used on the Illumina HumanHT-12 v3 Expression BeadChip, targeting more than 25 000 annotated genes using 48 000 probes. Expression data from Ad. RFP WT-R, S52R-R, H338R-R, WT-S, and H338R-S were compared to Ad. Emp (Fig. 2.9). Genes with altered expression levels due to mutant Spn4A polymers were compiled. Only FCs with a threshold cut-off of 1.7 fold (p<0.05) were considered significant and used for analysis. Using these criteria, 71 genes in S52R-R, 74 genes in H338R-R, and 45 genes in 26     H338R-S expressing cells reached significance. Next, these genes were compared to those significantly regulated by the WT Spn4A (Fig. 2.9). We found that a total of 18 genes were unique to the mutants while 76 genes overlapped with the WT (Fig. 2.9). These two different gene lists were separately analyzed (sections 2.3.7 and 2.3.8) and the altered genes from the expression of retained and secreted Spn4A WT will be discussed in Chapter 3. 2.3.7  Spn4A WT and mutant overexpression induces ER stress genes The expression of Spn4A, regardless of inhibitory function or polymer formation,  induced a response in 76 genes (Appendix 5, Supplemental Table A5.1). Gene ontology analysis was performed using DAVID. From 76 similarly regulated genes, 35 upregulated and 41 downregulated genes were separately submitted to DAVID Functional Annotation Clustering analysis. Genes were classified into gene ontology groups, enrichment for each annotation term was calculated, and annotation terms further grouped according to highly similar annotation terms. Notably, the most enriched cluster (ES = 4.3) from the upregulated genes was the response to ER stress (Table 2.3). Other enriched clusters with ES above two included annotations for heat shock proteins (ES = 2.5) and ER membranes (ES = 2.4), among others. The enrichment scores for the downregulated gene list were not as high as the upregulated, and are shown in Appendix 3, Supplemental Table A3.1. Notably, seven ER stress genes (Table 2.4) were among the top 12 most upregulated genes in response to WT-R, S52R, and H338R-S expression, but not WT-S. These genes are common to the UPR and include chaperones, a transcription factor controlling ER stress and cell cycle genes, a component of the ER-associated degradation system, and a regulator of ERinduced apoptosis. All seven genes were similarly regulated by Spn4A WT-R, the two retained mutants, and Spn4A H338R-S, except for secreted Spn4A WT-S, which did not induce the upregulation of DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP) as all others had. We tested the mRNA levels of the ERAD component HERPUD1 (Homocysteineinducible, ER stress-inducible, ubiquitin-like domain member 1), transcription factor DDIT3, and ER chaperone HSPA5 (heat shock 70 kDa protein 5, also known as BiP) by qPCR. We confirmed that DDIT3 and HSPA5 were all significantly upregulated by Spn4A WT or mutant expression, and that HERPUD1 is upregulated in cells expressing S52R-R (Fig. 2.10). Further, HSP5A was significantly more upregulated by H338R-R expression, compared to WT-R. We 27     could not validate the upregulation of HERPUD1 in response to WT-R and H338R-R (Fig. 2.10), as suggested by the microarray. We predicted that our analysis method may have a false discovery rate of approximately 25%. 2.3.8  Retained and secreted non-inhibitory and polymeric Spn4A mutants affect few genes in the H4 transcriptome Comparisons of Spn4A WT-R or WT-S with polymeric mutants identified 18 genes that  were differentially expressed in the presence of secreted and retained mutants (Fig. 2.9). Unique gene expression changes in each mutant group were identified by comparisons against each other (Fig. 2.11, Table 2.5). We reasoned that the response to non-inhibitory and polymeric Spn4A buildup in the ER should be detected in both S52R-R and H338R-R mutants as well as the secreted H338R-S. Surprisingly, KRT81 (keratin 81), which codes for fibrous structural proteins (96), was the only gene induced by all three mutants. Further, the H338R-S secreted mutant would reveal effects stemming from Golgi transit and extracellular polymer accumulation when compared with the retained mutants. We found that six genes were uniquely regulated by the secreted mutant (Table 2.5). Additionally, five genes were differentially regulated by both retained mutants (Fig. 2.11, Table 2.5). Using DAVID functional annotation chart, we grouped all 18 genes according to their ontology (Table 2.6). Functional annotations included cell adhesion, aminotransferase activity, carbohydrate binding, cartilage development, and calcium binding. Also, polymorphisms existed for most genes. There was no clear trend in the regulation for each annotation and how they relate to the presence of polymers in a cell was unclear. The most downregulated gene was MGP (matrix gla protein), which encodes for an extracellular matrix protein that binds calcium and may function in the migration of glioblastoma cells (97). The most upregulated gene was SPRR2D (small proline-rich protein 2D) in the retained mutants. SPRR2D is normally expressed in the cytosol of keratinocytes and becomes crosslinked to the membrane resulting in a protective barrier beneath the membrane (98). We have validated the SPRR2D upregulation in the mutants, but were unable to show that expression was unique to mutants (Fig. 2.13). As detected by microarray analysis, RFP WT-R upregulated SPRR2D by 2.00 fold, but was excluded from the analysis based on its p-value (p = 0.07), which did not reach the cut-off. By qPCR, SPRR2D had a FC of 30.31 (p<0.01) and was not significantly different from the mutants (Fig. 2.12). 28     2.4  Discussion  2.4.1  Molecular and cellular comparisons of Drosophila Spn4A and human neuroserpin mutants Despite the divergent functions of Drosophila Spn4A and hNS, both proteins were  closely related compared to other serpin clade prototypes by phylogenetic analysis and have brain-specific expression. Importantly, disease-linked amino acid residues are conserved in both serpins. Thus, we have evaluated the ability of Spn4A mutants to form polymers in order to study the cellular pathogenesis and organismal impacts of the serpinopathies. Following the biochemical and cellular work on hNS mutants, we examined the ability of mutant Spn4A to form polymers. We used hNS WT and two mutants as positive controls. Under our experimental conditions, hNS mutants formed polymers intracellularly and extracellularly, detected by western blots and immunofluorescence, as previously reported (57-60). However, low levels of monomeric mutant hNS were detected intracellularly. This is in contrast to native PAGE results in COS7 cells (61), where hNS S49P monomers were detected extracellularly, but not intracellularly. The discrepancy may be due to differences in cell types or different cell lysis conditions. Nonetheless, we were successful in replicating ladder-like formations on native PAGE, punctate characteristics by confocal microscopy, and ER retention using SDS-PAGE and immunofluorescence. Next, we asked if Spn4A mutants encoding amino acid substitutions equivalent to those found in hNS FENIB-causing mutants also formed polymers. As a negative control, we engineered the Spn4A S49A mutant, whose equivalent mutation in hNS has not been associated with disease. Our PAGE analysis confirmed that S49A failed to form polymers and was secreted, like WT Spn4A. In contrast, mutants with disease-associated residue substitutions formed polymers intracellularly and extracellularly, and were not secreted to WT levels. RFP tag and cytoplasmic localization did not affect the ability of mutants to form polymers. We noticed that while immunofluorescence images were similar between Spn4A and hNS mutants, their oligomeric states may be different as detected by native PAGE. In the extracellular samples, Spn4A mutants formed low and high order oligomers while human neuroserpin mutants only formed high molecular weight species. Perhaps Spn4A mutants formed polymers slower compared to human neuroserpin so that we were able to capture different 29     oligomeric species. By incubating mutant Spn4A for a longer period of time before native PAGE loading, we can test if lower molecular weight oligomers are lost. If they can still be detected, we suggest that Spn4A mutants can form different intramolecular linkages. Precedence for different structural multimer formation is best exemplified by antithrombin, which exhibits selfterminating dimers as well as higher order polymers (99;100). We also found that the Spn4A G392E mutants were barely detectable by native PAGE. However, intracellular and extracellular proteins levels were detected by SDS-PAGE at levels comparable to other Spn4A mutants. Further, punctate staining patterns were detected by immunofluorescence for Spn4A G392E mutants. The reason for the loss of visible bands on native PAGE is unclear, but may be due to a different polymer fold that concealed the FLAG tag, and was therefore undetectable with FLAG antibodies under native conditions. The use of antibodies directed at other epitopes may be useful in clarifying whether polymers of a different conformation are formed. Spn4A G392E may have different molecular properties to the hNS G392E mutant, which forms high order oligomers (61). Lastly, we studied the inhibitory activities of the Spn4A mutants. Functional studies of hNS mutants have revealed that hNS S49P is partially active (57). The ability of Spn4A S49P to form an EI complex in the extracellular environment, unlike the other mutants, indicates that it may be similar to its human counterpart. It also suggests that the mutation did not compromise catalytic activity. The identity of the high molecular weight band is unclear. Unlike the media samples from WT-S and S49A-S expressing cells, the extracellular furin levels did not decrease in the presence of Spn4A S49P-S (Fig. 2.7C). It is possible that S49P-S inhibited and formed SDS-stable EI complexes with another extracellular protease. The use of furin antibodies may clarify the identity of the high molecular weight band. Further, the ability of Spn4A S49P mutants to complex with furin may be assessed using recombinant furin. We did not detect intracellular EI complexes in S49P-S expressing cells (Fig. 2.7C). The lack of complexes may be due to the retention of Spn4A polymers in the ER and the localization of target proteases to the Golgi and extracellular space. For example, since furin localizes mainly to the Golgi or is shed to the extracellular space (92;101), insufficient levels of Spn4A S49P may reside in the Golgi (due to ER retention) at the steady state for complexes to form and be detected.    30     2.4.2  Cellular model development We have developed a new mammalian cell-based system to study serpinopathies using  Spn4A mutants. Our model recapitulates the known features of mutant neuroserpin including polymer formation, ER retention, and lack of inhibitory activity. Our cell-based system has three advantages over current cellular models. First, we have developed RFP fusions of serpins and demonstrated that the tag does not affect polymer formation. The RFP tag has facilitated imaging of serpins in cells and flies (Supplemental Fig. A3.3). Importantly, the tag will allow us to visualize Spn4A trafficking, formation of polymers, and/or interactions with cellular proteins over time in live cells. For many proteins associated with neurodegenerative diseases, FRAP (fluorescence recovery after photobleaching), FLIP (fluorescence loss in photobleaching), and time-lapse microscopy have been successfully applied to visualize the dynamic process of protein aggregate remodeling and defective intracellular transport mechanism (for example, monitoring rates of vesicle transport), among others (102). Second, we have developed an adenovirus delivery system to allow titration of mutant serpin expression using different MOIs. Polymer expression levels are important for pathogenesis as demonstrated in hemizygous versus homozygous transgenic mice expressing the hNS G392E mutation (80). We now have the molecular tools to study the gene dosagedependent accumulation of mutant serpin. Third, we have localized polymers to different compartments of a cell. Spn4A is naturally ER-retained. We showed that deletion of C-terminal HDEL allowed secretion of Spn4A and removal of the N-terminus signal peptide targeted Spn4A to the cytoplasm. We demonstrated polymer formation in the cytoplasm, ER, and extracellular space. The current hypothesis is that polymer formation in the ER leads to ER stress-induced changes followed by cell dysfunction and death (103). In our model systems, we may address if mutant serpins in the Golgi and extracellular space have additional cellular effects compared to ER-retained Spn4 variants. Further, we showed that polymer formation is not exclusive to the secretory pathway and can occur regardless of the ER environment. Interestingly, cytoplasmic polymers were not observed at the same frequency or amount as secretory pathway polymers. We suggest that while polymerization is not the result of the ER environment, confinement of mutant serpins to a membrane enclosed space aggravates polymer formation.  31     2.4.3  Transcriptome profiling of retained and secreted Spn4A mutants In order to better understand the cellular response to polymer accumulation, we used the  adenovirus delivery system to express secreted and retained Spn4A WT and mutants in H4s for microarray analysis. Our assumption was that Spn4A polymers would regulate the same cellular stress pathways as human neuroserpin polymers in mammalian cells. Experimental conditions were chosen based on the expression of Spn4A in all cells and visible punctate inclusions as visualized by confocal microscopy. We hypothesized that the expression of polymers would specifically activate cellular stress genes. However, a general response to the overexpression of Spn4A was ER stress, regardless of protein activity or polymer formation. Using a MOI of 50, more than one infectious virus particle was expected to enter one cell, and thus a very high Spn4A expression was expected. It is likely that proteins overwhelmed the ER by overexpression and induced ERassociated gene expression, masking possible stress responses to polymer formation. HSPA5 (also known as BiP) is a chaperone that regulates the UPR and was upregulated in response to retained Spn4A-R WT and mutants. Under our experimental conditions, we suggest that UPR was a general response to Spn4A overexpression, regardless of localization, function, or oligomerization. From our qPCR assay, we detected upregulation of HERPUD1 in response to the retained S52R-R mutant but not WT-R, in contradiction to the microarray data. We believe that qPCR, which can detect small amounts of DNA through amplification, may be a more sensitive detection method compared to hybridization on a microarray. Currently, we estimate a false discovery rate of 25% (we validated nine of 12 genes tested). HERPUD1 codes for a regulator of ERAD, which promotes proteasome-mediated protein degradation. ERAD is implicated in the clearance of hNS polymeric mutants, as proteasome inhibition results in a buildup of mutant hNS but not WT hNS (78). Further investigation into the regulation of HERPUD1 and proteasomemediated degradation of Spn4A polymers is needed. Microarray comparisons showed that DDIT3 (also known as CHOP) was specifically upregulated in H338R-S, but not WT-S. Signaling from DDIT3 may induce apoptosis (63), and whether the gene regulation difference is significant needs to be validated by qPCR.  32     Last, we analyzed genes that were specifically regulated in response to Spn4A mutants. We did not observe specific changes in genes related to UPR, EOR, autophagy, or apoptosis. However, we did identify 18 significantly changed genes in response to polymer accumulation. Unfortunately, we could not validate by qPCR that SPRR2D was specifically upregulated in response to retained Spn4A polymers. The validation of the rest of the genes is of immediate interest. 2.4.4  Conclusions and future directions FENIB is characterized by destabilizing mutations in neuroserpin leading to pathogenic  polymer formation, decreased extracellular levels, and inclusion body formation. We showed that the Drosophila brain-specific serpin, Spn4A, has equivalent amino acids to its human homolog that when mutated, recapitulated the same cellular signatures. Further, we demonstrated that polymer formation is not an ER-specific event. We used this cellular model for transcriptome analysis of the cellular response to the accumulation of polymeric Spn4A. However, we found that ER stress was a general response to Spn4A overexpression and few genes were regulated specifically in response to Spn4A polymer-forming mutants. We have performed qPCR experiments to test the expression levels of ER stress genes using retained Spn4A WT-R and mutants. We suggest that for future qPCR assays, comparisons between secreted WT-S and H338R-S will more clearly reveal differences in ER stress regulation, since WT-R already induced high ER stress levels. Of interest are HERPUD1 and DDIT3, which may be upregulated in the presence of polymeric mutants but not the WT, as suggested by qPCR and microarray experiments. Functional assays will be necessary to evaluate the consequences. To test if ERAD is activated, we will compare the quantity of Spn4A WT and mutants with and without proteasome inhibition. A buildup of protein after inhibition would suggest that the proteasome is normally activated to degrade the proteins. Further, DDIT3 is a transcriptional regulator whose activity can be monitored using a promoter-luciferase reporter assay. However, it is unlikely that DDIT3 signaling leads to apoptosis in the neuroglioma cells as we have grown them for up to five days without toxic effects. Further, cells transiently transfected with Spn4A polymeric mutants did not activate caspase 3 after 48 hours (data not shown). The 17 changes in gene expression that were regulated in response to Spn4A polymers, but not the WT, will need to be validated by qPCR. Once true targets have been confirmed,  33     western blotting and functional assays can be designed to evaluate the cellular response to the polymers. The transcriptome analysis did not reveal obvious toxic effects in response to the polymers. Indeed, previous studies performed in cell lines were unable to detect toxic effects (61). Perhaps H4 neuroglioma cells may tolerate serpin polymer expression due to their proliferative ability as a cancer cell line. It may be beneficial to use primary neuron cultures for further examination of the toxic effects of serpin polymers. Lastly, transgenic Drosophila will help decipher the effects of Spn4A variants in vivo. In collaboration with Dr. Carl Hashimoto, we generated transgenic Drosophila overexpressing retained and cytoplasmic RFP Spn4A WT and RFP Spn4A H338R. The secreted counterparts will be generated for future studies. Currently, RFP expression in fly pupae has been confirmed (Supplemental Fig. A3.3), but no detectable phenotypic changes in the flies generated were found (personal communication). Further characterization of the transgenic Drosophila to ascertain the effects of Spn4A polymer-forming mutants in vivo is of great interest.  34     Figure 2.1. Unfolded protein response signaling pathways. The chaperone BiP binds IRE1, ATF6 and PERK under non-stressed conditions. Accumulation of unfolded proteins causes BiP to dissociate with the sensors leading to their activation. Activated IRE1 alternatively splices XBP1 mRNA, ATF6 is activated by proteolysis, and PERK phosphorylates eIF2. Together, UPR signaling leads to upregulation of ERAD, chaperones, lipid synthesis, anti-oxidative stress response, amino acid metabolism, and/or apoptotic genes. Reprinted from Seminars in Cell & Developmental Biology, 18, Malhotra JD and Kaufman RJ , The endoplasmic reticulum and the unfolded protein response (62), 716-731, © 2007, with permission from Elsevier.  35     Figure 2.2. Serpin variants used in this study. We have three wild type Spn4A constructs with different predicted cellular localization, with and without tagged-RFP, and one human neuroserpin (hNS) construct, without RFP. Construct names for Spn4A indicate predicted localization (R for ER-retained, S for secreted, C for cytoplasmic) as indicated. Key characteristics of the constructs include SP (signal peptide) for targeting to the secretory pathway, HF (his and FLAG tag) or F (FLAG tag), RFP, RSL (reactive site loop), and HDEL ER-retention signal. From these wild type constructs, we have made single point mutations at sites S36, S39, H317, and G374 (triangles) in Spn4A, according to the human neuroserpin serpinopathy-associated residues (S49P, S52R, H338R, and G392E), and single point mutations in human neuroserpin at positions S49 and H338.  36     Figure 2.3. Phylogenetic relationship of Drosophila Spn4A to one archetypal member of each serpin clade (A-I) that contains human serpins. The tree is a majority rule consensus of the set of trees resulting from a Bayesian search method. Triticum aestivum Serpin Z1A was used as an outgroup. The values on the internal nodes reflect group support. Label prefixes “Hs”, “Dm”, and “Ta” were used for genes of human, Drosophila, and wheat, respectively. The bracketed letter is the clade for which the serpin belongs to. The lineage leading to Spn4A is bolded. The scale bar represents the number of substitutions per site.  37     Figure 2.4. Sequence alignment between human nueroserpin and Spn4A. Residues are fully conserved (highlighted), have high identity similarities (red text), or are not conserved (black text). Serpinopathy-associated residues (S49P, S52R, H338R, and G392E) are asterisked. Sequences were aligned using Espript 2.2 (104).  38     Figure 2.5. Polymer formation studies of human nueroserpin and Spn4A WT and mutants and quantification of intracellular and extracellular levels of Spn4A WT and mutants. A) Native PAGE and SDS-PAGE analysis of hNS WT and mutants. Human neuroserpin, empty vector, and wildtype are abbreviated as hNS, EV, and WT respectively. H4 cells were transfected as labeled. Cultured media were collected (extracellular samples) and cells were lysed (intracellular samples) after 48 hours, ran on a native PAGE to detect polymer formation and SDS-PAGE to quantify total amounts. Western blot analysis was performed using anti-FLAG antibodies for Spn4A (green) and anti-Hsp47 antibodies for Hsp47 (red) as a loading control. B) Native PAGE and SDS-PAGE analysis of secreted Spn4A WT-S and mutants, as described above. C) Quantification of intracellular and extracellular amounts of secreted Spn4A WT-S and mutants. Using SDS-PAGE such as in (B), we normalized the band intensities of Spn4A to Hsp47, and calculate the percent expressed compared to the WT. Percentages are averages ± SEM (standard error of the mean), and p-values have been calculated with a two-tailed student ttest from three experiments performed in duplicates. With the exception of the S49A mutant, the other four mutants had significantly increased amounts of intracellular protein (p<0.05*) and decreased amounts of extracellular protein (p<0.0001**) compared to the WT. D) and E) Native PAGE and SDS-PAGE analysis of secreted RFP Spn4A WT-S and mutants (D) and cytoplasmic Spn4A WT-C and mutants, with and without RFP (E), as described in (A).  39     40     Figure 2.6. Localization and staining patterns of human neuroserpin and Spn4A WT and mutants. H4 cells were transfected with the WT or mutants of A) FLAG-tagged hNS, B) RFP Spn4A-S, C) and D) RFP Spn4A-R, or E) RFP Spn4A-C, as labeled. After 48 hours, they were fixed and probed for hNS (red) with anti-FLAG antibodies, ER (green) with anti-calnexin antibodies, Golgi (green) with anti-Gm130, or tubulin (green) with anti-tubulin, and stained for nuclei (blue) with Hoechst dye. See Appendix 3, Supplemental Fig. A3.1 for enlarged figures.  41     A)  42     B)  43     C)  44     D)  45     E)  46     Figure 2.7. Heat and SDS-stable complex formation assay between furin and Spn4A WT and mutants. A) Branched pathway mechanism of serpins. Serpins (I) encounter protease (E) and form a reversible complex, EI. Cleavage of the serpin leads to the short-lived enzyme intermediate [EI’]. Either the inhibitory pathway proceeds to a stable EI* complex or the substrate pathway continues with proteolysis of the serpins. B) In vitro EI* complex formation assay. H4 cells were transfected with Spn4A WT-R or H338R-R for 48 hours. Cells were harvested, lysed, and incubated for 10 minutes with His-tagged recombinant furin at 30°C. The reaction was quenched with 50 mM EDTA, ran on an SDS-PAGE, and probed with anti-His antibodies for Spn4A and furin (green) and anti-tubulin antibodies (red), as a loading control, for western blot analysis. C) In cellulo EI* complex formation assay of secreted Spn4A WT-S and mutants. FLAG-tagged furin overexpressing HEK293-C4 cells were transfected as labeled for 48 hours. Empty vector and wildtype are abbreviated as EV and WT, respectively. Media was collected (extracellular), cells were lysed (intracellular), and loaded onto a native PAGE or boiled for 10 minutes in SDS loading buffer for SDS-PAGE. Western blot analysis was performed using anti-FLAG antibodies for Spn4A and furin (green) and anti-Hsp47 antibodies for Hsp47 (red) as a loading control. D) In cellulo EI* complex formation assay for secreted RFP Spn4A WT-S and H338R-S, as described in (C).  47     Figure 2.8. Qualitative and quantitative measurements of Spn4A expression levels as delivered by recombinant adenoviruses and cell viability measurements of virus-induced toxicity. A) Western blot analysis of H4s infected at MOI 1 or 5. Cells were infected for 48 hours, lysed in SDS buffer, and loaded onto an 8% SDS-PAGE. For western blotting, anti-FLAG for Spn4A (green) and anti-Hsp47 (red) were used. B) Flow cytometry quantitation of RFP expressing H4s. Cells were infected for 48 hours with Ad. RFP WT-R or WT-S at different MOIs, or Spn4A mutants at MOI 50. Cells were fixed in 2% formaldehyde and RFP expressing cells were quantified on a flow cytometer. The number in the label represents MOI. Example raw data are shown in Appendix 3, Supplemental Fig. A3.2. C) Confocal analysis of infected cells expressing Spn4A H338R-S at MOI 1 and MOI 50. H4 cells were infected for 48 hours, fixed, labeled using anti-calnexin for ER (green), and stained for the nuclei (blue) using Hoechst dye. D) Confocal analysis of infected cells at MOI 50. H4 cells were infected as labeled and fixed after 48 hours. Cells were labeled using anti-calnexin for ER (green) or anti-Gm130 for the Golgi (green), and stained for the nuclei (blue) using Hoechst dye. E) MTS cell viability assay of noninfected cells (NI), infected cells, and STS (staurosporine) treated cells. Cells were treated with 500nM of STS for 16 hours before MTS was added. After 72 hours of infection, MTS was added, cells were incubated for 45 minutes at 37°C, and absorbance at 490 nm measured. Experiments were performed in triplicates in three independent experiments and one representative experiment is shown. Absorbance was normalized to the NI and standard deviations of the triplicates are shown.  48     49     50     51     Figure 2.9. Strategy for microarray analysis of H4 neuroglioma cells infected with recombinant adenovirus. H4 cells were infected with Ad. Emp, Ad. RFP WT-R, Ad. RFP S52R-R, Ad. RFP H338-R, Ad. RFP WT-S, or Ad. RFP H338R-S at MOI 50 and incubated for 48 hours before RNA extraction and analysis using the Illumina HumanHT-12 v3 Expression BeadChip. Adenovirus expressing Spn4A were compared to Ad. Emp and expression values with a 1.7 fold difference (p<0.05) were considered for analysis. The distribution of overlapping and unique genes over the threshold is shown in the venn diagram.  52     Figure 2.10. The mRNA levels of three ER stress genes in response to RFP Spn4A WT-R, S52R-R, or H338R-R determined by qPCR. RNA was collected from infected H4 neuroglioma cells, reverse transcribed to cDNA, and used for real-time PCR. FC represent comparisons of Ad. RFP WT-R, Ad. RFP S52R-R or Ad. RFP H338R-R to Ad. Emp. Significance (p<0.05*) was calculated from two-tailed Student’s t-test from three experiments performed in duplicates. Error bars represent SEM.  53     Figure 2.11. Distribution of genes differentially regulated in response to polymeric Spn4A mutants. The numbers are the number of genes up or down regulated.  54     Figure 2.12. The mRNA levels of small proline-rich protein 2D in response to RFP Spn4A WT-R, S52R-R, or H338R-R determined by qPCR. RNA was collected from infected H4 neuroglioma cells, reverse transcribed to cDNA, and used for real-time PCR. FC represent comparisons of Ad. RFP WT-R, Ad. RFP S52R-R or Ad. RFP H338R-R to Ad. Emp. Significance (p<0.05*) was calculated from two-tailed Student’s t-test from three experiments performed in duplicates. Error bars represent SEM.  55     Table 2.1. Serpin rate of polymer formation correlates with severity of disease. Mutants of neuroserpin are each associated with a rate of polymer formation, as demonstrated by in vitro biochemical polymer formation assays. Increased polymer formation and inclusion body deposition (evident in cross sections of cerebral cortex stained for inclusions) are associated with a decreased age of disease onset and increased disease severity. Reprinted from The International Journal of Biochemistry & Cell Biology, 40, Davies MJ and Lomas DA, The molecular aetiology of the serpinopathies (105), 1273-1286, © 2008, with permission from Elsevier.             56     Table 2.2. Serpin clades A to I, prototypical serpins belonging to the clades, and their amino acid sequence identity to Spn4A. Amino acid sequences were compared using CLUSTALW2 (87). Clade letter A B C D E F  Clade name (5)  Example  Antitrypsin-like Intracellular, ovalbumin-like Antithrombin Heparin cofactor II Plasminogen activator inhibitor  Alpha-1-antitrypsin Ovalbumin Antithrombin-III Heparin cofactor Plasminogen activator inhibitor 1 Pigment epitheliumderived factor Plasma protease C1 inhibitor Heat shock protein 47 Neuroserpin  G  Pigment epithelium-derived factor C1 inhibitor  H I  Heat shock protein 47 Neuroserpin  % amino acids identical to Spn4A 27 32 25 21 28 22 25 18 31  57     Table 2.3. Functional annotation clusters for genes upregulated by secreted or retained RFP Spn4A WT and mutants expression. DAVID functional annotation clustering was carried out using only the non unique upregulated genes and high stringency filter. Term represent annotation term, count is the number of genes involved in the individual term, p-value is calculated using a modified Fisher’s exact test and represent the significance of gene-term enrichment, fold enrichment measures the magnitude of enrichment against the complete array background, and annotation cluster enrichment score is the negative log transformation of the geometric mean of the p-values in the group.       58    Term  Count  p-value  Fold Genes Enrichment Annotation Cluster 1: Enrichment Score = 4.3 GO:0034976~response to 5 3.21E-07 80.41 HERPUD1,HSPA5,FAM129A,AC endoplasmic reticulum stress 023024.2,DDIT3 GO:0006984~ER-nuclear 4 2.97E-05 62.49 HERPUD1,HSPA5,AC023024.2,D signaling pathway DIT3 GO:0033554~cellular 5 1.52E-02 4.88 HERPUD1,HSPA5,FAM129A,AC response to stress 023024.2,DDIT3 Annotation Cluster 2: Enrichment Score = 2.5 3 2.18E-03 39.39 SM00271:DnaJ DNAJB9,DNAJB11,DNAJC3 IPR015609:Molecular 3 2.18E-03 41.70 chaperone, heat shock protein, Hsp40, DnaJ DNAJB9,DNAJB11,DNAJC3 3 3.06E-03 35.30 domain:J DNAJB9,DNAJB11,DNAJC3 IPR001623:Heat shock 3 3.12E-03 34.75 protein DnaJ, N-terminal DNAJB9,DNAJB11,DNAJC3 IPR018253:Heat shock 3 3.25E-03 34.04 protein DnaJ, conserved site DNAJB9,DNAJB11,DNAJC3 GO:0031072~heat shock 3 7.95E-03 21.29 protein binding DNAJB9,DNAJB11,DNAJC3 Annotation Cluster 3: Enrichment Score = 2.4 GO:0042175~nuclear 6 1.78E-04 10.52 envelope-endoplasmic HERPUD1,HSP90B1,SEC11C,NU reticulum network CB2,HSPA5,AC023024.2 GO:0005789~endoplasmic 5 1.61E-03 9.26 HERPUD1,HSP90B1,SEC11C,HS reticulum membrane PA5,AC023024.2 GO:0012505~endomembrane 6 1.52E-02 3.82 HERPUD1,HSP90B1,SEC11C,NU system CB2,HSPA5,AC023024.2 GO:0031090~organelle 6 5.13E-02 2.77 HERPUD1,HSP90B1,SEC11C,NU membrane CB2,HSPA5,AC023024.2 Annotation Cluster 4: Enrichment Score = 2.3 IPR000886:Endoplasmic 5 9.41E-07 63.19 HSP90B1,SDF2L1,PDIA6,PDIA4, reticulum, targeting sequence HSPA5 4 6.34E-04 22.47 GO:0042470~melanosome HSP90B1,PDIA6,PDIA4,HSPA5 GO:0048770~pigment 4 6.34E-04 22.47 granule HSP90B1,PDIA6,PDIA4,HSPA5 GO:0016023~cytoplasmic 4 8.69E-02 3.63 membrane-bounded vesicle HSP90B1,PDIA6,PDIA4,HSPA5 GO:0031988~membrane4 9.37E-02 3.51 bounded vesicle HSP90B1,PDIA6,PDIA4,HSPA5 GO:0031410~cytoplasmic 4 1.24E-01 3.10 vesicle HSP90B1,PDIA6,PDIA4,HSPA5 4 1.36E-01 2.97 GO:0031982~vesicle HSP90B1,PDIA6,PDIA4,HSPA5 Annotation Cluster 5: Enrichment Score = 2.3 GO:0070013~intracellular 10 4.15E-03 2.80 HYOU1,HSP90B1,DNAJB9,DNA organelle lumen JB11,ANG,SDF2L1,RP51103G7.7,PDIA6,PDIA4,HSPA5 GO:0043233~organelle 10 4.86E-03 2.74 HYOU1,HSP90B1,DNAJB9,DNA lumen JB11,ANG,SDF2L1,RP51103G7.7,PDIA6,PDIA4,HSPA5  59    Term GO:0031974~membraneenclosed lumen  Count  p-value  10  5.52E-03  Fold Enrichment 2.69  Genes  HYOU1,HSP90B1,DNAJB9,DNA JB11,ANG,SDF2L1,RP51103G7.7,PDIA6,PDIA4,HSPA5 Annotation Cluster 6: Enrichment Score = 2.1 GO:0006984~ER-nuclear 4 2.97E-05 62.49 HERPUD1,HSPA5,AC023024.2,D signaling pathway DIT3 GO:0042981~regulation of 5 4.83E-02 3.41 HERPUD1,HSP90B1,HSPA5,AC0 apoptosis 23024.2,DDIT3 GO:0043067~regulation of 5 4.98E-02 3.38 HERPUD1,HSP90B1,HSPA5,AC0 programmed cell death 23024.2,DDIT3 GO:0010941~regulation of 5 5.03E-02 3.37 HERPUD1,HSP90B1,HSPA5,AC0 cell death 23024.2,DDIT3  60     Table 2.4. ER stress genes, gene descriptions, and fold changes for WT-R, S52R-R, H338RR, WT-S, and H338R-S. These seven genes were within the top 11 most upregulated in response to RFP WT-R expressing, top 10 for retained mutants, and top 12 for the secreted H338R-S mutant. None of the genes listed were within the top 15 genes upregulated in response to WT-S. NS is not significant. All FCs reported had p<0.05.   Gene HERPUD 1  HYOU1 PDIA4 SDF2L1 DDIT3  CRELD2  HSPA5  Gene name and description Homocysteine-inducible, endoplasmic reticulum stressinducible, ubiquitin-like domain member 1 – upregulated during UPR, participates in ERAD Hypoxia upregulated 1 – upregulated during hypoxic stress, functions in protein folding and ER secretion Protein disulfide isomerase family A, member 4 – ER chaperone Stromal cell-derived factor 2-like 1 – ER chaperone DNA-damage-inducible transcript 3 (also known as CHOP) – transcription factor upregulated during ER stress or DNA damage, leading to in ER stress response and cell cycle arrest among others; may induce apoptosis Cysteine-rich with EGF-like domains 2 – upregulated during ER stress, functions in protein folding and trafficking Heat shock 70 kDa protein 5 (also known as BiP) – ER chaperone  WT-R  S52R-R  H338R-R  WT-S  H338R-S  3.79  3.73  3.78  1.94  2.41  3.50  3.54  3.75  2.63  2.88  3.45  3.50  3.28  2.52  3.14  3.23  3.76  3.45  1.80  2.46  3.19  2.69  3.01  NS  2.05  3.13  3.30  2.99  2.92  3.03  2.91  3.16  3.19  2.30  2.68  61     Table 2.5. Genes differentially regulated in response to polymeric serpins. The gene symbol, gene name, and fold changes (FC) for 18 genes differentially regulated in response to RFP S52RR, RFP H338R-R, or RFP H338R-S are listed. Some genes were not significant (NS).     Gene Gene name Symbol Downregulated MGP Homo sapiens matrix Gla protein VWA5A Homo sapiens von Willebrand factor A domain containing 5A KIAA036 PREDICTED: Homo sapiens KIAA0363 3 protein INDO Homo sapiens indoleamine-pyrrole 2,3 dioxygenase CDH6 Homo sapiens cadherin 6, type 2, K-cadherin (fetal kidney) SIPA1 Homo sapiens signal-induced proliferationassociated gene 1 DPYSL3 Homo sapiens dihydropyrimidinase-like 3 SIDT2 Homo sapiens SID1 transmembrane family, member 2 Upregulated PSAT1 Homo sapiens phosphoserine aminotransferase 1 INHBE Homo sapiens inhibin, beta E GFPT1 Homo sapiens glutamine-fructose-6-phosphate transaminase 1 LOC6430 PREDICTED: Homo sapiens similar to NADH 31 dehydrogenase subunit 5 CD34 Homo sapiens CD34 antigen WDR69 Homo sapiens WD repeat domain 69 CALR Homo sapiens calreticulin SLC35B1 Homo sapiens solute carrier family 35, member B1 KRT81 Homo sapiens keratin 81 SPRR2D Homo sapiens small proline-rich protein 2D  FC RFP S52R-R  FC RFP H338R-R  FC RFP H338R-S  -2.99 NS  -3.23 NS  NS -1.94  NS  -1.91  NS  NS  NS  -1.82  -1.80  -1.86  NS  -1.75  -1.95  NS  NS NS  -1.73 -1.70  NS NS  NS  1.73  NS  NS NS  NS 1.76  1.74 NS  NS  NS  1.77  NS NS 1.80 1.71  NS NS NS 1.85  1.78 1.78 NS NS  1.99 2.67  1.74 2.81  1.79 NS  62     Table 2.6. Functional annotations for genes differentially regulated by polymeric serpins expression. DAVID functional annotation chart was carried out using genes unique to the mutants. Red coloured genes are upregulated and green, downregulated.  Term cell-cell adhesion aminotransferase carbohydrate binding cartilage development polymorphism calcium binding  Genes CD34, MGP, CDH6 GFPT1, PSAT1 CD34, GFPT1, CALR GFPT1, MGP VWA5A, SIPA1, MGP, DPYSL3, INDO, SIDT2, KIAA0363, KRT81, SLC35B1, INHBE, SPRR2D, CD34, WDR69, PSAT1 MGP, CALR  63     Chapter 3: Transcriptome profiling of human neuroglioma cells expressing the proprotein convertase inhibitor, Spn4A 3.1  Introduction  3.1.1  Proprotein convertases The concept of enzyme maturation by proteolysis was first described in 1967 for sheep β-  lipotrophic hormone (106) and human insulin (107). However, the responsible protease would remain unidentified for nearly another two decades until the discovery of the precursor processing endoprotease, Kex2, in yeast (108). Shortly afterwards, in 1989, the mammalian homolog of Kex2, furin, was discovered (109). Within a year, furin was shown to convert a glycoprotein (110) and growth factor (111) to their mature forms. Today, nine structurally similar proprotein convertases (PCs) have been discovered in humans including furin, PC1/3, PC2, PC4, PACE4, PC5/6 (two isoforms), PC7 (also known as lymphoma PC [LPC]), SKI-1 (subtilisin/kexin-like isozyme-1, also known as site-1 protease [S1P]) and PCSK9 (proprotein convertase subtilisin kexin 9, also known as neural apoptosis regulated convertase-1 [NARC-1]). Furin, SKI-1, and PC7 are ubiquitously expressed (112), while tissue-specific PCs include PC1/3 and PC2 in neuroendocrine cells (113;114), PC4 in germ cells (115), and PCSK9 in brain, liver, and intestinal cells (116). Their physiological roles as enzyme regulators and aberrant expression in disease are of great biological and clinical significance (discussed in 3.1.2). 3.1.1.1 Structural features Several structural features are important for the targeting, activation, and localization of PCs (Fig. 3.1). An N-terminal signal peptide targets PCs to the endoplasmic reticulum (ER). A prodomain follows the signal peptide cleavage site and acts as an intramolecular chaperone that facilitates folding in the ER, transport, and activation (117;118). Immediately downstream is a set of basic amino acids that make up the autoproteolytic cleavage site for excision of the propeptide, a catalytic domain containing the catalytic triad, and lastly, a P domain which regulates the pH and calcium dependence of the protein (101;119). Additionally, furin, PC7, the isoform PC5/6B, and SKI-1 have transmembrane domains and C-terminal cytoplasmic domains for localization and sorting to the TGN (trans-Golgi network), endosomal system, and cell surface (120;121). While these four PCs exist as membrane bound proteins, additional cleavage 64     sequences near the C-terminus of furin and SKI-1 allow truncation of the PCs and generation of soluble and secreted forms (92;120). Other PCs are found in dense core granules or are constitutively secreted (112;122). 3.1.1.2 Activation and subcellular localization The activation of PCs is spatially regulated (Fig. 3.2). The first step is the autocatalytic cleavage of the prodomain in the ER for all convertases except PC2, which is processed in immature secretory granules (122). The propeptide remains associated with, and inhibits, the active site until the PC-propeptide heterodimer exits the ER to the Golgi, where a decrease in pH and increase in calcium promote the cleavage and release of the inhibitory propeptide (101). The exception is PCSK9, which is secreted as the inactive heterodimer (116;123). Specifically, furin, PC5/6, PACE4, and PC7 are activated in the TGN (118;124;125), PC1/3 and PC2 in secretory granules (126), and SKI-1 in the cis/medial-Golgi compartments (127). Once activated, functional PCs cycle between different cellular compartments (Fig. 3.3). Furin activation in the TGN has been recently contested with two papers, which report furin-mediated degradation of an ER-localized misfolded insulin proreceptor (128) and furin-mediated cleavage of the chimeric ER-retained hemojuvelin, and suggest that furin can be functional in the ERGIC (ER-Golgi Intermediate Compartment) pathway (129). 3.1.1.3 Substrate specificity and physiological substrates The family of PCs can be separated into two groups based on their substrate specificity. The first group consists of furin, PC1/3, PC2, PC4, PACE4, PC5/6, and PC7, which recognize basic amino acids and have the general consensus sequence (K/R)-(X)n-(K/R)↓, where n = 0, 2, 4, or 6, X is any amino acid, and the down arrow denotes cleavage (122). Other substrate characteristics may form more favorable cleavage conditions specific for a PC. For example, the most efficient processing site for furin is R-X-(K/R)-R↓ with basic residues at P3, P5, and P6 sites, while the minimal is R-X-X-R (130-132). The first group differs from SKI-1 and PCSK9 which have the consensus motif (R/K)-X-(hydrophobic)-X↓ (133) and VFAQ↓ (123), respectively. Various protein precursors such as hormones, neuropeptides, growth factors and their receptors, cell surface receptors, enzymes, as well as bacterial and viral products have been identified as substrates for PCs, with the exception of PCSK9. The only substrate identified for 65     PCSK9 is the autocatalytic cleavage of its own prodomain. While substrates could be predicted from amino acid sequences and demonstrated in vitro, the discovery of bona fide endogenous substrates for each PC has been complicated by their similar specificity, which allows processing of some precursors by multiple convertases, and the spatial and temporal expression of PCs and predicted substrates. The most convincing evidence of their physiological roles comes from knockout mice studies. A wide range of phenotypes are detected with the knockout of each PC, and include embryonic lethality (furin (134), PC5/6 (135), or SKI-1 (136)), craniofacial abnormalities and some death (PACE4 (137)), hormonal and/or neuroendocrine deficiency (PC1/3 (138) or PC2 (139)), reduced fertility (PC4 (140)), and enhanced cholesterol uptake by the liver (PCSK9 (141)). Only PC7 knockout mice fail to show any abnormal phenotype (142). The phenotype from the loss of PC function would mimic that of the loss of matured substrate. Accordingly, substrates can be inferred. For example, the phenotypes of TGFβ1 (transforming growth factor beta 1), bone morphogenetic proteins, or α4-integrin deficient embryos mimic furin null embryos and are suggested as substrates (143;144). Different from null mice, conditional furin knockout in the liver is not lethal and predicted precursors are still processed, albeit with reduced efficiency (145). Likely, hepatocytes express convertases with redundant functions. The full list of substrates for furin and other PCs will not be discussed here, but included throughout the chapter as needed. 3.1.2  Furin as a therapeutic target PCs activate several bacterial toxin and viral glycoproteins and their aberrant expression  is associated with diseases such as cancer, atherosclerosis, neurodegenerative disorders, and dyslipidemia (146). We will focus our discussion on pathologies related to the ubiquitously expressed furin and for which furin-targeted intervention may be beneficial. 3.1.2.1 Bacterial and viral infections requiring furin-like enzymes Bacteria and viruses have hijacked the spatial and temporal regulation of protein maturation for their own protoxins or proproteins. These include cell surface activation of anthrax toxin protective antigen (PA) (147), Aeromonas hydrophila aerolysin toxin (148), and Clostridium septicum α-toxin (149). Proteolytically activated toxins form pores in cell membranes, allowing entry of the bacteria, and causing cell toxicity. Additionally, endocytosed 66     toxins such as Pseudomonas exotoxin A (PEA), diphtheria toxin, and shiga toxin are processed by furin in the endosome to enable translocation to the cytosol (101). Last, many pathogenic viruses including Ebola, highly pathogenic avian influenza virus, human immunodeficiency virus 1 (HIV-1), human cytomegalovirus (hCMV), Dengue virus, West Nile virus, and tick-borne encephalitis virus require furin or furin-like protease processing of viral glycoproteins (150-156). 3.1.2.2 Cancers associated with increased furin-like activity Aberrant expression of convertases is detected in many cancers. Furin is upregulated in head and neck (157), breast (158), and lung (159) cancers, as well as in a number of tumor cell lines such as glioblastomas (160) and gonadal cancer lines (161). Further, critical proteins upregulated in malignancy are either directly processed by PCs or are indirectly controlled by PC substrates. Consequently, inhibition of PCs can attenuate the malignancy phenotype in vivo (162165) (discussed in section 3.1.3). Tumorigenesis and metastasis are driven by multiple complex mechanisms, the details of which will not be covered here. Simply, uncontrolled growth of cells due to misregulation of the cell cycle, formation of blood vessels for attaining nutrients and oxygen, as well as invasion and migration of tumor cells by remodeling of the extracellular matrix (ECM) contribute to cancer pathology (166). We will discuss specific examples of PC-processed proteins that promote tumor malignancy: TGFβ, PDGF (platelet-derived growth factor), IGF1 (insulin growth factor 1), IGFR (IGF receptor), and MT1-MMP (membrane type 1 matrix metalloprotease). TGFβ is a cytokine that controls multiple cellular functions, including cell proliferation, differentiation, migration, and apoptosis, among others (167). TGFβ is a potent cell growth inhibitor in most cells. It is processed by furin intracellularly and secreted into the extracellular matrix as a latent protein. Once activated by proteases or integrins, TGFβ signals through TGFβR (TGFβ receptor) and primarily through Smad transcription factors (167). Downstream cell growth inhibition effects are mediated by upregulation of cell cycle inhibitors, the action of retinoblastoma protein (Rb) and E2F transcription factors to suppress the G1/S transition (Gap1/Synthesis), and downregulation of the proto-oncogene, c-myc (168-170). However, in mesenchymal cells, TGFβ can promote proliferation through upregulation of the growth factor PDGF (171).  67     Many tumor cells can escape the tumor-suppressive activities of TGFβ through various pathways such as increasing expression of oncoproteins or inactivation of TGFβ signaling pathways (170). Once they are resistant to the TGFβ tumor suppressor effects, TGFβ is upregulated in many cancers to confer proliferative, invasive and angiogenic ability by increasing the production of mitogenic growth factors PDGF (172;173) and fibroblast growth factor (FGF) (174), extracellular matrix-degrading enzymes for motility (175), and VEGF (176) (vascular endothelial growth factor) for angiogenesis. Maturation of both TGFβ and PDGF are mostly mediated by furin (177). IGF1 and its receptor IGFR are also highly expressed in cancer cells to stimulate cell proliferation and inhibit apoptosis (178). Through regulation of MMPs (matrix metalloproteases) expression (179;180), they can further mediate tumor invasion. Furin, and to a lesser extent PC6A and PC7, are responsible for processing IGF and IGFR (181;182). Last, furin processes many ECM proteins important for tumor invasion and metastasis. The ECM is composed of glycosaminoglycans and fibrous proteins such as proteoglycans, collagen, elastin, fibronectin, and laminin to mediate cell adhesion and support (175). One of the earliest MMPs that acts on the basement membrane is the collagenase MT1-MMP, which also cleaves and activates the gelatinase MMP2 (166;183). Many members of the MMP family have furin consensus sites and furin activation of MT1-MMP has been demonstrated (184;185). 3.1.3  Furin-targeted inhibitors The molecular targets of furin have spurred the development of inhibitors to treat  microbial diseases and cancer. Several lines of evidence suggest that furin knockdown would be a viable strategy. First, no obvious phenotype is seen in liver-specific furin knockouts, which suggest that related PCs could compensate for the loss of furin in specific cell types. Second, furin inhibitors protect against furin-mediated Pseudomonas toxicity in mice without adverse physiological effects (186). Last, double transgenic mice with salivary gland heterozygous furin knockout and PLAG-1 (pleiomorphic-adenoma gene 1) proto-oncogene overexpression show delayed salivary gland tumorigenesis (162). However, mice with homozygous furin knockdown in the salivary gland exhibit poor health.  68     Much progress has been made on the design of furin inhibitors. We will focus on small molecule, prodomain-based, peptide-based, and protein-based inhibitors, which are best characterized and show the most promise. 3.1.3.1 Small molecule inhibitors Small molecule inhibitors are attractive therapeutic strategies because they are easily produced through synthetic routes, may be metabolically and proteolytically stable, and can be administered easily. They are the newest inhibitors being developed for furin and only two have been recently discovered. From a small molecule screen, guanidinylated aryl 2,5dideoxystreptamine derivatives (GADDs) are found to potently and relatively specifically inhibit furin (Ki = 6nM) and protect RAW264.7 macrophage cells from anthrax PA by inhibiting its processing (187). Further, GADDs preferentially recognize furin and PC5/6B compared to PACE4 and PC7 (187). Another high-throughput screen identified B3, a cell permeable compound, which exhibits a Ki of 12 µM. B3 can inhibit furin-mediated processing of pro-MT1MMP, which leads to a reduction in cancer cell motility and invasiveness (188). The authors report that B3 may also inhibit other PCs. While small molecule inhibitors may have a promising future, prodomain-, peptide-, or protein-based inhibitors are much better characterized and importantly, are effective in vivo. 3.1.3.2 Prodomain-based inhibitors The next class of inhibitors is based on the PC natural prodomain, which tightly binds and inhibits the catalytic domain of PCs. Purified prodomain, vectors carrying the prodomain, or smaller synthetic prodomain peptides can inhibit PCs in the low nanomolar to high picomolar range (189-191). Tumor cells transfected with the furin prodomain and transplanted onto SCID (Severe Combined Immunodeficiency) mice display less proliferation and invasiveness compared to controls (163). However, the furin prodomain can inhibit PC5/6 ten-fold more potently than furin and thus, the selectivity of prodomain inhibitors would have to be improved to be useful (189). 3.1.3.3 Peptide-based inhibitors Many peptide inhibitors are substrate analogs (192-196). The most promising inhibitors are polyarginine peptides (192) and the TPRARRRKKRX peptide (195) for their potency and characterization in cell culture and mice. Specifically, D-hexa-arginine (Ki = 106 nM) protects 69     cultured cells and mice from PEA toxicity without side effects (186) and the TPRARRRKKRT peptide (Ki = 23 nM) protects mice from inhaled anthrax and injected PEA (195). These peptides are broad PC inhibitors. D-hexa-arginine also inhibits PACE4 and PC1 in the high nanomolar and low micromolar range (192) and TPRARRRKKRT inhibits PACE4, PC4, PC5/6, and PC7, in the high nanomolar range (195). 3.1.3.4 Protein-based inhibitors Bioengineered proteins and natural endogenous proteins constitute the last group of inhibitors. Several protein-based inhibitors have been designed by mutating the reactive sites of protease inhibitors to target furin, such as turkey ovomucoid third domain (197), α2macroglobulin (198), eglin C (199), and α1-antitrypsin (200). The α1-antitrypsin variant, known as α1-PDX (α1 antitrypsin Portland), has been studied extensively because of its potency, selectivity, and demonstrated application under pathological circumstances. In vitro, α1-PDX inhibits furin (Ki = 0.6 nM) and PC5/6B (Ki = 2.3 nM) (201), but in cellulo, it may target additional PCs in a cell-type specific manner (202). Application of α1-PDX to cells confers protection from furin-mediated activation of pore-forming proaerolysin (148), HIV-1 glycoprotein 160 (gp160) (200), PEA (201), and hCMV pro-glycoprotein B (gB) (203). Further, it is a powerful tool for tumorigenesis inhibition in cell culture and in vivo (204;205). Nude mice that are injected with colon carcinoma HT29 cells expressing α1-PDX showed decreased tumor size and incidence and delayed tumor development in comparison to control mice injected with HT29 cells (205). Further in vivo studies using transplantation of astrocytoma cells (164) or colon carcinoma CT26 cells (165) have shown that α1-PDX expressing cancer cells are less proliferative, invasive, and metastatic. Cancer cell lines treated with α1-PDX have unprocessed IGFR, TGFβ, and MT1-MMP (164;165;205). However, the effects of α1-PDX on cancer cells may be cell-type specific as breast cancer cells transfected with α1-PDX have increased cell motility, migration, and invasion due to increased MMP9 activity (206). Lastly, two naturally occurring serpins, the human proteinase inhibitor 8 (PI8) (207) and Drosophila Spn4A (34-36), inhibit furin. Human PI8 has a Ki of 54 pM, but its lack of an Nterminal signal peptide means its cytoplasmic localization prohibits interactions with furin. Furthermore, PI8 can also inhibit other types of proteases, such as trypsin and chymotrypsin (208). In comparison to all other furin inhibitors, Spn4A is the most potent (Ki = 13pM) (36). 70     Also, Spn4A has been shown to inhibit Drosophila PC2 (Ki = 3.5 nM) (36), which suggests it may have a broad specificity for PCs. Spn4A naturally has a C-terminal HDEL ER retention signal, whose deletion leads to Spn4A secretion to the extracellular milieu. Our lab has demonstrated the effectiveness of secreted Spn4A (Spn4A-S) in blocking furin-mediated processing of HIV-1 gp160, influenza A hemagglutinin (HA), and hCMV gB (unpublished data). Further, the ER-retained Spn4A (Spn4A-R) also inhibited maturation of hCMV gB, implying that furin may be actively processing enzymes in the ERGIC pathway and can be inhibited. 3.1.4  Project rationale, hypothesis, and experimental approach The involvement of furin in a myriad of pathologies underlies the intense development of  furin inhibitors. However, many issues have to be resolved before they are used in a clinical setting. Most fundamentally, the cellular effects of furin inhibition need to be comprehensively studied, including those that ameliorate the disorders as well as unwanted side effects. As a first step, we profiled the transcriptome of neuroglioma cells expressing the most potent furin inhibitor, Spn4A. In the United States, approximately 25 000 people are diagnosed with malignant brain tumors annually, two thirds of which are high grade gliomas (HGG) (209). There are currently no effective therapies to treat HGG and it is associated with poor prognosis. Five year survival rates are as low as 10% (210). Hallmarks of the disease are local invasion, angiogenesis, and a lack of metastasis (211). While furin expression levels have not been investigated in primary brain tumors, it is upregulated in many glioblastoma cell lines (160). Further, α1-PDX expression in astrocytoma (164) and glioma cells (160) inhibit furin-mediated substrate processing and reduce cancer cell proliferation and invasion. Since furin may be considered a target for glioblastoma therapy, a complete and unbiased study of the cellular response to PC inhibition on glioma cells is essential. The compartments in which furin is active have come under scrutiny (section 3.1.1.2). Since Spn4A is naturally ER-retained, and the deletion of the ER retention motif results in a secreted variant, we were able to assess how compartmentalization of the inhibitor affects furin activity. Lastly, the possibility of using Spn4A as a therapeutic agent will ultimately need to be assessed in vivo. As fusion fluorescent proteins can facilitate animal studies, we examined if 71     Spn4A tagged with RFP (91), a monomeric red fluorescent protein, can inhibit furin and regulate the same pathways in neuroglioma cells. In summary, we addressed the questions: How does Spn4A affect the transcriptome of neuroglioma cells? Is the cellular response to Spn4A specific on its compartmentalization? Can fluorescently-tagged Spn4A inhibit furin and regulate the same pathways? Our approach was to express Spn4A-S, RFP Spn4A-S, and RFP Spn4A-R in H4 human neuroglioma cells and profile the transcriptome via microarray. We hypothesized that we could identify the downstream effects of the loss of functional PCs such as furin and their substrates. Specifically, the loss of growth factor processing may lead to changes in genes related to cell proliferation, invasion, and angiogenesis. Additionally, the microarray data would reveal if expression of Spn4A had stress or apoptotic effects on mammalian cells. Since active furin is usually localized to the TGN and cell surface, we predicted that ER localized Spn4A would still inhibit furin, but to a lesser extent than secreted Spn4A. Last, we hoped the addition of a RFP tag would not affect inhibitory activity.  72     3.2  Methods Methods were performed as described in Chapter 2: Transfection and infection conditions  (2.2.5), SDS-PAGE, native PAGE, and western blot (2.2.6), Furin complex formation assay (2.2.8), RNA extraction and cDNA synthesis (2.2.11), Microarray and data analysis (2.2.12), and Real-time PCR (2.2.13). The microarray was performed using Ad. Emp, Ad. RFP WT-S and Ad. RFP WT-R as described in Chapter 2. In addition, H4 neuroglioma cells were infected with adenovirus expressing Spn4A-S (Ad. WT-S, no RFP tag) and RNA extracted for microarray experiments. Ad. WT-S was generated previously by the Jean lab. Microarray data analysis was performed using DAVID v6.7 (93) and Ingenuity Pathway Analysis (IPA v8.5, Ingenuity® Systems, www.ingenuity.com). DAVID analysis was performed as described (2.2.12). Further, differentially regulated genes (threshold cut-off of 1.7 fold, p<0.05) were uploaded into IPA for functional analysis, network generation, and canonical pathway analysis. The functional analysis identified the molecular and cellular functions that were most significant to the data set. Right-tailed Fisher’s exact test was used to calculate a pvalue determining the probability that each biological function and/or disease assigned to that data set is due to chance alone. For network generation, genes were overlaid onto a global molecular network developed from information contained in Ingenuity’s Knowledge Base. Networks were then algorithmically generated based on their connectivity and biological functions and/or diseases that were most significant to the genes in the network identified. Righttailed Fisher’s exact test was used to calculate a p-value determining the probability that the identified biological function assigned to the network is due to chance. Last, canonical pathway analysis identified pathways from the IPA library of canonical pathways that were most significant to the uploaded data set. Fisher’s exact test was used to calculate a p-value.  73     3.3  Results and discussion  3.3.1  RFP Spn4A inhibitory activities In order to use RFP-tagged Spn4A in future transgenic animal studies, we addressed if  RFP-tagged Spn4A could still inhibit furin. Normally, for inhibition to occur, a heat- and SDSstable complex needs to form between the serpin and protease. We performed complex formation assays using HEK293-C4 cells, a human embryonic kidney cell line overexpressing FLAGtagged furin (92) and confirmed that the enzyme-inhibitor complex still formed (Fig. 3.4). 3.3.2  Experimental procedures and microarray strategy To study the response of neuroglioma cells to Spn4A, we used an adenovirus delivery  system to overexpress Spn4A in human H4 neuroglioma cells. Development of the expression system is described in section 2.3.5. Briefly, infections performed at MOI 50 resulted in infections of nearly 100% of the cells, as detected through flow cytometry and confocal microscopy. Viral infections were not toxic. Since furin is localized to the Golgi and endosomal compartments, we used Ad. WT-S for inhibition of PCs in the secretory pathway. Further, RFPtagged Spn4A would be useful for live cell imaging or transgenic animal studies. Therefore, we also studied the gene expression profile of Ad. RFP WT-S. Last, we addressed the compartmental specificity of Spn4A function by comparing the profile of secreted RFP WT-S to ER-retained RFP WT-R. Experimentally, H4 cells were infected with Ad. Emp, Ad. WT-S, Ad. RFP WT-S, and Ad. RFP WT-R. After 48 hours, RNA was extracted and used on the Illumina HumanHT-12 v3 Expression BeadChip, which targets more than 25 000 annotated genes using 48 000 probes. Expression data from Ad. WT-S, Ad. RFP WT-S, or Ad. RFP WT-R were compared to Ad. Emp for fold change and p-value calculation. Genes with altered expression levels due to the presence of inhibitory Spn4A were compiled. Only fold changes with a threshold cut-off of 1.7 fold (p<0.05) were considered significant and used for analysis. Further, off-target effects were excluded by a comparison of non-inhibitory Spn4A mutants (Chapter 2). Using these criteria, 643 genes in Ad. WT-S, 383 genes in Ad. RFP WT-S, and 214 genes in Ad. RFP WT-R infected cells reached significance. To identify over-represented biological themes and affected pathways and networks from the gene list, we used DAVID Functional Annotation Clustering analysis and IPA. Annotations or  74     pathways were considered significant if their enrichment scores (ES) or the negative log of the pvalues were greater than two. 3.3.3  Ontological, pathway, and network analysis of genes regulated by Spn4A in the secretory pathway In response to the expression of Spn4A-S, 643 genes were differentially regulated using  the described criteria. Gene ontology and enrichment analysis was performed by separately submitting the 468 downregulated and 175 upregulated genes to the DAVID Functional Annotation Clustering software. Genes were classified into gene ontology groups, enrichment for each annotation term calculated, and annotation terms further grouped according to highly similar annotation terms. Gene ontology and enrichment analysis revealed 20 significant annotation clusters (Table 3.1, genes in Supplemental Table A5.2), with the highest cluster annotating cell division, cell cycle process, and nuclear division (ES = 23.6). Of the top ten clusters, eight were directly related to cell cycle progression including annotations for kinetochore and centromere structural proteins (ES = 13.3), different groups of DNA binding proteins (ES = 7.3, 4.7, 4.3, 3.6), motor proteins for mitosis (ES = 3.7), and cyclins (ES = 3.4). Other categories with significant changes included sprouty development genes (ES = 4.0), regulation of phosphorylation (ES = 3.3), response to nutrient (ES = 2.2), and coagulation (ES = 2.1). Through a gene ontology search of the upregulated genes, five significant gene ontology clusters were identified (Table 3.2, genes in Supplemental Table A5.2). All clusters annotated to extracellular matrix components (ES = 6.7) such as collagen (ES = 2.7), glycosaminoglycans (ES = 2.3), cysteine-rich secretory proteins (ES = 2.3), and leucine-rich proteoglycans (ES = 2.1). Molecular and cellular function analysis from IPA largely agreed with the biological themes identified by DAVID. Top functions included those described above as well as cell death, cellular movement, cellular compromise, cell morphology, and amino acid metabolism (Fig. 3.5, genes in Supplemental Tables A5.3 and A5.4). To identify well-characterized cell signaling pathways that were the most relevant to these 643 genes, Canonical Pathway Analysis from IPA was used. Notably, seven of the eight statistically enriched pathways identified affected cell cycle progression and cancer signaling (Fig. 3.6, genes listed in Supplemental table A5.5). Five pathways affecting cell cycle included two cell cycle checkpoint regulation pathways, two DNA damage response pathways, and one mitosis transition pathway. Additionally, several genes associated with breast and pancreatic 75     cancer signaling pathways were identified. Last, aryl hydrocarbon receptor (AhR) signaling, which is a response to xenobiotics (212), was also identified as an affected pathway. To further explore the relationships between the regulated genes, we used IPA network analysis to visualize the interactions between molecules. The list of significant networks and the functions associated with molecules of the network are summarized in Table 3.3. The top two networks were similarly annotated with cell cycle, cellular assembly and organization, DNA replication, recombination, and repair, and gene expression (Table 3.3, Fig. 3.7). The most powerful network contained 33 focus genes out of a maximum of 35. The network converged on MYC (v-myc myelocytomatosis viral oncogene homolog), an oncogene coding for a transcription factor that regulates cell cycle progression, apoptosis, and cellular transformation (213) (Fig. 3.7A). Further, several downregulated genes were connected to MIR124 (Fig 3.7A). The overexpression of the microRNA MIR124 depresses the expression of those target genes (214) in Fig. 3.7A, and promotes differentiation and inhibits proliferation in glioma cells (215). Similarly, most genes were downregulated in the second most powerful network, which converged on E2F2 (E2F transcription factor 2), a regulator of S-phase entry in cell cycle (216) (Fig. 3.7B). The downregulation of MYC and E2F2, and expression of MIR124 could play a crucial role in the control of cell proliferation. The global profiling of the transcriptome revealed several biological themes that were affected in response to Spn4A WT-S. To investigate how PC inhibition may affect tumor malignancy, we focused on the regulation of specific genes implicated in cell proliferation, cell movement, and angiogenesis. 3.3.4  Regulation of specific genes implicated in cell proliferation Previous studies have demonstrated an inhibition of cell proliferation in the presence of  furin inhibitors (160;164). Our global analysis now provides the genetic basis for inhibition. Approximately 160 genes annotating to cellular proliferation were differentially regulated (Supplemental Table A5.3). As visualized in the network analysis, MYC, MIR124, and E2F2 are likely some key regulators of cell growth and proliferation. Further, we summarized genes whose products directly affected cell proliferation in Fig. 3.8 (FCs in Supplemental Table A4.1). First, we scanned the list of genes annotated with cell cycle from DAVID (Supplemental Table A5.2) and IPA (Supplemental Table A5.3) for kinases, cyclins, and their regulators that control the phases of the cell cycle. Notably, many were downregulated, with the exception of CDKN1C 76     (cyclin-dependent kinase inhibitor 1C), which codes for an inhibitor of CDK2 (cyclin-dependent kinase 2)/cyclinE complexes, and CDKN2C, an inhibitor of G1 kinases CDK4 and CDK6 (217). CDC25A (cell division cycle 25) is an enzyme that removes phosphates from CDK2/cyclinE, which activates the complex and allows for G1 to S transition (218). We observed downregulation of CDC25A, CDK2, CCNE2 (cyclin E2), CDC2, CCND1, and CCNB1, important for G1/S and G2/M (Gap2/Mitosis) transitions (Fig. 3.8). Next, we mapped genes important for proliferation and growth, such as those coding for DNA replication and mitosis proteins onto Fig. 3.8 (FCs in Supplemental Table A4.1). ORCs (origin of replication complex) and MCMs (mini chromosome maintenance) for formation of the pre-initiation replication complex (219), as well as DNA polymerase components were all downregulated, suggesting the inhibition of chromosome duplication. We also identified the differential regulation of several genes coding for components or regulators of mitotic progression and chromosome segregation. SMCs (structural maintenance of chromosome) for chromosome condensation and sister chromatid cohesion (220), centromere proteins that function in sister chromatid adhesion and kinetochore formation, and several kinases (NEK2 [never in mitosis gene A-related kinase 2], AURKA [aurora kinase A], AURKB) regulating mitotic processes (221;222) were downregulated (Fig. 3.8, Supplemental Table A4.1). These genes represented the most proximal causes for cell proliferation inhibition observed in glioma cells expressing PC inhibitors (160;164). Notably, many of these gene, such as CDKs, CDC25, and the aurora kinases, are overexpressed in cancer cells and are targets for therapy (218;222;223). 3.3.5  Regulation of specific genes implicated in cell movement Tumor cells invade surrounding tissues by a dynamic process of detachment and  adhesion to the ECM and degradation of the ECM (224). The loss of invasive ability was reported in glioma cells expressing PC inhibitors (160;164). The invasiveness of H4 neuroglioma cells has not been tested and the expression levels of adhesion proteins and ECM proteases are unknown. Nonetheless, cell movement was a significant theme identified in IPA function analysis (Fig. 3.5). To assess motility and ECM remodeling, we analyzed the top ten most downregulated and upregulated genes annotated with cell movement (Table 3.4, full list in Supplemental Table A5.4). Among the downregulated were the chemokine IL8 (Interleukin-8), adhesion proteins ITGA2 (integrin alpha 2), AJAP1 (adherens junctions associated protein 1), 77     and ITGA6, transcription factor ETV5 (ets variant 5), growth factors TGFα and GDF15, the ECM protein SPP1 (secreted phosphoprotein 1, also known as osteopotin), and signaling molecules ERRFI1 (ERBB receptor feedback inhibitor 1) and RGS1 (regulator of G-protein signaling 1). Among the upregulated were regulators of the cytoskeleton C5ORF13 (chromosome 5 open reading frame 13, also known as P311) and S100A4 (S100 calcium binding protein A4), signaling molecule SOCS2 (suppressor of cytokine signaling 2) and GUCY1A3 (guanylate cyclase 1, soluble, alpha 3), phosphatases ALPP (alkaline phosphatase, placental [regan isozyme]) and PPAP2A (phosphatidic acid phosphatase type 2A), adhesion molecules CD24 (CD24 molecule), CD36 (CD36 molecule, thrombospondin receptor), and OLR1 (oxidized low density lipoprotein [lectin-like] receptor 1), and the ECM protein DCN (decorin). We focused on cell adhesion molecules, ECM proteins, and regulators of the cytoskeleton, which have immediate implications in tumor cell invasion. Integrins, osteopotin, and AJAP1 (downregulated) can mediate adhesion (Table 3.4). First, integrins, which are transmembrane heterodimers composed of α and β chains, bind ECM ligands to induce cytoskeletal reorganization and cell motility signaling cascades (224). While several integrins are overexpressed in tumor cells to assist invasion (224), IGTA2 and IGTA6 have not yet been reported as such. Second, osteopotin is an ECM ligand for integrins and CD44 (downregulated by 1.8 fold, Supplemental Table A5.4) and is highly expressed in several malignancies to induce inhibition of apoptosis and activation of various ECM proteases (225). Last, the transmembrane adhesion protein, AJAP1, can also modulate tumor cell migration and invasion, although contradictory roles are reported (226;227). Invasiveness is attenuated in glioma cells (227) but enhanced in epithelial and mesenchymal cells (226) that overexpress AJAP1. Six upregulated genes can affect the regulation of growth factors, adhesion, angiogenesis, and cytoskeletal organization. The most upregulated gene was decorin, an ECM component that may enhance cellular structural stability (228) as well as inhibit TGFβ activity and EGFR (epidermal growth factor receptor) activation (229;230). Its overexpression inhibits malignancy in vivo (229;230). Next, two adhesion molecules OLR1 and CD24 were upregulated (231;232). While OLR1 has thus far not been directly implicated in cancer, CD24 was shown to promote invasion in glioma cells in vivo when overexpressed (232). CD36 is a receptor that negatively regulates angiogenesis in human endothelial cells (233). Its function in human neurons is unclear, but it plays a role in detecting pheromones in insects and responding to fatty acids in 78     rodents when expressed in sensory neurons (233). Last, two regulators of the cytoskeleton, P311 and S100A4, are highly implicated in glioma cell invasion. P311 is overexpressed in invading glioma cells and its interaction with Filamin A is hypothesized to promote cytoskeletal reorganization (234). S100A4, also overexpressed in glioma cells (235), bind to myosin IIA at the leading edge to promote protrusions (236). Further, S100A4 also promotes expression of MMPs (237;238) for degradation of the ECM. Notably, we did not observe genes coding for ECM proteases and protease inhibitors within the top ten most down or upregulated genes (Table 3.4). Scanning for these genes in the full list (Supplemental Table A5.4) revealed that only MMP3 was differentially regulated (downregulated by -2.2 fold). MMP3 (also called stromelysin-1) can degrade many ECM substrates, including collagens, laminins, fibronectin, and proteoglycans as well as release cell surface molecules, such as heparin-binding EGF-like growth factor, and activate other MMPs (239). Its overexpression in mammary epithelial cells promotes tumorigenesis (239). Further, MMP3 has a furin consensus site and was demonstrated to be an in vitro substrate (240); thus post-translational MMP3 activity may also be modulated through inhibition of furin. Plasmin is another extracellular protease that is activated by tissue plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Plasmin, the activated form, can degrade ECM components as well as activate MMPs (241). PLAT (tissue type plasminogen activator) and PLAUR (urokinase plasminogen activator receptor), whose gene product activates tPA and localizes uPA, were downregulated by -1.8 and -2.1 fold, respectively (Supplemental Table A5.4). We have reported the differential regulation of several genes that may affect invasive ability. As current data suggest that PC inhibitors block invasiveness in neuroglioma cells, the downregulation of integrins, osteopotin, MMP3, PLAT, and PLAUR as well as the upregulation of DCN may be additional mechanisms important in the loss of invasive ability in furin-inhibited cancer cells. Conflictingly, the downregulation of AJAP1 and upregulation of CD24, P311, and S100A4 could promote invasion. Functional assays (discussed later) will need to be performed to study the invasive ability of neuroglioma cells expressing Spn4A. The phenotype can then be related to these gene products to determine which more readily influences invasive ability. 3.3.6  Regulation of specific genes implicated in angiogenesis Although no confirmatory data has been reported, our microarray profiling suggested that  PC inhibitors may also indirectly affect tumor-induced angiogenesis. The ability to form new 79     blood vessels requires growth signaling. We found that two key angiogenic factors, VEGFA and IL8 (209), were downregulated by -2.5 and -5.3 fold, respectively. Other angiogenic factors PDGF and FGF (209) were not differentially regulated. Notably, furin, and other PCs, cleave and activate PDGF (177). It is possible that PC inhibitors can directly inhibit processing of PDGF and indirectly affect expression levels of VEGFA and IL8 to block angiogenic signaling to endothelial cells. 3.3.7  Regulation of specific genes implicated in stress and apoptotic responses To evaluate if Spn4A expression would be pathogenic, we looked for induction of  cellular stress and apoptosis. Unfortunately, overexpression of Spn4A, regardless of function, upregulated ER stress responses (Chapter 2). IPA and DAVID GO analysis did not identify enrichment for genes in response to stress specifically induced by inhibitory Spn4A WT. Cell death was a significant cellular function identified by IPA (Fig 3.2). However, most genes that were annotated with cell death overlapped with those of cell cycle and proliferation. Therefore, we specifically looked for the differential regulation of genes from three major players of apoptosis: the caspases, TNF (tumor necrosis factor) superfamily, and BCL2 (B cell lymphoma) superfamily. All apoptotic signaling pathways converge on caspases, a family of cysteine proteases, which were not differentially regulated transcriptionally. However, they are made as zymogens and activated by cleavage or dimerization (242). Therefore, apoptosis mediated by caspases may not be apparent in microarray analysis. We further investigated apoptotic genes mediated through transmembrane death receptors of the TNF receptor superfamily. Only one gene, TNFRSF10B, was affected (downregulated by -1.77 fold). Last, we asked if members of the BCL2 families were differentially regulated. The BCL2 superfamily of proteins has both pro-apoptotic and anti-apoptotic members. We observed differential gene regulation of BCL6 (downregulated by -1.75 fold), BCL11A (upregulated by 1.97 fold), and BMF (BCL2 modifying factor, upregulated by 2.76 fold). The role of BCL11A in apoptosis is unclear, but it regulates axonal branching in glial cells (243). Notably, BCL6 is anti-apoptotic (244) and BMF is pro-apoptotic (245). Normally, BMF is sequestered by association with myosin, and apoptosis can only be activated if BMF is released and binds BCL2 (245). Whether or not it is activated in the presence of Spn4A will have to be examined. We have grown infected cells expressing Spn4A for up to five days and have not detected cell death (data not shown). Further,  80     cells transiently transfected with Spn4A did not exhibit activated caspase 3 after 48 hours (data not shown). 3.3.8  Ontological, pathway, and network analysis of genes regulated by RFP-tagged Spn4A in the secretory pathway To investigate if an RFP tag may affect the function of Spn4A, we analyzed the global  transcriptional changes for secreted RFP-tagged Spn4A-S. Notably, fewer genes were regulated compared to Spn4A without RFP. From a total of 383 differentially regulated genes, 236 were downregulated and 147 upregulated. Gene ontology and enrichment analysis of the downregulated genes revealed nine significant clusters including sprouty assisted development (ES = 5.1), cell cycle progression (several clusters), regulation of phosphorylation (two clusters), blood vessel development (ES = 2.9), and coagulation (ES = 2.4) as listed in Appendix 4, Supplementary Table A4.2. Compared to non-RFP-tagged Spn4A, these clusters had lower enrichment scores but shared the same annotations. Gene ontology of the upregulated genes annotated to extracellular matrix components (Supplemental Table A4.3). In addition, molecular and cellular functional analysis for genes regulated in the presence of RFP Spn4A was performed using IPA. Gene functions largely agreed with the DAVID analysis, and were very similar to the functional annotations for Spn4A (Supplemental Fig. A4.1). Pathway analysis performed using Ingenuity revealed ten significant canonical pathways (Supplemental Fig. A4.2). Cell cycle progression pathways as well as metabolism of xenobiotics, coagulation, hepatic fibrosis/hepatic stellate cell activation, and arginine and proline metabolism were recognized. Several networks were found to be significant (Supplemental Table A4.4). Most genes of the top two networks mapped to cancer, cardiovascular system development and function, embryonic development, dermatological disease and conditions, and cell cycle functions. We noted that MYC, identified as a key regulator in response to Spn4A-S, was not differentially regulated in cells expressing RFP Spn4A-S. Further, not as many genes associated with DNA replication and mitosis components were differentially regulated (Supplemental Table A4.1). To evaluate the gene ontology similarities and differences in response to RFP-tagged or non-tagged Spn4A, each unique or overlapped gene list was separately analyzed by DAVID and summarized in Figure 3.9. Full cluster annotations are reported in Supplemental Tables A4.5 to A4.9. In general, cell cycle and extracellular matrix annotations were found in the genes unique 81     to WT-S and in the overlapping region, which suggested that while WT-S and RFP WT-S regulated many similar pathways, WT-S induced transcriptional changes in many more genes of that pathway. No gene annotations reached significance from the unique and downregulated RFP WT-S gene list. One cluster reached significance using the list unique for RFP WT-S upregulated genes, and involved chloride ion binding (ES = 2.1). However, only three molecules were associated with the annotation, which may call its significance into question. The decrease in differentially regulated genes may be due to the reduced ability of RFPtagged Spn4A-S to inhibit furin. Although we were able to detect a complex between RFP Spn4A and furin (Fig. 3.1), it is probable that the tag slowed down the kinetics of binding, which may have allowed some furin to escape the complex. Alternatively, the tag may have destabilized the final complex and allowed faster hydrolysis and release of the protease. 3.3.9  Ontological, pathway, and network analysis of genes regulated by RFP-tagged Spn4A retained in the ER Last, we asked whether or not the effects of Spn4A function are compartment-specific by  profiling the transcriptome of ER-localized RFP Spn4A-R and comparing the profile to that of RFP WT-S and WT-S. We found that pathways and functions altered by expression of RFP WTR were very similar, but with a lower number of gene expression changes. Of 214 differentially regulated genes, 158 were downregulated and 56 upregulated. DAVID gene ontology analysis of down and upregulated genes (Supplemental Tables A4.10 and A4.11), IPA molecular and cellular functional analysis (Supplemental Fig. A4.3), and IPA network analysis (Supplemental A4.12) pointed to cellular proliferation as the most significantly altered gene function and network. In comparison to RFP WT-S, 83 genes were uniquely regulated by RFP WT-R (58 downregulated, 25 upregulated) and 131 were shared (100 downregulated, 31 upregulated) (Fig. 3.10, full annotation in Supplemental Table A4.13 to A4.16). All enriched clusters of downregulated genes for either unique or overlapped groups showed that DNA replication and cell cycle were affected. Analysis of the unique and upregulated genes demonstrated a difference between secreted and retained Spn4A. The response to RFP WT-R expression did not involve upregulation of genes coding for extracellular matrix proteins, which was an annotation enriched in RFP WT-S (ES = 3.2).  82     With the exception of ECM related genes, ER-retained Spn4A-R affected similar genes compared to secreted Spn4A-S, suggesting that their cellular mechanism of action may be the same. Furin activity is believed to be inhibited in the ER and early Golgi compartments due to its association with its pro-peptide, which is cleaved and released in the Golgi due to the acidic environment (101). We suggest that in the slightly acidic environments of the ERGIC and cisGolgi compartments, activated furin may encounter ER-retained Spn4A-R. Spn4A-R may move by anterograde transport to the ERGIC and cis-Golgi compartments, where some could inhibit furin, while others move by retrograde transport back to the ER. Precedence of ER-retained proteins that cycle in the ERGIC and cis-Golgi compartments is demonstrated by the KDEL (Lys-Asp-Glu-Leu) receptor, which transport ligands with KDEL motifs back to the ER. Optimal binding of receptor to ligand is at pH 5.5 in vitro, suggesting that binding of ligands occur in the ERGIC and cis-Golgi compartments (246). Spn4A-R may inhibit the same PCs as secretory pathway Spn4A-S, but its usual ER localization would hamper the extent of inhibition. Thus, fewer cell cycle genes and ECM related genes would be regulated in response to Spn4A-R expression. To visualize the differences between the regulation of genes in the presence of WT-S, RFP WT-S, or RFP WT-R, we compared the absolute number of genes differentially regulated in cell growth and proliferation as well as cell movement (Fig. 3.11). These two functions were among the most significant in all three analyses and represented two themes especially important in tumorigenesis and metastasis. Cells differentially regulated the most number of genes in response to WT-S and the fewest number to RFP WT-R. The RFP tag and compartmentalization affected Spn4A function, although neither could totally abolish its function. 3.3.10 Real-time PCR validation We measured the mRNA levels of three cell cycle genes, CDK2, CDC2, and PCNA, and the chemokine IL8, using qPCR (real-time PCR) for cells infected with Ad. RFP WT-R compared to those infected with Ad. Emp (Fig. 3.12). We validated PCNA, CDK2, and IL8 downregulation. These genes were not significantly and differentially regulated in cells expressing non-inhibitory Spn4A mutants (Chapter 2), RFP S52R-R and RFP H338R-R (data not shown).  83     3.4  Conclusions and future directions We and others have hypothesized that PCs represent a novel class of therapeutic targets  for bacterial and viral diseases as well as cancer (40;112;196;247;248). Here, we evaluate the applicability of Spn4A as a PC inhibitor in neuroglioma cells. We used gene expression analysis to evaluate potential effects on tumor progression and invasion as well as to uncover stress or apoptotic pathways. Previously, two studies featuring furin inhibition in glioma cells demonstrated decreased tumor proliferation and invasive ability due to the loss of matured IGF-1R, TGFβ, MT1-MMP, and MMP-2 (160;164). Results presented here corroborate those findings and uncover additional genes that could explain the loss of the malignancy phenotype. First, we suggest that the loss of cell proliferative ability is likely due to upregulation of G1 kinase/cyclin inhibitors and downregulation of kinases, cyclins, DNA replication machinery, and mitosis components. Second, we report changes in the regulation of genes implicated in cell adhesion and migration and ECM degradation, which may contribute to the loss of malignancy. However, we have also uncovered genes that promote invasion, which are regulated by Spn4A expression. Third, our results demonstrated a new potential antiangiogenic role for PC inhibitors in their ability to downregulate VEGFA and IL8. Further, our microarray data did not reveal specific induction of cellular stress and apoptosis by Spn4A. Last, the results demonstrate that an RFP tag as well as ER compartmentalization will affect the role of Spn4A in mediating the above effects. Kinetic studies will have to be performed to evaluate if the RFP tag slows the serpin inhibitory mechanism. We have demonstrated the ability of Spn4A to change the transcriptome of neuroglioma cells. We will first validate more genes by qPCR to measure the robustness of the data. Of interest are the cell proliferation, cell movement, and angiogenesis genes discussed. Next, we will validate the results with functional assays targeting changes in cell cycle and invasive ability. We predict that Spn4A would induce cell cycle arrest, which we will test through CFSE (carboxyfluorescein succinimidyl ester) staining. CFSE is a dye that enters the cytoplasm of cells and is passed to daughter cells following division. Therefore, cell proliferation can be visualized using flow cytometry analysis. Next, the ability of Spn4A to affect cell adhesion and ECM remodeling will be assessed. As the invasiveness of H4 neuroglioma cells has not been tested, we will use an already established invasive glioma cell line such as U87MG in parallel. Invasive 84     ability may be tested by monitoring a scratch wound on a cell monolayer or in a Matrigel invasion assay, in which cells are plated in an invasion chamber coated with Matrigel and allowed to invade. Last, we will use antibodies and western blotting to evaluate if the angiogenic factors VEGFA and IL8 are downregulated on the protein level. As a negative control, we have constructed non-inhibitory Spn4A hinge-mutants (Appendix 1), which are fully processed by furin. Further, a better understanding of how Spn4A exerts its biological influence is needed. Primarily, its physiological targets need to be clarified. Spn4A is highly specific for furin, but has not been tested against other PCs. To detect Spn4A targets, we will immunoprecipitate Spn4A and use antibodies specific for the PCs to identify the co-immunoprecipitated complexes on western blots. Alternatively, we may target furin with siRNA and test if the same mRNAs are differentially regulated. If Spn4A does primarily inhibit furin, we hypothesize that inhibition of furin-mediated processing of TGFβ, IGFR, and PDGF may lead to the changes observed. Western blots to test for processing of furin targets and the ability of recombinant processed substrates to restore malignancy will be necessary to show the mechanism underlying Spn4A cellular effects. Although the inhibition of PCs by Spn4A is the most likely cause for the transcriptome changes, we cannot rule out other functional domains of serpins, beyond its inhibitory domain, that may contribute to the described effects. For example, non-inhibitory serpins, such as ovine uterine serpin and maspin, have blocked cell cycle progression through unknown mechanisms when exogenously supplied or expressed (22;249;250). Further, the serpins’ cleaved C-terminal tails, which are released upon serpin-protease complex formation, may have activity. Exogenously synthesized peptides corresponding to the 26 residue C-terminus of α1-antitrypsin has been shown to increase the proliferation of hepatoma and breast cancer cells, but not kidney or skin cells (251). Other functional domains of Spn4A, beyond its inhibitory activity, have not been reported. Non-inhibitory Spn4A (Appendix 1) and synthesized peptides corresponding to the C-terminal tail of Spn4A are important controls to understand the contribution of additional domains to transcriptome changes. In summary, we have expressed a furin inhibitor, Spn4A, in a neuroglioma cell line and conducted a transcriptome analysis. We found several patterns of gene expression suggesting changes in cell proliferation and cell mobility, which collectively support previous studies 85     demonstrating loss of tumor growth and invasive ability (160;164). Further, we provide new mechanistic hypotheses as to how treatment with furin inhibitors may lead to the loss of invasive ability and angiogenesis.  86     Figure 3.1. Structural features of PCs. The structural features of human furin and six other PCs, yeast Kex2p, and bacterial subtilisin E are shown. PC5/6 is expressed as two isoforms, which are generated by alternative splicing. The diagonally dashed line links two halves of the PC5/6B isoform. The bold labels D, H, S highlight the active site residues while N and D highlight the oxyanion hole residues. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology, Thomas G, Furin at the cutting edge: From protein traffic to embryogenesis and disease (101), © 2002.  87     Figure 3.2. Activation of furin is spatially regulated. In the ER, the furin prodomain acts as an intramolecular chaperone (IMC) to facilitate folding of the inactive catalytic domain (pink) into the active conformation (red oval). Furin undergoes autoproteolytic intramolecular excision of the propeptide at Arg107 (blue). However, the propeptide still remains associated with the mature domain as a potent inhibitor during transport to the Golgi. The acidic pH of the transGolgi network promotes the autoproteolytic intramolecular cleavage of the propeptide at Arg75 (blue) and subsequent disassociation of the fragments from furin. Adapted with permission from Anderson E, Molloy SS, Jean F, Fei H, Shimamura S, and Thomas G (117), © 2002 American Society for Biochemistry and Molecular Biology. Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology, Thomas G, Furin at the cutting edge: From protein traffic to embryogenesis and disease (101), © 2002.  88     Figure 3.3. Cellular localization of the PCs. PCs can be found in the Golgi, the trans-Golgi network (TGN), secretory granules (SG), endosomes (END), at the plasma membrane (PM) as integral membrane proteins, in the extracellular matrix (ECM) and at the cell surface (CS), and/or in the extracellular space (ECS). Reprinted by permission from Informa UK Ltd: Expert Opinion, Chretien M, Seidah NG, Basak A and Mbikay M, Proprotein convertases as therapeutic targets (40), © 2008.  89     Figure 3.4. Spn4A and furin EI complex formation assay. HEK293-C4 cells overexpressing FLAG-tagged furin were transfected as labeled for 48 hours. Cells (C) were lysed and media (M) was collected, boiled for 10 minutes in SDS loading buffer, and proteins separated on a SDSPAGE. Western blot analysis was performed using anti-FLAG antibodies for Spn4A and furin (green) and anti-Hsp47 (red) as a loading control.  90     Figure 3.5. Top 10 most significant cellular and molecular functions for genes differentially regulated by Spn4A WT-S expression identified by Ingenuity Pathway Analysis. Ingenuity Pathway Analysis software was used to associate cellular and molecular functions to differentially regulated genes in response to Spn4A WT-S. Significance was calculated from Fisher’s exact test. Genes associated with cellular growth and proliferation and cellular movement are in Appendix 5, Supplemental Tables A5.3 and A5.4.  20 15 10 5  Amino Acid Metabolism  Cellular Assembly and Organization  Cell Morphology  Cellular Compromise  Cellular Movement  Cellular Development  DNA Replication, Recombination, and Repair  Cellular Growth and Proliferation  Cell Death  0 Cell Cycle  -log (significance)  25  91     Figure 3.6. The most significant canonical pathways deduced from genes differentially regulated by Spn4A WT-S expression. Ingenuity Pathway Analysis software was used to generate canonical pathways regulated in response to Spn4A WT-S. Significance was calculated from Fisher’s exact test (p<0.01). Genes associated with each canonical pathway are listed in Supplemental Table A5.5.  8  6 5 4 3 2 1 Pancreatic Adenocarcinoma Signaling  Hereditary Breast Cancer Signaling  ATM Signaling  Mitotic Roles of Polo-Like Kinase  Role of BRCA1 in DNA Damage Response  Cell Cycle: G1/S Checkpoint Regulation  Aryl Hydrocarbon Receptor Signaling  0 Role of CHK Proteins in Cell Cycle Checkpoint Control  -log (significance)  7  92     Figure 3.7. The top two most significant networks of genes regulated by Spn4A WT-S expression. A) and B) Networks were identified by Ingenuity Pathway Analysis. Different shapes identify with different types of molecular classes and the lines connecting the molecules indicate their relationships. The fold changes of upregulated genes (red) and downregulated genes (green) are labeled underneath the molecule. Colour denotes intensity of regulation.  93     A)  94     B)  95     Figure 3.8. Points of the cell cycle where genes are differentially regulated in response to Spn4A-S expression. The cell cycle consists of G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). The amount of DNA (coloured lines) and size of cell are illustrated in each stage. Differentially regulated genes that act on transitions or at stages of the cell cycle are labelled. Full gene names and fold changes are in Supplemental Table A4.1.  96     Figure 3.9. Summary of DAVID gene ontology analysis for each list of regulated genes unique to or shared between WT-S and RFP WT-S. The list of differentially regulated genes in response to WT-S expression were compared to those of RFP WT-S and summarized into a venn diagram. Downregulated (green circles) or upregulated (red circles) genes for each unique or shared list were separately analyzed on DAVID functional annotation clustering and one representative term from each cluster is shown. The numbers in brackets are enrichment scores for the clusters. Only significant clusters (enrichment score > 2) are shown, up to ten clusters. Full gene annotations are in Appendix 4, Supplemental Tables A4.5 to A4.9.  97     Figure 3.10. Summary of DAVID gene ontology analysis for each list of regulated genes unique to or shared between RFP WT-S and RFP WT-R. The list of differentially regulated genes in response to RFP WT-S expression were compared to those of RFP WT-R and summarized into a venn diagram. Downregulated (green circles) or upregulated (red circles) genes for each unique or shared list were separately analyzed on DAVID functional annotation clustering and one representative term from each cluster is shown. The numbers in brackets represent enrichment score for the cluster. Only significant clusters (enrichment score > 2) are shown. Full gene annotations are in Appendix 4, Supplemental Tables A4.13 to A4.16.  98     Figure 3.11. Comparison of the number of genes annotated with cell growth and proliferation or cell movement that are regulated in response to WT-S, RFP WT-S, and RFP WT-R. Genes annotated by Ingenuity Pathway Analysis as cell growth and proliferation or cell movement were counted and summarised.  99     Figure 3.12. The mRNA levels of three cell cycle genes and IL8 in response to RFP WT-R determined by microarray and qPCR. RNA was collected from H4 neuroglioma cells infected with Ad. Emp or Ad. RFP WT-R, reverse transcribed to cDNA, and used for real-time PCR. FC represent comparisons of Ad. RFP WT-R to Ad. Emp. Significant values (p<0.05*) are as denoted (two-tailed Student’s t-test). Error bars represent SEM (standard error of the mean).  0.0  RFP WT-R/Emp FC  -0.5 -1.0 -1.5 -2.0  *  *  -2.5 -3.0 -3.5  *  PCNA  CDC2  CDK2  IL8  Microarray  -1.7  -1.8  -2.0  -2.8  qRT-PCR  -1.7  -2.3  -1.9  -3.2  100     Table 3.1. DAVID GO functional annotation clusters for genes downregulated by Spn4A WT-S expression. DAVID functional annotation clustering was carried out using high stringency for clustering. Term represent annotation term, count is the number of genes involved in the individual term, p-value is calculated using a modified Fisher’s exact test and represent the significance of gene-term enrichment, fold enrichment measures the magnitude of enrichment against the complete array background, and annotation cluster enrichment score is the negative log transformation of the geometric mean of the p-values in the group. Genes from one annotation for each cluster, up to ten clusters, are listed in Supplemental Table A5.2.  Term  Count p-value Annotation Cluster 1: Enrichment Score = 23.6 cell division 45 2.13E-25 GO:0022402~cell cycle process 66 7.44E-25 GO:0000278~mitotic cell cycle 54 8.53E-25 GO:0007067~mitosis 43 1.02E-24 GO:0000280~nuclear division 43 1.02E-24 mitosis 38 1.53E-24 GO:0000087~M phase of mitotic cell cycle 43 2.15E-24 GO:0022403~cell cycle phase 56 3.65E-24 GO:0048285~organelle fission 43 5.37E-24 GO:0000279~M phase 50 9.50E-24 GO:0051301~cell division 46 3.40E-22 Annotation Cluster 2: Enrichment Score = 13.3 kinetochore 20 1.23E-16 GO:0000779~condensed chromosome, centromeric region 20 1.53E-15 GO:0000777~condensed chromosome kinetochore 17 5.70E-13 GO:0000776~kinetochore 17 5.12E-11 Annotation Cluster 3: Enrichment Score 11.9 GO:0031981~nuclear lumen 85 4.43E-14 GO:0031974~membrane-enclosed lumen 96 1.16E-12 GO:0043233~organelle lumen 94 2.61E-12 GO:0070013~intracellular organelle lumen 91 1.20E-11 Annotation Cluster 4: Enrichment Score = 7.3 IPR018525:DNA-dependent ATPase MCM, conserved site 7 3.54E-09 domain:MCM 7 3.83E-09 IPR001208:DNA-dependent ATPase MCM 7 1.04E-08 SM00350:MCM 7 1.54E-08 IPR012340:Nucleic acid-binding, OB-fold 8 1.63E-04      Fold Enrichment 7.42 4.47 5.58 7.46 7.46 9.02 7.33 5.19 7.16 5.82 5.94 13.72 11.97 11.57 8.83 2.34 2.06 2.06 2.04 38.45 37.99 34.17 31.77 6.76  101    Term  Count p-value Annotation Cluster 5: Enrichment Score = 4.7 nucleotide phosphate-binding region:ATP 49 3.09E-07 GO:0005524~ATP binding 65 1.14E-06 GO:0001882~nucleoside binding 69 1.28E-06 GO:0032559~adenyl ribonucleotide binding 65 1.83E-06 GO:0030554~adenyl nucleotide binding 67 2.52E-06 atp-binding 58 3.09E-06 GO:0001883~purine nucleoside binding 67 4.25E-06 GO:0032555~purine ribonucleotide binding 66 4.92E-04 GO:0032553~ribonucleotide binding 66 4.92E-04 GO:0017076~purine nucleotide binding 68 5.72E-04 nucleotide-binding 59 1.07E-03 GO:0000166~nucleotide binding 73 3.43E-03 Annotation Cluster 6: Enrichment Score: 4.3 GO:0006268~DNA unwinding during replication 6 2.04E-05 GO:0032392~DNA geometric change 6 7.99E-05 GO:0032508~DNA duplex unwinding 6 7.99E-05 Annotation Cluster 7: Enrichment Score = 4.0 IPR007875:Sprouty 5 8.66E-06 domain:SPR 5 9.11E-06 GO:0043407~negative regulation of MAP kinase activity 5 1.44E-02 Annotation Cluster 8: Enrichment Score = 3.7 domain:Kinesin-motor 9 4.33E-06 IPR001752:Kinesin, motor region 8 3.39E-05 IPR019821:Kinesin, motor region, conserved site 8 3.39E-05 SM00129:KISc 8 5.14E-05 GO:0003777~microtubule motor activity 10 8.27E-05 GO:0003774~motor activity 11 2.15E-03 motor protein 10 3.39E-03 Annotation Cluster 9: Enrichment Score = 3.6 GO:0005663~DNA replication factor C complex 5 5.74E-06 PIRSF004274:phage T4 DNA polymerase accessory protein 44 4 5.65E-05 GO:0003689~DNA clamp loader activity 4 1.33E-04 GO:0033170~protein-DNA loading ATPase activity 4 1.33E-04 IPR013748:Replication factor C 3 1.52E-03 GO:0008094~DNA-dependent ATPase activity 5 4.79E-02      Fold Enrichment 2.23 1.84 1.79 1.82 1.78 1.91 1.75 1.51 1.51 1.49 1.53 1.37 16.21 12.61 12.61 31.38 31.01 5.25 9.30 8.57 8.57 7.97 5.45 3.23 3.30 32.90 40.65 33.16 33.16 43.94 3.64  102    Term  Count p-value Annotation Cluster 10: Enrichment Score = 3.4 IPR004367:Cyclin, C-terminal 6 9.91E-06 PIRSF001771:cyclin, A/B/D/E types 5 9.98E-05 IPR014400:Cyclin, A/B/D/E 5 1.12E-04 PIRSF001771:Cyclin_A_B_D_E 5 1.47E-04 IPR006671:Cyclin, N-terminal 6 8.24E-04 cyclin 7 9.83E-04 IPR013763:Cyclin-related 6 1.41E-03 IPR006670:Cyclin 6 2.01E-03 SM00385:CYCLIN 6 2.69E-03 Annotation Cluster 11: Enrichment Score = 3.3 GO:0005663~DNA replication factor C complex 5 5.74E-06 GO:0006297~nucleotide-excision repair, DNA gap filling 6 5.90E-05 hsa03420:Nucleotide excision repair 6 9.30E-03 GO:0006289~nucleotide-excision repair 6 1.36E-02 Annotation Cluster 12: Enrichment Score = 3.3 GO:0019220~regulation of phosphate metabolic process 28 1.77E-04 GO:0051174~regulation of phosphorus metabolic process 28 1.77E-04 GO:0042325~regulation of phosphorylation 27 2.33E-04 GO:0045859~regulation of protein kinase activity 21 8.34E-04 GO:0043549~regulation of kinase activity 21 1.28E-03 GO:0051338~regulation of transferase activity 21 2.08E-03 Annotation Cluster 13: Enrichment Score = 3.1 GO:0051303~establishment of chromosome localization 5 5.15E-04 GO:0050000~chromosome localization 5 5.15E-04 GO:0051310~metaphase plate congression 4 2.56E-03 Annotation Cluster 14: Enrichment Score = 3.0 GO:0007126~meiosis 10 9.58E-04 GO:0051327~M phase of meiotic cell cycle 10 9.58E-04 GO:0051321~meiotic cell cycle 10 1.11E-03 Annotation Cluster 15: Enrichment Score = 2.6 GO:0031577~spindle checkpoint 5 1.99E-04 GO:0045841~negative regulation of mitotic metaphase/anaphase transition 4 2.56E-03 GO:0007094~mitotic cell cycle spindle assembly checkpoint 4 2.56E-03 GO:0045839~negative regulation of mitosis 4 3.35E-03 GO:0051784~negative regulation of nuclear division 4 3.35E-03 GO:0030071~regulation of mitotic metaphase/anaphase transition 4 1.93E-02      Fold Enrichment 18.83 18.48 18.31 16.94 7.99 6.05 7.12 6.59 6.13 32.90 13.35 4.55 4.20 2.21 2.21 2.21 2.30 2.22 2.13 12.61 12.61 13.75 3.94 3.94 3.86 15.76 13.75 13.75 12.61 12.61 6.88  103    Term  Count p-value Annotation Cluster 16: Enrichment Score = 2.3 GO:0048754~branching morphogenesis of a tube 8 1.56E-03 GO:0001763~morphogenesis of a branching structure 8 3.30E-03 GO:0035239~tube morphogenesis 9 1.92E-02 Annotation Cluster 17: Enrichment Score = 2.2 GO:0005658~alpha DNA polymerase:primase complex 4 3.01E-04 GO:0030894~replisome 4 4.72E-03 GO:0043601~nuclear replisome 4 4.72E-03 GO:0043596~nuclear replication fork 4 5.79E-03 GO:0032993~protein-DNA complex 5 1.62E-01 Annotation Cluster 18: Enrichment Score = 2.2 domain:BRCT 1 4 2.27E-03 domain:BRCT 2 4 2.27E-03 IPR001357:BRCT 4 1.65E-02 SM00292:BRCT 4 1.98E-02 Annotation Cluster 19: Enrichment Score = 2.2 GO:0007584~response to nutrient 11 3.96E-03 GO:0009991~response to extracellular stimulus 14 5.55E-03 GO:0031667~response to nutrient levels 12 1.50E-02 Annotation Cluster 20: Enrichment Score = 2.1 GO:0007596~blood coagulation 9 4.89E-03 GO:0050817~coagulation 9 4.89E-03 GO:0007599~hemostasis 9 6.92E-03 GO:0050878~regulation of body fluid levels 9 2.97E-02  Fold Enrichment 4.65 4.09 2.68 26.32 11.28 11.28 10.53 2.35 14.47 14.47 7.32 6.81 2.97 2.41 2.30 3.40 3.40 3.21 2.47  104     Table 3.2. DAVID GO functional annotation clusters for genes upregulated by Spn4A WTS expression. Parameters are as described in Table 3.1.  Term  Count p-value Annotation Cluster 1: Enrichment Score = 6.7 GO:0031012~extracellular matrix 17 1.03E-08 GO:0005578~proteinaceous extracellular matrix 16 2.67E-08 extracellular matrix 14 2.85E-08 GO:0044421~extracellular region part 20 1.57E-04 Annotation Cluster 2: Enrichment Score = 2.7 GO:0005581~collagen 5 1.68E-04 trimer 4 9.31E-04 hydroxylysine 4 1.57E-03 triple helix 4 1.57E-03 GO:0030934~anchoring collagen 3 2.20E-03 hydroxyproline 4 2.62E-03 collagen 5 5.18E-03 IPR008160:Collagen triple helix repeat 4 2.61E-02 Annotation Cluster 3: Enrichment Score = 2.3 GO:0005539~glycosaminoglycan binding 6 3.80E-03 GO:0030247~polysaccharide binding 6 5.55E-03 GO:0001871~pattern binding 6 5.55E-03 Annotation Cluster 4: Enrichment Score = 2.3 IPR018244:Allergen V5/Tpx-1 related, conserved site 3 3.08E-03 IPR001283:Allergen V5/Tpx-1 related 3 5.77E-03 IPR014044:SCP-like extracellular 3 5.77E-03 SM00198:SCP 3 7.71E-03 Annotation Cluster 5: Enrichment Score = 2.1 IPR000372:Leucine-rich repeat, cysteine-rich flanking region, N-terminal 6 8.27E-04 SM00013:LRRNT 6 1.56E-03 repeat:LRR 6 7 4.36E-03 IPR001611:Leucine-rich repeat 7 5.31E-03 repeat:LRR 5 7 6.97E-03 repeat:LRR 7 6 9.30E-03 repeat:LRR 4 7 1.06E-02 repeat:LRR 3 7 2.10E-02 repeat:LRR 1 7 2.69E-02 repeat:LRR 2 7 2.73E-02 leucine-rich repeat 7 2.81E-02  Fold Enrichment 6.20 6.30 7.80 2.61 17.66 20.48 17.17 17.17 41.20 14.39 7.16 6.22 5.73 5.24 5.24 35.18 25.80 25.80 22.06  8.15 6.97 4.55 4.36 4.13 4.66 3.78 3.24 3.06 3.05 3.02  105     Table 3.3. Top 10 networks generated from genes differentially regulated by Spn4A WT-S expression as identified by Ingenuity Pathway Analysis. Networks were generated using Ingenuity Pathway Analysis. Score is the negative log of the significance, which is based on number of focus molecules and size of network. The most annotated functions from molecules in the network are listed. ID  Score  1  49  Focus Molecules 33  2  39  29  3  37  28  4  31  25  5 6  31 29  26 24  7  29  24  8  23  21  9  23  24  10  22  20  Top Functions Cell Cycle, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair Cellular Assembly and Organization, DNA Replication, Recombination, and Repair, Gene Expression DNA Replication, Recombination, and Repair, Infection Mechanism, Gene Expression Cardiovascular System Development and Function, Embryonic Development, Tissue Development Cell Cycle, Embryonic Development, Small Molecule Biochemistry Cell Cycle, Cellular Assembly and Organization, DNA Replication, Recombination, and Repair Connective Tissue Disorders, Organismal Injury and Abnormalities, Cardiovascular System Development and Function Cell-To-Cell Signaling and Interaction, Reproductive System Development and Function, Cardiovascular Disease Nervous System Development and Function, Organ Development, Gene Expression Dermatological Diseases and Conditions, Genetic Disorder, Immunological Disease     106     Table 3.4. Top 10 downregulated or upregulated genes associated with cell movement as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S expression. Gene symbol, gene names, and fold changes are listed. The full list is in Supplemental Table A5.4. Gene Symbol Downregulated IL8 ITGA2 ETV5 TGFA GDF15 SPP1 ERRFI1 AJAP1 ITGA6 RGS1 Upregulated C5ORF13 ALPP CD24 S100A4 SOCS2 PPAP2A GUCY1A3 CD36 OLR1 DCN  Gene Name  Fold Change  interleukin 8 integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) ets variant 5 transforming growth factor, alpha growth differentiation factor 15 secreted phosphoprotein 1 ERBB receptor feedback inhibitor 1 adherens junctions associated protein 1 integrin, alpha 6 regulator of G-protein signaling 1  -5.319 -4.695 -4.348 -3.846 -3.236 -2.959 -2.933 -2.924 -2.857 -2.710  chromosome 5 open reading frame 13, also known as P311 alkaline phosphatase, placental (Regan isozyme) CD24 molecule S100 calcium binding protein A4 suppressor of cytokine signaling 2 phosphatidic acid phosphatase type 2A guanylate cyclase 1, soluble, alpha 3 CD36 molecule (thrombospondin receptor) oxidized low density lipoprotein (lectin-like) receptor 1 decorin  2.203 2.206 2.283 2.364 2.643 2.717 3.054 3.329 3.630 4.215     107     Chapter 4: Conclusion The goals of this thesis were to study the cellular response to both the disease-causing and therapeutic roles of serpins using Drosophila Spn4A. Accordingly, we developed polymerforming Spn4A mutants and expressed Spn4A WT and mutants in H4 neuroglioma cells to simultaneously address their cellular effects via microarray. Fig. 4.1 summarizes the key findings and future work.  4.1  Implications for cellular and animal models of serpinopathies Current cellular models expressing hNS mutants reveal that polymers induce an EOR  response (73). However, pathways activating EOR and its downstream effects leading to disease are not clear. To address the cellular pathways leading to pathology, we expressed Spn4A polymer-forming mutants in H4 neuroglioma cells and identified genes, via microarray, that are differentially regulated in their presence. The validation of the 17 differentially regulated genes is of immediate interest. Further, the question of whether or not human neuroserpin mutants regulate the same genes will need to be addressed. These genes that are specifically regulated in the presence of polymers may provide insight into mutant serpin pathogenesis. Identification and characterization of the proteins regulated by serpin polymers may serve as potential targets for effective therapy. It is possible that none of the genes identified can be validated or their gene products linked to cytotoxicity. In fact, we and others have not observed or reported toxic effects of serpin polymers in cell culture systems, suggesting that studies performed in cancer cells may not be appropriate. Non-cancerous neuronal cells or transgenic animals may be more appropriate models. Such systems would allow us to address, for example, if apoptosis may result from NFκB activation that drives proliferation (74) of post-mitotic neurons. The adenovirus constructs that we have developed can be applied to most cell systems efficiently, which will aid the transition for future studies. Last, in collaboration with Dr. Carl Hashimoto, we are making transgenic Drosophila overexpressing Spn4A WT and mutants. Our future studies will include characterization of the transgenic flies. Using the Drosophila model, we will evaluate if Spn4A polymers may mediate disease like hNS mutants. If the transgenic flies recapitulate the neurological dysfunction associated with FENIB, we will characterize disease pathways in vivo. Alternatively, if the 108     polymers do not cause neurological disorders, we will examine the mechanisms underlying how polymers are cleared or pathways leading to restoration of neuronal function.  4.2  Implications for the use of PCs as therapeutic targets We and others have hypothesized that PCs may represent therapeutic targets for cancer  and infectious diseases (40;112;196;247;248). It was previously shown that PC inhibitors ameliorate the malignancy phenotype in glioma cells (160;164), as well as several other cancer cell lines (163;165;206). Now, we have profiled the transcriptome of neuroglioma cells expressing Spn4A, a potent inhibitor of furin and identified additional genes that may mediate tumor proliferation and invasion, as well as angiogenesis. Our data suggest that Spn4A expression can, on the transcriptome level, modulate multiple pathways contributing to malignancy and therefore, may represent a good therapeutic agent. Spn4A directly inhibits the maturation (160;164) and may indirectly affect the expression of several proteins currently being evaluated as therapeutic targets. For example, inhibitors for MMPs (252), VEGFA (253), and Aurora kinases (254) are currently in clinical trials. However, targeted inhibition of several MMPs and MT1-MMP in phase II or III clinical trials were ineffective (255) and resistance to VEGFA inhibitors by upregulation of other angiogenic factors (IL8, FGF, PDFG) or recruitment of vascular progenitors inevitably occurs (209). We propose that using a PC inhibitor such as Spn4A may be advantageous as it simultaneously disrupts multiple genes and proteins involved in tumorigenesis and metastasis (Fig. 4.2). The microarray results will be valuable for researchers proposing to use PC inhibitors for proliferative diseases. Although the microarray was performed on neuroglioma cells, similar pathways may be regulated in other types of cancer cells. Others have demonstrated that furin inhibition can suppress the metastatic potential in multiple cell types such as colon carcinoma (165), breast cancer cells (206), and head and neck squamous carcinoma cells (163). However, regulation is dependent on the inhibitor used. For example, in breast cancer cells, α1-PDX promotes invasiveness while the prosegment of furin inhibits the process (206). Therefore, the ability of Spn4A to reduce the malignancy phenotype will need to be assessed in a cell specific manner. The applicability of Spn4A in fighting infectious disease in normal cells will have to be evaluated based on our new data. Since Spn4A expression may affect cellular proliferation, 109     motility, and cardiovascular systems, systematic treatment using furin inhibitors would not be possible, at least in the long term. We did not observe induction of cellular stress and apoptosis specific to Spn4A WT, but we detected differential regulation of genes of the BCL2 family. While no apparent apoptosis was observed under our experimental conditions, we cannot rule out that the cells may be sensitized to stress. The use of Spn4A in short term therapy for acute diseases in non-proliferating cells may be possible with minimal effects.  4.3  Closing remarks We have developed a cell-based system expressing Spn4A WT and polymer-forming  mutants to assess their effects on the cellular transcriptome. Importantly, we identified genes that may reflect the downstream effects of the loss of functional PC substrates or of polymer buildup. The proposed future experiments are targeted at elucidating the underlying mechanisms and/or consequences of Spn4A WT and mutants. Defining the mechanism underlying the cellular changes in response to Spn4A WT will be essential in the development of PC inhibitors as therapeutic agents. Further, understanding the genes modulated by polymers may provide important insight into disease mechanisms. Taken together, our study lays the foundation for future studies aimed at elucidating the serpins’ roles as a therapeutic and in disease.     110     Figure 4.1. Flow chart of key findings and future directions in this thesis project.  111     Figure 4.2. PCs as anti-cancer treatment targets. 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We hypothesized that glycine may disrupt the inhibitory action of serpins because glycine is found in non-inhibitory maspin (Supplemental Fig. A1.1). Methods were as described in Chapter 2: Cell lines (2.2.2), Plasmid constructs (2.2.3) and primers (Appendix 2), Recombinant adenovirus constructs and production (2.2.4), Transfection and infection conditions (2.2.5), SDS-PAGE, native PAGE, and western blot (2.2.6), Immunofluorescence (2.2.7), and Furin complex formation assay (2.2.8). Results and Discussion We first substituted mutations at Spn4A T328D, T328G, and T328K into Spn4A-R (Supplemental Fig. A1.2A) and transfected them into H4 neuroglioma cells for in vitro complex formation assays with furin. H4 cell lysates containing Spn4A WT-R or the T328G variant could form a heat and SDS-stable complex with recombinant human furin (Supplemental Fig. A1.3A). However, the amount of SDS-stable complex formed by Spn4A T328G-R is clearly less than the WT and a large buildup of cleaved Spn4A T328G-R was observed. This suggests that P14 glycine affects the inhibitory mechanism, but does not completely abolish inhibitory function. Aspartic or lysine substitutions at T328 rendered Spn4A completely non-inhibitory, as no SDSstable complex was detected (Supplemental Fig. A1.3A). The hinge mutants likely slowed down the rate of loop insertion so the serpin follows the substrate pathway. Using immunofluorescence, we ensured that the single point mutations did not affect protein folding or cause aggregation (Supplemental Fig. A1.3B). We further engineered the T328D mutation into the secreted Spn4A-S and both secreted and retained RFP-tagged Spn4A (Supplemental Fig. A1.2A) and tested their activities in cellulo using HEK293-C4 cells that overexpress FLAG-tagged furin. Transfected cells were analyzed 133     for complex formation by SDS-PAGE. We found that intracellularly, secreted Spn4A WT-S were found as free serpins or as complexes while T328D mutants were mostly cleaved by furin and formed no inhibitory complexes (Supplemental Fig. A1.4A). In the media, complexes were detected in both WT-S and T328D-S, and not ER-retained T328D-R. The T328D-R should not be trafficked to the extracellular space. The presence of extracellular cleavage products suggests that some Spn4A-R may have escaped the ER retention because of the loss of the C-terminus from furin cleavage. Complex and cleavage product were both detected in the secreted T328D-S variant, suggesting that uncleaved Spn4A T328D-S are trafficked to the extracellular space where it can undergo both inhibitory and substrate pathways. In comparison, the lack of intracellular EI complexes suggests that the rate of loop insertion has been compromised, but could still occur in specific environments. We confirmed that secreted and retained RFP Spn4A T328D variants do not form intracellular complexes and has increased cleavage product (Fig. A1.4B). Cleaved Spn4A T328D-R was not observed in the media, suggesting that the RFP tag may play a role in the retention, trafficking, or degradation of cleaved products. The RFP tag does not affect the secretion of Spn4A T328D-S, as extracellular EI complex and cleavage product were both observed in the T328D-S mutant, similar to the non-tagged Spn4A. Further, RFP tag Spn4A mutants exhibited similar staining to those without the RFP, as detected through confocal microscopy (data not shown). Last, adenoviruses of furin-directed hinge mutants Spn4A T328D-R and T328D-S, and SKI-1 directed (RSL changed from RRKR to RRLL to inhibit SKI-1) hinge mutant Spn4A T328D RRLL-S and T328D RRLL-R (Supplemental Fig. A1.2B), were produced. These variants were used as negative controls for my own resesearch as well as others who study furin and SKI1 processing of viral proteins during infections. The adenovirus expression of the hinge mutants were tested in HEK293-C4 cells (Supplemental Fig. A1.5). Spn4A T328D-R and T328D-S did not form an EI complex in cellulo unlike the WT forms (Supplemental Fig. A1.5). No EI complexes were detected with SKI-1 directed Spn4A RRLL WT or hinge mutant, as expected. Further work from Vanessa Silva (Jean Lab, technician) demonstrated that unlike Spn4A WT-S, the hinge mutant Spn4A T328D-S was not effective at blocking furin-processing of influenza HA. Andrea Olmstead (Jean Lab, phD student) confirmed that Ad. Spn4A T328D RRLL-S did not form a heat- and SDS-stable complex with SKI-1 and was not effective at blocking SKI-1 activation of sterol regulatory element-binding proteins. 134     Supplemental Figure A1.1. Sequence alignment of the hinge and reactive site loop region of Drosophila Spn4A and human maspin. The P14 amino acid from the RSL (reactive site loop) is in blue text.  135     Supplemental Figure A1.2. Serpin hinge mutant constructs. The mutations T328D (blue triangles) were engineered into A) furin-directed Spn4A-R, RFP Spn4A-R, Spn4A-S, and RFP Spn4A-S, and B) SKI-1 directed Spn4A RRLL-R and Spn4A RRLL-S. RRKR and RRLL are the amino acid pseudo-substrate sequences targeting furin and SKI-1, respectively. Other key characteristics of the constructs include SP (signal peptide) for targeting to the secretory pathway, HF (His and FLAG tag), RFP (Red fluorescent protein), and HDEL ER retention signal.  A)  B)  136     Supplemental Figure A1.3. In vitro EI complex formation assay and immunofluorescence of Spn4A hinge mutants. A) H4 cells were transfected with Spn4A WT-R and H338R-R for 48 hours. Cells were harvested, lysed, and incubated for 10 minutes with His-tagged recombinant furin at 30°C. The reaction was quenched with 50 mM EDTA, ran on an SDS-PAGE, and probed with anti-His antibodies for Spn4A and furin (green) and anti-tubulin antibodies (red), as a loading control, for western blot analysis. B) H4 cells were transfected as labeled. After 48 hours, they were fixed and probed for Spn4A (red) with anti-FLAG antibodies and ER (green) with anti-calnexin antibodies, and stained for nuclei (blue) with Hoechst dye.  137     A)  B)  138     Supplemental Figure A1.4. In cellulo EI complex formation assay with Spn4A hinge mutants. A) and B) HEK293-C4 cells that overexpress FLAG-tagged furin were transfected as labeled. After 48 hours, media (M) was collected, cells (C) were lysed, boiled for 10 minutes in SDS loading buffer, and proteins separated on a SDS-PAGE. Western blot analysis was performed using anti-FLAG antibodies for Spn4A and furin (green) and anti-Hsp47 (red) as a loading control.  A)  B)  139     Supplemental Figure A1.5. In cellulo EI complex formation assay with adenovirus expressing Spn4A hinge mutants. HEK293-C4 cells that overexpress FLAG-tagged furin were infected as labeled at MOI 5 as labeled for 48 hours. Cells (C) were lysed, boiled for 10 minutes in SDS loading buffer, and proteins separated on a SDS-PAGE. Western blot analysis was performed using anti-FLAG antibodies for Spn4A and furin (green) and anti-Hsp47 (red) as a loading control.  140     Appendix 2: Supplemental methods and materials  Supplemental Table A2.1. Primer names and sequences used for cloning. For mutagenesis primers, only the forward primer sequences are provided as the reverse primer is just the reverse complement of the forward.  Primer Name Primer Sequence Primers for cloning cytoplasmic Spn4A F-KpnI-HF Spn4A ATCGGGTACC ATG CAC CAC CAC CAC CAC CAC GAC R-Not1-Spn4A-S ATCG GCGGCCGC TCACTCGCTGGAGGCGAAGGTATTTTC F-EcoR1-tagRFP ATCG GAATTC ATG GTG TCT AAG GGC GAA GAG Sp4s.AS GCGGCCGCTCACTCGCTGGAGGCGAAGG Mutagenesis primers for Spn4A Spn4A S49A-F GAGAACATCGTCTTCGCGCCCTTTTCCATCC Spn4A S49P-F GAGAACTCGTCTTCCCGCCCTTTTCCATCC Spn4A S52R F CGTCTTCTCGCCCTTTCGCATCCAGACTTGTGC Spn4A H338R- F GTGTCAGCCATCATACGCAAGGCCTTCATTGAAG Spn4A G392E-F CTGCCATTGTTTTGGGAGTCAGTTGTGCGGCTCG F-Spn4A T328G CAACGAGGAGGGAGGGGAGGCTGCGGCC F-Spn4A-T328K CAACGAGGAGGGAAAGGAGGCTGCGGCC F-Spn4A-T328D GTCAACGAGGAGGGAGACGAGGCTGCGGCCACG Primers for cloning hNS F-BamH1- hNS ATCGGGATCCATGGACTACAAAGATGACGATGACAAGATGGCTT TCCTTGGACTCTTCT R-Not1-hNS ATCG GCGGCCGC TTAAAGTTCTTCGAAATCATGTCCAC F-QC-FhNS1 GTACCGAGCTCGGATCGTTGGACTACAAAGATGACG F-QC-FhNS2 GGACTACAAAGATGACGTTGACAAGATGGCTTTCC F-QC-FhNS3 GTTGGACTACAAAGTTGACGTTGACAAGATGGCTTTCC F-SP-F-hNS /5PHOS/GACGATGACAAGTTCCCTGAGGAAGCCATTG R-SP-F-hNS /5PHOS/ATCTTTGTAGTCAGTGGCCCCTGTAGCCATACT hNS S49P-F GATGAAAATATTCTCTTCCCTCCATTGAGTATTGCTCTTGC hNS H338R-F GATTTTTCTTTCCAAAGCAATTCGCAAGTCCTTCCTAGAGG  141     Supplemental Table A2.2. Titers of Adenovirus constructs.  Adenovirus construct Titer Empty 2.90E+06 RFP WT-R 5.10E+07 RFP S52R - R 8.10E+05 RFP H338R - R 6.80E+05 RFP WT-S 8.72E+07 RFP H338R-S 1.34E+08 WT-S 5.45E+07 RFP T328D-S* 1.20E+08 RFP H338R-C* 1.64E+08 * RFP T328D-S and RFP H338R-C constructs were made and titered for use in the future.  142     Supplemental Table A2.3. Primers and probes for qPCR.  Primer name F-HERPUD1 R-HERPUD1 F-DDIT3 R-DDIT3 F-Hspa5 R-Hspa5 F-SPRR2D R-SPRR2D F-CDK2 R-CDK2 F-PCNA R-PCNA F-CDC2 R-CDC2 F-bAct425 R-bAct502  Sequence AGGGACTTGCTTCCAAAGGT CTGTCCCCGATTAGAACCAG CAGAGCTGGAACCTGAGGAG TGGATCAGTCTGGAAAAGCA CAGCCTGGCGACAAGAGT CCTTGGGCAGTATTGGATTC GGTACTCTAGCACCGATCTGCT TGCACTGCTGCTGTTGATAA CCTCCTGGGCTGCAAATA CAGAATCTCCAGGGAATAGGG TGGAGAACTTGGAAATGGAAA GAACTGGTTCATTCATCTCTATGG TGGATCTGAAGAAATACTTGGATTCTA CAATCCCCTGTAGGATTTGG CCAACCGCGAGAAGATGA CCAGAGGCGTACAGGGATAG  Roche probe # 82 82 9 9 39 39 18 18 50 50 69 69 79 79 64 64  143     Appendix 3: Chapter 2 supplemental figures and tables  Supplemental Figure A3.1. Enlarged figures of RFP Spn4A WT-S and H338R-S. Experiments were as described in Fig. 2.6.  RFP Spn4A WT-S     RFP Spn4A H338R-S     144     Supplemental Figure A3.2. Example flow cytometry graphs used to calculate percentage of cells expressing RFP Spn4A. Experiments were as described in Fig. 2.8.  MOI 0  RFP WT-R MOI 10  1200 100  0.011  12.3  87.7  300  # Cells  # Cells  900  200  600  100  300  0  0 10  0  10  1  2  10 FL2-H  10  3  10  4  10  0  10  1  2  10 FL2-H  10  3  10  4  145     Supplemental Figure A3.3. Immunofluorescent images of transgenic Drosophila pupae expressing RFP Spn4A WT-R, RFP Spn4A T328D-R, and RFP Spn4A H338R-R. UAS (upstream activating sequence) transgenic Drosophila expressing RFP Spn4A WT-R, T328D-R, or H338R-R were generated and salivary gland expression was driven by Ptc-Gal4. Sample names start with YCL, and a and b denote two different transgenic lines. The non-inhibitory and monomeric Spn4A T328D-R mutant is described in Appendix 1. Drosophila images were from Dr. Carl Hashimoto (Yale University).  146     Supplemental Table A3.1. Functional annotation clusters for genes downregulated by secreted or retained RFP Spn4A WT and mutants expression. DAVID functional annotation clustering was carried out using only the non unique downregulated genes and high stringency filter. Term represent annotation term, count is the number of genes involved in the individual term, p-value is calculated using a modified Fisher’s exact test and represent the significance of gene-term enrichment, fold enrichment measures the magnitude of enrichment against the complete array background, and annotation cluster enrichment score is the negative log transformation of the geometric mean of the p-values in the group.  Term  Count p-value Annotation Cluster 1: Enrichment Score = 2.4 GO:0009725~response to hormone stimulus 6 2.35E-03 GO:0009719~response to endogenous stimulus 6 3.59E-03 GO:0010033~response to organic substance 7 9.50E-03 Annotation Cluster 2: Enrichment Score = 2.1 GO:0042493~response to drug 5 2.17E-03 GO:0007584~response to nutrient 4 5.58E-03 GO:0048545~response to steroid hormone stimulus 4 1.32E-02 GO:0031667~response to nutrient levels 4 1.42E-02 GO:0009991~response to extracellular stimulus 4 1.90E-02  Fold Enrichment 6.15 5.57 3.66 8.76 10.71 7.81 7.61 6.82  147     Appendix 4: Chapter 3 supplemental figures and tables  Cellular Assembly and Organization  Cell-To-Cell Signaling and Interaction  DNA Replication, Recombination, and Repair  Cell Morphology  Cellular Compromise  Cellular Development  Cell Death  Cell Cycle  Cellular Movement  10 9 8 7 6 5 4 3 2 1 0 Cellular Growth and Proliferation  -log (significance)  Supplemental Figure A4.1. Top 10 most significant cellular and molecular functions for genes differentially regulated by RFP Spn4A WT-S expression identified by Ingenuity Pathway Analysis. Ingenuity Pathway Analysis software was used to associate cellular and molecular functions to differentially regulated genes. Significance was calculated from Fisher’s exact test.       148  Pancreatic Adenocarcinoma Signaling  Mitotic Roles of Polo-Like Kinase  Arginine and Proline Metabolism  Hepatic Fibrosis / Hepatic Stellate Cell Activation  Cell Cycle Regulation by BTG Family Proteins  Coagulation System  Metabolism of Xenobiotics by Cytochrome P450  Cell Cycle: G1/S Checkpoint Regulation  Role of CHK Proteins in Cell Cycle Checkpoint Control  Aryl Hydrocarbon Receptor Signaling  -log (significance)     Supplemental Figure A4.2. The most significant canonical pathways deduced from differentially regulated genes in response to RFP Spn4A WT-S. Ingenuity Pathway Analysis software was used to generate canonical pathways regulated in response to RFP Spn4A WT-S. Significance was calculated from Fisher’s exact test (p<0.01).  7  6  5  4  3  2  1  0  149     Supplemental Figure A4.3. Top 10 most significant cellular and molecular functions for genes differentially regulated by RFP Spn4A WT-R expression identified by Ingenuity Pathway Analysis. Ingenuity Pathway Analysis software was used to associate cellular and molecular functions to differentially regulated genes. Significance was calculated from Fisher’s exact test.  8 6 5 4 3 2 1 Small Molecule Biochemistry  Lipid Metabolism  Cell Morphology  Cell-To-Cell Signaling and Interaction  Cell Cycle  DNA Replication, Recombination, and Repair  Cellular Development  Cell Death  Cellular Growth and Proliferation  0 Cellular Movement  -log (significance)  7     150     Supplemental Table A4.1. Fold changes of genes discussed in Figure 3.8. Genes symbols, gene names, and fold changes (FC) are listed. NS is not significant. Gene Gene name symbol CELL CYCLE REGULATOR CDK2 Homo sapiens cyclin-dependent kinase 2 CDC2 Homo sapiens cell division cycle 2, G1 to S and G2 to M CDC25A Homo sapiens cell division cycle 25 homolog A (S. pombe) CCNE2 Homo sapiens cyclin E2 CCND1 Homo sapiens cyclin D1 CCNB1 Homo sapiens cyclin B1 CDKN1C Homo sapiens cyclin-dependent kinase inhibitor 1C CDKN2C Homo sapiens cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) E2F2 Homo sapiens E2F transcription factor 2 MYC Homo sapiens v-myc myelocytomatosis viral oncogene homolog DNA REPLICATION ORC6L Homo sapiens origin recognition complex, subunit 6 like (yeast) MCM4 Homo sapiens minichromosome maintenance complex component 4 MCM3 Homo sapiens minichromosome maintenance complex component 3 MCM7 Homo sapiens minichromosome maintenance complex component 7 MCM6 Homo sapiens minichromosome maintenance complex component 6 MCM8 Homo sapiens minichromosome maintenance complex component 8 MCM2 Homo sapiens minichromosome maintenance complex component 2 MCM5 Homo sapiens minichromosome maintenance complex component 5 MCM10 Homo sapiens minichromosome maintenance complex component 10 POLA1 Homo sapiens polymerase (DNA directed), alpha 1, catalytic subunit POLA2 Homo sapiens polymerase (DNA directed), alpha 2 (70kD subunit) POLE2 Homo sapiens polymerase (DNA directed), epsilon 2 (p59 subunit) RFC4 Homo sapiens replication factor C (activator 1) 4, 37kDa RFC2 Homo sapiens replication factor C (activator 1) 2, 40kDa RFC5 Homo sapiens replication factor C (activator 1) 5, 36.5kDa RFC3 Homo sapiens replication factor C (activator 1) 3, 38kDa PRIM1 Homo sapiens primase, DNA, polypeptide 1 (49kDa)      FC WT-S  FC RFP WT-S  FC RFP WT-R  NS -2.02 -1.82 -2.81 -2.52 -1.92 2.58 1.91  NS -1.81 -1.99 -2.07 -2.51 NS 2.66 NS  -1.96 NS NS -1.87 -2.50 NS NS NS  -2.53 -1.96  -2.43 NS  NS NS  -1.81  NS  NS  -2.10  -1.71  NS  -2.15  -1.95  -1.83  -1.70  NS  NS  -1.70  NS  NS  -1.71  NS  NS  -1.71  NS  -1.70  -1.72  NS  NS  -2.18  -1.82  NS  -1.71  NS  NS  -2.00  NS  NS  -2.18  -2.21  NS  -2.16 -1.93 -2.11 -2.41 -2.04  NS -2.06 NS -1.95 NS  -1.77 NS -1.79 -1.80 -1.80  151    Genes MITOSIS CENPO CENPK CENPE CENPA MLF1IP RAD21 NEK2 AURKA AURKB SMC2 SMC3    Gene name  FC WT-S  FC RFP WT-S  FC RFP WT-R  Homo sapiens centromere protein O Homo sapiens centromere protein K Homo sapiens centromere protein E, 312kDa Homo sapiens centromere protein A Homo sapiens MLF1 interacting protein Homo sapiens RAD21 homolog (S. pombe) Homo sapiens NIMA (never in mitosis gene a)-related kinase 2 Homo sapiens aurora kinase A (AURKA), transcript variant 3, mRNA. Homo sapiens aurora kinase B Homo sapiens structural maintenance of chromosomes 2 Homo sapiens structural maintenance of chromosomes 3  -1.87 -1.83 -1.91 -2.03 -1.74 -1.73 -1.89  NS NS NS NS NS NS NS  NS -1.71 NS NS NS NS NS  -2.04  -1.89  -2.18  NS -1.75 -1.75  -2.11 NS NS  -1.74 NS NS     152     Supplemental Table A4.2. DAVID GO functional annotation clusters for genes downregulated by RFP Spn4A WT-S expression. DAVID functional annotation clustering was carried using high stringency filter. Term represent annotation term, count is the number of genes involved in the individual term, p-value is calculated using a modified Fisher’s exact test and represent the significance of gene-term enrichment, fold enrichment measures the magnitude of enrichment against the complete array background, and annotation cluster enrichment score is the negative log transformation of the geometric mean of the p-values in the group.  Term  Count p-value Annotation Cluster 1: Enrichment Score = 5.1 IPR007875:Sprouty 5 5.59E-07 domain:SPR 5 5.88E-07 GO:0043407~negative regulation of MAP kinase activity 5 1.37E-03 Annotation Cluster 2: Enrichment Score = 4.3 GO:0000278~mitotic cell cycle 17 3.85E-05 GO:0022403~cell cycle phase 18 4.01E-05 GO:0007067~mitosis 13 4.19E-05 GO:0000280~nuclear division 13 4.19E-05 GO:0000087~M phase of mitotic cell cycle 13 5.01E-05 mitosis 11 5.08E-05 GO:0048285~organelle fission 13 6.19E-05 GO:0022402~cell cycle process 21 7.11E-05 GO:0000279~M phase 15 1.39E-04 Annotation Cluster 3: Enrichment Score = 3.4 GO:0045859~regulation of protein kinase activity 15 2.58E-04 GO:0019220~regulation of phosphate metabolic process 18 2.86E-04 GO:0051174~regulation of phosphorus metabolic process 18 2.86E-04 GO:0043549~regulation of kinase activity 15 3.64E-04 GO:0051338~regulation of transferase activity 15 5.49E-04 GO:0042325~regulation of phosphorylation 17 5.53E-04 Annotation Cluster 4: Enrichment Score = 3.4 GO:0031974~membrane-enclosed lumen 42 1.61E-04 GO:0031981~nuclear lumen 35 2.21E-04 GO:0043233~organelle lumen 40 4.67E-04 GO:0070013~intracellular organelle lumen 38 1.19E-03 Annotation Cluster 5: Enrichment Score = 3.4 IPR004367:Cyclin, C-terminal 5 1.50E-05 cyclin 6 2.68E-04 IPR014400:Cyclin, A/B/D/E 4 2.98E-04 PIRSF001771:cyclin, A/B/D/E types 4 4.08E-04 IPR006671:Cyclin, N-terminal 5 5.18E-04 PIRSF001771:Cyclin_A_B_D_E 4 5.39E-04 IPR013763:Cyclin-related 5 8.07E-04      Fold Enrichment 62.26 61.59 10.18 3.41 3.23 4.37 4.37 4.29 5.22 4.20 2.75 3.38 3.19 2.75 2.75 3.08 2.96 2.70 1.79 1.91 1.73 1.69 31.13 10.37 29.05 25.65 13.21 23.51 11.78  153    Term IPR006670:Cyclin SM00385:CYCLIN  Count p-value 5 1.09E-03 5 1.74E-03 Annotation Cluster 6: Enrichment Score = 2.9 GO:0001944~vasculature development 12 6.11E-04 GO:0001568~blood vessel development 11 1.83E-03 GO:0048514~blood vessel morphogenesis 10 2.27E-03 Annotation Cluster 7: Enrichment Score = 2.4 GO:0050817~coagulation 7 2.38E-03 GO:0007596~blood coagulation 7 2.38E-03 GO:0007599~hemostasis 7 3.19E-03 GO:0050878~regulation of body fluid levels 7 1.13E-02 Annotation Cluster 8: Enrichment Score = 2.2 GO:0006270~DNA replication initiation 4 1.21E-03 IPR018525:DNA-dependent ATPase MCM, conserved site 3 3.47E-03 domain:MCM 3 3.55E-03 IPR001208:DNA-dependent ATPase MCM 3 4.42E-03 SM00350:MCM 3 5.71E-03 IPR012340:Nucleic acid-binding, OB-fold 3 1.19E-01 Annotation Cluster 9: Enrichment Score = 2.1 GO:0006469~negative regulation of protein kinase activity 7 1.17E-03 GO:0033673~negative regulation of kinase activity 7 1.39E-03 GO:0051348~negative regulation of transferase activity 7 1.94E-03 GO:0043086~negative regulation of catalytic activity 8 8.21E-02 GO:0044092~negative regulation of molecular function 9 8.39E-02      Fold Enrichment 10.90 9.51 3.52 3.31 3.49 5.13 5.13 4.84 3.72 18.33 32.69 32.33 29.05 25.36 5.03 5.90 5.70 5.35 2.13 1.98     154     Supplemental Table A4.3. DAVID GO functional annotation clusters for genes upregulated by RFP Spn4A WT-S expression. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.2 GO:0005578~proteinaceous extracellular matrix 10 3.54E-04 GO:0031012~extracellular matrix 10 6.18E-04 extracellular matrix 8 1.32E-03 Annotation Cluster 2: Enrichment Score = 2.4 domain:GST N-terminal 4 3.87E-04 IPR004045:Glutathione S-transferase, N-terminal 4 4.93E-04 IPR010987:Glutathione S-transferase, C-terminal-like 4 1.13E-03 IPR017933:Glutathione S-transferase/chloride channel, Cterminal 4 1.25E-03 domain:GST C-terminal 4 1.49E-03 PIRSF000503:glutathione transferase 3 5.36E-03 IPR012335:Thioredoxin fold 5 5.69E-03 GO:0004364~glutathione transferase activity 3 9.58E-03 IPR004046:Glutathione S-transferase, C-terminal 3 9.69E-03 GO:0016765~transferase activity, transferring alkyl or aryl (other than methyl) groups 3 4.98E-02 hsa00480:Glutathione metabolism 3 5.15E-02 Annotation Cluster 3: Enrichment Score = 2.0 GO:0030247~polysaccharide binding 6 5.55E-03 GO:0001871~pattern binding 6 5.55E-03 GO:0030246~carbohydrate binding 8 1.58E-02 GO:0005539~glycosaminoglycan binding 5 2.01E-02      Fold Enrichment 4.47 4.14 4.84 27.46 25.27 19.17 18.53 17.47 26.36 6.95 19.90 19.86 8.29 7.96 5.24 5.24 3.02 4.77     155     Supplemental Table A4.4. Top 10 networks generated from genes differentially regulated by RFP Spn4A WT-S expression as identified by Ingenuity Pathway Analysis. Networks were generated using Ingenuity Pathway Analysis. Score is the negative log of the significance, which is based on number of focus molecules and size of network. The most annotated functions from molecules in the network are listed. ID  Score  1  40  Focus Molecules 27  2 3  39 31  26 23  4  29  21  5  27  20  6  27  20  7 8 9 10  23 18 18 18  18 16 16 15  Top Functions Cancer, Cardiovascular System Development and Function, Embryonic Development Cancer, Dermatological Diseases and Conditions, Cell Cycle Cancer, Gastrointestinal Disease, Hematological System Development and Function Cancer, Cardiovascular System Development and Function, Cell Morphology Cellular Growth and Proliferation, Hematological System Development and Function, Cell Death Connective Tissue Disorders, Organismal Injury and Abnormalities, Cardiovascular System Development and Function Cell Morphology, Cellular Development, Cellular Movement Cellular Movement, Hepatic System Disease, Tissue Development Embryonic Development, Tissue Development, Gene Expression Genetic Disorder, Neurological Disease, DNA Replication, Recombination, and Repair          156     Supplemental Table A4.5. DAVID GO functional annotation clusters of unique and downregulated genes in response to Spn4A WT-S compared to RFP Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.  Term  Count p-value Annotation Cluster 1: Enrichment Score = 20.4 GO:0022402~cell cycle process 48 4.59E-23 GO:0007067~mitosis 32 5.72E-22 GO:0000280~nuclear division 32 5.72E-22 GO:0000278~mitotic cell cycle 39 7.72E-22 GO:0000087~M phase of mitotic cell cycle 32 9.97E-22 GO:0000279~M phase 37 1.17E-21 GO:0048285~organelle fission 32 1.96E-21 GO:0022403~cell cycle phase 40 4.16E-21 mitosis 28 1.91E-20 cell division 31 2.55E-19 GO:0051301~cell division 32 3.81E-18 Annotation Cluster 2: Enrichment Score = 13.1 Term Count PValue kinetochore 17 1.74E-16 GO:0000779~condensed chromosome, centromeric region 17 1.29E-15 GO:0000777~condensed chromosome kinetochore 14 2.03E-12 GO:0000776~kinetochore 14 7.78E-11 Annotation Cluster 3: Enrichment Score = 10.0 GO:0031981~nuclear lumen 56 1.45E-11 GO:0043233~organelle lumen 62 1.48E-10 GO:0070013~intracellular organelle lumen 61 1.74E-10 GO:0031974~membrane-enclosed lumen 62 3.19E-10 Annotation Cluster 4: Enrichment Score = 8.1 GO:0015630~microtubule cytoskeleton 34 3.26E-12 GO:0044430~cytoskeletal part 37 7.81E-08 GO:0005856~cytoskeleton 43 2.49E-06 Annotation Cluster 5: Enrichment Score =6.8 IPR018525:DNA-dependent ATPase MCM, conserved site 6 2.03E-08 domain:MCM 6 2.33E-08 IPR001208:DNA-dependent ATPase MCM 6 4.52E-08 SM00350:MCM 6 5.16E-08 IPR012340:Nucleic acid-binding, OB-fold 7 5.92E-05 Annotation Cluster 6: Enrichment Score =5.54 GO:0001882~nucleoside binding 48 7.55E-08 GO:0005524~ATP binding 45 1.22E-07 GO:0032559~adenyl ribonucleotide binding 45 1.81E-07 GO:0030554~adenyl nucleotide binding 46 2.94E-07 GO:0001883~purine nucleoside binding 46 4.55E-07  Fold Enrichment 5.78 9.88 9.88 7.17 9.70 7.66 9.49 6.60 11.25 8.65 7.35 Fold Enrichment 19.73 17.21 16.13 12.31 2.61 2.29 2.31 2.25 4.21 2.67 2.12 56.78 55.35 50.47 48.62 10.19 2.24 2.30 2.26 2.20 2.16  157    Term Count p-value nucleotide phosphate-binding region:ATP 34 6.98E-07 atp-binding 41 1.38E-06 GO:0032553~ribonucleotide binding 45 3.90E-05 GO:0032555~purine ribonucleotide binding 45 3.90E-05 GO:0017076~purine nucleotide binding 46 5.23E-05 GO:0000166~nucleotide binding 51 6.51E-05 nucleotide-binding 41 3.07E-04 Annotation Cluster 7: Enrichment Score = 4.1 GO:0006268~DNA unwinding during replication 5 4.13E-05 GO:0032508~DNA duplex unwinding 5 1.20E-04 GO:0032392~DNA geometric change 5 1.20E-04 Annotation Cluster 8: Enrichment Score = 4.0 GO:0007126~meiosis 9 8.62E-05 GO:0051327~M phase of meiotic cell cycle 9 8.62E-05 GO:0051321~meiotic cell cycle 9 9.97E-05 Annotation Cluster 9: Enrichment Score = 3.1 GO:0031577~spindle checkpoint 5 2.09E-05 GO:0045841~negative regulation of mitotic metaphase/anaphase transition 4 4.81E-04 GO:0007094~mitotic cell cycle spindle assembly checkpoint 4 4.81E-04 GO:0045839~negative regulation of mitosis 4 6.34E-04 GO:0051784~negative regulation of nuclear division 4 6.34E-04 GO:0030071~regulation of mitotic metaphase/anaphase transition 4 3.98E-03 GO:0010639~negative regulation of organelle organization 5 3.19E-02 Annotation Cluster 10: Enrichment Score = 3.0 IPR001752:Kinesin, motor region 6 1.91E-04 IPR019821:Kinesin, motor region, conserved site 6 1.91E-04 SM00129:KISc 6 2.17E-04 domain:Kinesin-motor 6 2.43E-04 GO:0003777~microtubule motor activity 7 5.19E-04 GO:0003774~motor activity 8 2.80E-03 motor protein 7 9.15E-03 GO:0007018~microtubule-based movement 6 2.36E-02 Annotation Cluster 11: Enrichment Score = 2.6 GO:0050000~chromosome localization 4 1.27E-03 GO:0051303~establishment of chromosome localization 4 1.27E-03 GO:0051310~metaphase plate congression 3 1.10E-02 Annotation Cluster 12: Enrichment Score = 2.2 GO:0032886~regulation of microtubule-based process 6 7.54E-04 GO:0070507~regulation of microtubule cytoskeleton organization 5 3.34E-03 GO:0051493~regulation of cytoskeleton organization 5 1.37E-01      Fold Enrichment 2.62 2.28 1.85 1.85 1.81 1.72 1.80 24.03 18.69 18.69 6.31 6.31 6.18 28.04 24.47 24.47 22.43 22.43 12.24 4.15 11.08 11.08 10.67 10.54 6.87 4.23 3.91 3.67 17.95 17.95 18.35 8.24 8.01 2.51  158     Supplemental Table A4.6. DAVID GO functional annotation clusters of unique and upregulated genes in response to Spn4A WT-S compared to RFP Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.6 GO:0031012~extracellular matrix 8 3.12E-05 GO:0005578~proteinaceous extracellular matrix 7 1.89E-04 GO:0044421~extracellular region part 9 3.46E-03 Annotation Cluster 2: Enrichment Score: 2.3 domain:Laminin EGF-like 1 3 1.90E-03 domain:Laminin EGF-like 2 3 2.05E-03 laminin egf-like domain 3 2.52E-03 IPR002049:EGF-like, laminin 3 4.63E-03 SM00180:EGF_Lam 3 7.53E-03 IPR013032:EGF-like region, conserved site 4 3.59E-02    Fold Enrichment 8.33 7.87 3.36 45.30 43.62 39.36 28.79 22.06 5.37          159     Supplemental Table A4.7. DAVID GO functional annotation cluster of unique and downregulated genes in response to RFP Spn4A WT-S compared to Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 2.0 chloride 3 6.39E-03 GO:0031404~chloride ion binding 3 9.84E-03 GO:0043168~anion binding 3 1.39E-02    Fold Enrichment 24.19 19.13 15.98            160     Supplemental Table A4.8. DAVID GO functional annotation clusters of shared and downregulated genes in response to RFP Spn4A WT-S and Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 5.4 IPR007875:Sprouty 5 3.04E-07 domain:SPR 5 3.13E-07 GO:0043407~negative regulation of MAP kinase activity 5 8.42E-04 Annotation Cluster 2: Enrichment Score = 4.2 GO:0022403~cell cycle phase 17 2.82E-05 GO:0000278~mitotic cell cycle 16 3.06E-05 GO:0022402~cell cycle process 20 3.55E-05 GO:0007067~mitosis 12 5.84E-05 GO:0000280~nuclear division 12 5.84E-05 GO:0000087~M phase of mitotic cell cycle 12 6.88E-05 mitosis 10 8.14E-05 GO:0048285~organelle fission 12 8.41E-05 GO:0000279~M phase 14 1.34E-04 Annotation Cluster 3: Enrichment Score = 3.8 GO:0045859~regulation of protein kinase activity 15 6.33E-05 GO:0043549~regulation of kinase activity 15 9.12E-05 GO:0051338~regulation of transferase activity 15 1.41E-04 GO:0019220~regulation of phosphate metabolic process 17 1.90E-04 GO:0051174~regulation of phosphorus metabolic process 17 1.90E-04 GO:0042325~regulation of phosphorylation 16 4.02E-04 Annotation Cluster 4: Enrichment Score = 3.6 IPR004367:Cyclin, C-terminal 5 8.23E-06 cyclin 6 1.29E-04 IPR014400:Cyclin, A/B/D/E 4 1.90E-04 PIRSF001771:cyclin, A/B/D/E types 4 2.72E-04 IPR006671:Cyclin, N-terminal 5 2.91E-04 PIRSF001771:Cyclin_A_B_D_E 4 3.59E-04 IPR013763:Cyclin-related 5 4.56E-04 IPR006670:Cyclin 5 6.16E-04 SM00385:CYCLIN 5 9.62E-04 Annotation Cluster 5: Enrichment Score = 3.37 GO:0005654~nucleoplasm 25 2.11E-05 GO:0031981~nuclear lumen 31 2.77E-04 GO:0031974~membrane-enclosed lumen 36 4.68E-04 GO:0043233~organelle lumen 34 1.44E-03 GO:0070013~intracellular organelle lumen 32 3.84E-03      Fold Enrichment 72.44 72.02 11.61 3.48 3.65 2.99 4.60 4.60 4.52 5.55 4.42 3.60 3.63 3.51 3.37 2.96 2.96 2.89 36.22 12.13 33.81 29.40 15.37 26.95 13.70 12.68 11.14 2.61 1.98 1.79 1.72 1.66  161    Term  Count p-value Annotation Cluster 6: Enrichment Score = 3.3 GO:0001944~vasculature development 12 1.97E-04 GO:0001568~blood vessel development 11 6.71E-04 GO:0048514~blood vessel morphogenesis 10 9.09E-04 Annotation Cluster 7: Enrichment Score = 2.7 GO:0050817~coagulation 7 1.22E-03 GO:0007596~blood coagulation 7 1.22E-03 GO:0007599~hemostasis 7 1.65E-03 GO:0050878~regulation of body fluid levels 7 6.08E-03 Annotation Cluster 8: Enrichment Score = 2.4 GO:0006469~negative regulation of protein kinase activity 7 5.86E-04 GO:0033673~negative regulation of kinase activity 7 7.01E-04 GO:0051348~negative regulation of transferase activity 7 9.85E-04 GO:0044092~negative regulation of molecular function 9 4.54E-02 GO:0043086~negative regulation of catalytic activity 8 4.68E-02 Annotation Cluster 9: Enrichment Score = 2.2 GO:0033273~response to vitamin 6 1.11E-03 GO:0009991~response to extracellular stimulus 9 4.78E-03 GO:0007584~response to nutrient 7 6.52E-03 GO:0031667~response to nutrient levels 7 3.03E-02      Fold Enrichment 4.01 3.77 3.98 5.85 5.85 5.52 4.24 6.73 6.50 6.09 2.26 2.42 7.60 3.42 4.18 2.97  162     Supplemental Table A4.9. DAVID GO functional annotation clusters of shared and upregulated genes in response to RFP Spn4A WT-S and Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term Annotation Cluster 1: Enrichment Score = 2.9 extracellular matrix GO:0005578~proteinaceous extracellular matrix GO:0031012~extracellular matrix GO:0044421~extracellular region part Annotation Cluster 1: Enrichment Score = 2.1 GO:0005539~glycosaminoglycan binding GO:0001871~pattern binding GO:0030247~polysaccharide binding Annotation Cluster 1: Enrichment Score = 2.0 IPR015590:Aldehyde dehydrogenase IPR016162:Aldehyde dehydrogenase, N-terminal GO:0016620~oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor IPR016160:Aldehyde dehydrogenase, conserved site GO:0006081~cellular aldehyde metabolic process nucleotide phosphate-binding region:NAD  Count  p-value  Fold Enrichment  8 9 9 11  2.47E-04 3.45E-04 5.74E-04 3.63E-02  6.38 5.06 4.69 2.05  5 5 5  6.69E-03 9.12E-03 9.12E-03  6.57 6.01 6.01  3 3 3  4.16E-03 4.64E-03 6.75E-03  30.54 28.93 23.82  3 3 3  9.93E-03 1.05E-02 5.96E-02  19.63 19.00 7.51            163     Supplemental Table A4.10. DAVID GO functional annotation clusters of genes downregulated by RFP Spn4A WT-R expression. Parameters are as described in Supplemental Table A4.2.      Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.8 cell division 12 8.61E-06 GO:0000279~M phase 13 9.87E-05 mitosis 9 1.09E-04 GO:0022402~cell cycle process 17 1.40E-04 GO:0051301~cell division 12 1.68E-04 GO:0022403~cell cycle phase 14 2.19E-04 GO:0000278~mitotic cell cycle 13 2.98E-04 GO:0000280~nuclear division 10 3.35E-04 GO:0007067~mitosis 10 3.35E-04 GO:0000087~M phase of mitotic cell cycle 10 3.83E-04 GO:0048285~organelle fission 10 4.50E-04 Annotation Cluster 2: Enrichment Score = 3.3 GO:0006268~DNA unwinding during replication 4 3.27E-04 GO:0032508~DNA duplex unwinding 4 7.11E-04 GO:0032392~DNA geometric change 4 7.11E-04 Annotation Cluster 3: Enrichment Score = 2.3 GO:0006270~DNA replication initiation 4 4.95E-04 domain:MCM 3 1.73E-03 IPR018525:DNA-dependent ATPase MCM, conserved site 3 1.80E-03 IPR001208:DNA-dependent ATPase MCM 3 2.31E-03 SM00350:MCM 3 3.09E-03 IPR012340:Nucleic acid-binding, OB-fold 3 6.76E-02 GO:0044454~nuclear chromosome part 4 1.09E-01 Annotation Cluster 4: Enrichment Score = 2.1 GO:0005663~DNA replication factor C complex 4 1.66E-05 PIRSF004274:phage T4 DNA polymerase accessory protein 44 3 5.37E-04 GO:0006297~nucleotide-excision repair, DNA gap filling 4 5.97E-04 GO:0033170~protein-DNA loading ATPase activity 3 7.29E-04 GO:0003689~DNA clamp loader activity 3 7.29E-04 hsa03430:Mismatch repair 4 2.63E-03 hsa03420:Nucleotide excision repair 4 1.64E-02 GO:0006289~nucleotide-excision repair 4 1.67E-02 GO:0008094~DNA-dependent ATPase activity 3 8.71E-02 GO:0006281~DNA repair 5 3.04E-01 GO:0016887~ATPase activity 4 5.51E-01 GO:0042623~ATPase activity, coupled 3 6.84E-01  Fold Enrichment 5.69 3.98 6.14 3.02 4.07 3.41 3.53 4.56 4.56 4.48 4.38 28.40 22.09 22.09 24.85 46.44 45.47 40.41 34.60 6.99 3.43 69.24 77.09 23.39 68.74 68.74 13.92 7.27 7.36 6.03 1.78 1.38 1.27  164     Supplemental Table A4.11. DAVID GO functional annotation clusters of genes upregulated genes by RFP Spn4A WT-R expression. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Fold Enrichment Annotation Cluster 1: Enrichment Score = 2.2 GO:0004033~aldo-keto reductase activity 3 1.06E-03 60.01 nadp 4 9.44E-03 9.01 hsa00980:Metabolism of xenobiotics by cytochrome P450 3 2.53E-02 11.46  165     Supplementary Table A4.12. Top 10 networks generated from genes differentially regulated by RFP Spn4A WT-R expression as identified by Ingenuity Pathway Analysis.  Networks were generated using Ingenuity Pathway Analysis. Score is the negative log of the significance, which is based on number of focus molecules and size of network. The most annotated functions from molecules in the network are listed.  ID  Score  1  38  Focus Molecules 23  2 3  38 35  23 22  4  32  21  5 6 7  27 27 19  19 18 14  8  18  14  9  14  11  10  7  7  Top Functions DNA Replication, Recombination, and Repair, Embryonic Development, Drug Metabolism Lipid Metabolism, Small Molecule Biochemistry, Behavior Cell-To-Cell Signaling and Interaction, Tissue Development, Cellular Movement Cellular Growth and Proliferation, Cellular Development, Cell Cycle Cell Cycle, Cancer, Cell Death Infectious Disease, Cell Death, Cell Morphology Genetic Disorder, Skeletal and Muscular Disorders, Cellular Growth and Proliferation Cellular Development, Connective Tissue Development and Function, Skeletal and Muscular System Development and Function Cancer, Dermatological Diseases and Conditions, Gene Expression Cellular Assembly and Organization, Nervous System Development and Function, Infectious Disease          166     Supplemental Table A4.13. DAVID GO functional annotation clusters of unique and downregulated genes in response to RFP Spn4A WT-S compared to RFP Spn4A WT-R. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.5 GO:0031974~membrane-enclosed lumen 28 9.97E-05 GO:0043233~organelle lumen 27 1.94E-04 GO:0031981~nuclear lumen 22 7.81E-04 GO:0070013~intracellular organelle lumen 25 8.62E-04 Annotation Cluster 2: Enrichment Score = 2.9 GO:0022403~cell cycle phase 11 1.08E-03 GO:0022402~cell cycle process 13 1.09E-03 GO:0000278~mitotic cell cycle 10 1.89E-03 Annotation Cluster 3: Enrichment Score = 2.2 GO:0000279~M phase 9 3.35E-03 GO:0000280~nuclear division 7 6.40E-03 GO:0007067~mitosis 7 6.40E-03 GO:0000087~M phase of mitotic cell cycle 7 6.98E-03 GO:0048285~organelle fission 7 7.75E-03       Fold Enrichment 2.14 2.10 2.15 1.99 3.50 3.02 3.55 3.60 4.17 4.17 4.10 4.01     167     Supplemental Table A4.14. DAVID GO functional annotation clusters of unique and upregulated genes in response to RFP Spn4A WT-S compared to RFP Spn4A WT-R. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.2 GO:0005578~proteinaceous extracellular matrix 9 2.81E-04 GO:0031012~extracellular matrix 9 4.70E-04 extracellular matrix 7 1.71E-03 Annotation Cluster 2: Enrichment Score = 2.6 domain:GST N-terminal 4 1.80E-04 IPR004045:Glutathione S-transferase, N-terminal 4 2.32E-04 IPR010987:Glutathione S-transferase, C-terminal-like 4 5.35E-04 IPR017933:Glutathione S-transferase/chloride channel, C-terminal 4 5.92E-04 domain:GST C-terminal 4 7.06E-04 PIRSF000503:glutathione transferase 3 3.24E-03 hsa00980:Metabolism of xenobiotics by cytochrome P450 4 4.25E-03 hsa00982:Drug metabolism 4 4.66E-03 IPR004046:Glutathione S-transferase, C-terminal 3 5.91E-03 GO:0004364~glutathione transferase activity 3 6.04E-03 IPR012335:Thioredoxin fold 4 1.79E-02 hsa00480:Glutathione metabolism 3 3.09E-02 GO:0016765~transferase activity, transferring alkyl or aryl (other than methyl) groups 3 3.22E-02 Annotation Cluster 3: Enrichment Score = 2.4 cell adhesion 9 1.75E-03 GO:0007155~cell adhesion 11 6.04E-03 GO:0022610~biological adhesion 11 6.10E-03      Fold Enrichment 5.21 4.83 5.47 35.50 32.58 24.71 23.89 22.59 33.89 11.59 11.21 25.60 25.20 7.17 10.43 10.50 4.00 2.73 2.73  168     Supplemental Table A4.15. DAVID GO functional annotation clusters of unique and downregulated genes in response to RFP Spn4A WT-R compared to RFP Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 4.2 hsa03030:DNA replication 5 9.79E-06 dna replication 5 1.45E-04 GO:0005657~replication fork 4 1.94E-04 Annotation Cluster 2: Enrichment Score = 2.9 GO:0022402~cell cycle process 9 7.46E-04 GO:0000279~M phase 7 1.01E-03 GO:0022403~cell cycle phase 7 3.18E-03       Fold Enrichment 33.34 18.44 34.33 4.40 5.89 4.69     169     Supplemental Table A4.16. DAVID GO functional annotation clusters of shared and downregulated genes in response to RFP Spn4A WT-R and RFP Spn4A WT-S. Parameters are as described in Supplemental Table A4.2.     Term  Count p-value Annotation Cluster 1: Enrichment Score = 3.1 dna replication 6 7.71E-05 GO:0006260~DNA replication 8 1.66E-04 GO:0006259~DNA metabolic process 8 3.62E-02 Annotation Cluster 2: Enrichment Score = 2.3 Term Count PValue GO:0051174~regulation of phosphorus metabolic process 10 3.24E-03 GO:0019220~regulation of phosphate metabolic process 10 3.24E-03 GO:0042325~regulation of phosphorylation 9 8.71E-03 Annotation Cluster 3: Enrichment Score= 2.3 GO:0032103~positive regulation of response to external stimulus 5 7.08E-04 GO:0032101~regulation of response to external stimulus 6 2.98E-03 GO:0048584~positive regulation of response to stimulus 5 6.09E-02 Annotation Cluster4: Enrichment Score= 2.2 GO:0009967~positive regulation of signal transduction 8 2.56E-03 GO:0010740~positive regulation of protein kinase cascade 6 4.00E-03 GO:0010627~regulation of protein kinase cascade 6 2.01E-02 Annotation Cluster 5: Enrichment Score= 2.1 GO:0033273~response to vitamin 5 7.96E-04 GO:0007584~response to nutrient 5 1.19E-02 GO:0009991~response to extracellular stimulus 6 1.28E-02 GO:0031667~response to nutrient levels 5 3.62E-02  Fold Enrichment 13.56 6.76 2.54 Fold Enrichment 3.25 3.25 3.04  12.21 6.05 3.34 4.25 5.65 3.79 11.84 5.58 4.26 3.97       170     Appendix 5: Gene lists and annotations Supplemental Table A5.1. Lists of genes downregulated and upregulated by secreted or retained RFP Spn4A WT and mutants. Differentially regulated genes in response to RFP Spn4A WT (RFP WT-S and RFP WT-R) were compared against mutants (RFP H338R-S, RFP S52R-R, and RFP H338R-R) and the shared genes reported.  171    Downregulated genes PTGS2 PLAU ACOX2 RGS2 F2RL1 TFAP2C HOXA5 PTPRE WDR79 VGF ARHGAP19 TRIB2 PPAP2B MSX1 SLC35A2 TMEM158 CD55 COL8A1 ALDH1A3 CDH11 TMEM100 RDH10 CCL2 SEMA3A C10orf58 CBX1 FBXO32 TGFBR2 ICAM3 TNFRSF11B SNHG5 PAPPA COL4A1 ST8SIA4 GNG2 NOV SYTL2 AADACL1 GCHFR ADAM19 S1PR3  Upregulated genes AKNA ARMET SDF2L1 C1orf24 TMEM50B FAM129A SELS PDIA6 PLAC8 HERPUD1 FICD DNAJB9 WARS HYOU1 DNAJB11 NUCB2 TRA1P2 HSP90B1 AKR1C2 SEC11C DNAJC3 PDIA3P HSPA5 CRELD1 SASS6 TRIB3 CDK2AP2 PDIA4 ANG TAC1 CRELD2 BEX2 DDIT3 S100P CDH15  172     Supplemental Table A5.2. List of genes downregulated and upregulated by Spn4A WT-S that are associated with DAVID GO annotations. Genes from Table 3.1 and Table 3.2 are reported. One annotation term with the most number of genes from each cluster, up to ten clusters, are listed.       173    Cluster 1  Downregulated annotation GO:0022402~cell cycle process  2  kinetochore  3  GO:0031974~membraneenclosed lumen  4  IPR012340:Nucleic acidbinding, OB-fold GO:0001882~nucleoside binding  5  6 7 8 9 10    GO:0006268~DNA unwinding during replication IPR007875:Sprouty domain:Kinesin-motor GO:0005663~DNA replication factor C complex cyclin  Genes ADCY3,KIF23,RP11-553N16.2,KNTC1,TTK,PKMYT1, AURKA,TGFB2,ACVR1B,CCNE1,C18ORF24,RAD21,CDCA2 ,DAQB-126H3.5,C11ORF82,CDCA5,MYC,ASPM,DAAP57C1.3,KIF11,RP11-42J4.2,CDC2,SGOL2,SGOL1, CCNF, KIF15,TPX2,NUSAP1,MND1,PBK,RBBP8,CCND1,MAD2L1, TIMELESS,HAUS8,NUP43,CCDC99,NEK2,TIPIN,POLA1,AN LN,SPC24,SPC25,RP11-262H5.1,NCAPG2,CENPA, FBXO5, HELLS,ERCC6L,IL8,RP11-101P17.12,BIRC5, CENPE,GAS1, CDC25C,SMC2,SMC3,CDC25A,BRCA1,CCNB1,AC132812.1; KPNA2,PSMC4,KIF20B,RP5-1163J1.2,RAD54B,BARD1 CENPO,CCDC99,CTA250D10.18,NEK2,SGOL2,SGOL1,KNTC1,RP11101P17.12,NUP85,CENPE,CENPK,MLF1IP,SPC24,SPC25,RP1 1-262H5.1,C18ORF24,MAD2L1,CENPA,NUP43,ERCC6L KIF23,E2F2,RP11-475E11.4,GRPEL2,MRPS35,RP4686C3.1,LYAR,RP1-261G23.1,EZH2,PDLIM3,RP4686B20.1,PKMYT1,MCM10,TGFB2,CDT1,CCNE2,PRIM1,CC NE1,MCM8,MCM7,PTGES,FANCI,FOXF1,AEN,DDX21,MY C,EXOSC8,EXOSC9,CDC2,USP1,ENC1,TPX2,NUSAP1,TOP BP1,MCM2,MCM3,MCM4,HMGA1,MCM5,MCM6,RFC5,CC ND1,RFC3,RFC4,RFC2,NAV2,NOL11,KIF4A,HMGB2,LMNB 1,NEK2,TH1L,NOC3L,POLA1,BOP1,POLA2,HNRNPA3,MRP L11,HNRNPL,RP11-262H5.1,DDX46,KDELC2 ,CDC45L,POLE2,RP11-230F18.1,CACYBP,BRIX1,ORC6L, FBXO5,ETV4,SRGN,PHLDA1,XPOT,CEBPB,BIRC5,CDC25C ,WWTR1,HNRNPA1,SMC3,CDC25A,BRCA1,SF3A3,FXR1,C CNB1,DUSP4,TXNDC12,AC132812.1;KPNA2,ATF4,PLK4,C DKN2AIP,PHF15,KIF20B,PCNA,TBL1X,PARP2,CHAF1B MCM8,YARS,MCM7,MCM2,MCM3,MCM4,MCM5,MCM6 ADCY3,KIF23,GRPEL2,RP5-1054A22.2,PFTK1,FIGNL1,RP4686B20.1,PKMYT1,TTK,AURKA,ACVR1B,MCM8,MCM7,RP 5-902P8.8,MKKS,DDX21,MLKL,DAQB-126H3.5,DDX10 ,KIF14,IRAK2,DAAP-57C1.3,YARS,KIF11,CDC2,KIF15, TPX2,PBK,MCM2,PRKCE,MCM3,MCM4,RP118N6.1,MCM5,MCM6,RFC5,RFC3,RFC4,RFC2,NAV2,FLAD1, UBE2T,PRPS2,KIF4A,NEK2,DCK,POLA1,ASNS,NP,PFAS,T RIB1,EPHB2,DDX46,KIF21B,HELLS,ERCC6L,MARS,TGFB R2,ATAD2,CENPE,SMC2,SMC3,GART,TXNDC12,PLK4,PS MC4,KIF20B,RAD54B,MYO19 HMGB2,MCM7,MCM2,MCM4,HMGA1,MCM6 SPRY2,SPRY1,SPRED2,SPRED1,SPRY4 KIF23,KIF14,KIF4A,KIF11,KIF15,KIF20B,CENPE,DAQB126H3.5,KIF21B RFC5,RFC3,RFC4,RFC2,PCNA CCNE2,CCNB1,CCNE1,CCND1,RP5973N23.3,CACYBP,CCNF     174    Cluster 1  Upregulated annotation GO:0044421~extracellular region part  2 3  GO:0005581~collagen GO:0005539~glycosamino glycan binding IPR018244:Allergen V5/Tpx-1 related, conserved site leucine-rich repeat  4 5  Genes HAPLN1,LUM,RP11157P1.6,COL3A1,MGP,POSTN,DCN,SPARC,COL16A1,NTN3 ,RP1154O7.13,METRN,CRISPLD2,KAL1,SFRP4,COL6A1,SEPP1,E PYC,MFAP4,RP1-238D15.1 LUM,COL3A1,COL6A1,COL16A1,RP1-238D15.1 HAPLN1,CRISPLD2,POSTN,DCN,GPNMB,EPYC CRISPLD1,CRISPLD2,PI15 LUM,LRRN3,LRRC17,DCN,LRRC4C,EPYC,SLITRK5  175     Supplemental Table A5.3. Genes associated with cell growth and proliferation as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S. Gene symbols, gene names, and fold changes are listed.  176    Gene Symbol IL8 ITGA2 TGFA GDF15 EMP1 SPP1 MT2A ITGA6 PHLDA1 ENC1 F3 E2F2 CCND1 VEGFA PSMC4 CDCA7 TFAP2C JAG1 GPRC5C SH2B3 MT1A CDT1 HES1 IER3 DCBLD2 TGM2 IRF1 PRNP FHL2 PLA2G4A TPX2 GAS1 PTGES SPRY2 PNP PTGER4 PBK ATF4 MCM3 HMGA1 PHLDA2 HEY1 KIF23 DDX21 STIL TFPI2 KIF20B PLAUR ADORA2B IL11  Gene Name interleukin 8 integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) transforming growth factor, alpha growth differentiation factor 15 epithelial membrane protein 1 secreted phosphoprotein 1 metallothionein 2A integrin, alpha 6 pleckstrin homology-like domain, family A, member 1 ectodermal-neural cortex (with BTB-like domain) coagulation factor III (thromboplastin, tissue factor) E2F transcription factor 2 cyclin D1 vascular endothelial growth factor A proteasome (prosome, macropain) 26S subunit, ATPase, 4 cell division cycle associated 7 transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) jagged 1 (Alagille syndrome) G protein-coupled receptor, family C, group 5, member C SH2B adaptor protein 3 metallothionein 1A chromatin licensing and DNA replication factor 1 hairy and enhancer of split 1, (Drosophila) immediate early response 3 discoidin, CUB and LCCL domain containing 2 transglutaminase 2 (C polypeptide, protein-glutamine-gammaglutamyltransferase) interferon regulatory factor 1 prion protein four and a half LIM domains 2 phospholipase A2, group IVA (cytosolic, calcium-dependent) TPX2, microtubule-associated, homolog (Xenopus laevis) growth arrest-specific 1 prostaglandin E synthase sprouty homolog 2 (Drosophila) purine nucleoside phosphorylase prostaglandin E receptor 4 (subtype EP4) PDZ binding kinase activating transcription factor 4 (tax-responsive enhancer element B67) minichromosome maintenance complex component 3 high mobility group AT-hook 1 pleckstrin homology-like domain, family A, member 2 hairy/enhancer-of-split related with YRPW motif 1 kinesin family member 23 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21 SCL/TAL1 interrupting locus tissue factor pathway inhibitor 2 kinesin family member 20B plasminogen activator, urokinase receptor adenosine A2b receptor interleukin 11  Fold Change -5.319 -4.695 -3.846 -3.236 -3.106 -2.959 -2.950 -2.857 -2.778 -2.667 -2.545 -2.532 -2.519 -2.500 -2.494 -2.481 -2.481 -2.469 -2.451 -2.404 -2.392 -2.381 -2.315 -2.315 -2.288 -2.278 -2.268 -2.262 -2.252 -2.247 -2.212 -2.203 -2.193 -2.193 -2.169 -2.165 -2.155 -2.146 -2.146 -2.128 -2.114 -2.110 -2.110 -2.101 -2.083 -2.083 -2.075 -2.070 -2.058 -2.049  177    Gene Symbol SF3A3 AURKA NAMPT BARD1 CCNE1 MAD2L1 SMOX ATAD2 EFNB2 BCAR3 EIF2C2 PTPRF MYC BMP7 TFPI CDK1 CDCA7L ADCY3 CCNB1 TGFB2 FOXM1 NEK2 PMEPA1 SPRED1 BST2 DCK DTL PTPN13 TFDP1 UHRF1 AK3L1 CDC45L ATP6V0E2 BRCA1 CTSC IDO1 TRIB1 TSC22D1 CDC25A VLDLR CDKN2AIP SERPINE2 TIPIN ORC6L CD44 EZR PLAT TTK PLIN2  Gene Name splicing factor 3a, subunit 3, 60kDa aurora kinase A nicotinamide phosphoribosyltransferase BRCA1 associated RING domain 1 cyclin E1 MAD2 mitotic arrest deficient-like 1 (yeast) spermine oxidase ATPase family, AAA domain containing 2 ephrin-B2 breast cancer anti-estrogen resistance 3 eukaryotic translation initiation factor 2C, 2 protein tyrosine phosphatase, receptor type, F v-myc myelocytomatosis viral oncogene homolog (avian) bone morphogenetic protein 7 tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) cyclin-dependent kinase 1 cell division cycle associated 7-like adenylate cyclase 3 cyclin B1 transforming growth factor, beta 2 forkhead box M1 NIMA (never in mitosis gene a)-related kinase 2 prostate transmembrane protein, androgen induced 1 sprouty-related, EVH1 domain containing 1 bone marrow stromal cell antigen 2 deoxycytidine kinase denticleless homolog (Drosophila) protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)associated phosphatase) transcription factor Dp-1 ubiquitin-like with PHD and ring finger domains 1 adenylate kinase 3-like 1 CDC45 cell division cycle 45-like (S. cerevisiae) ATPase, H+ transporting V0 subunit e2 breast cancer 1, early onset cathepsin C indoleamine 2,3-dioxygenase 1 tribbles homolog 1 (Drosophila) TSC22 domain family, member 1 cell division cycle 25 homolog A (S. pombe) very low density lipoprotein receptor CDKN2A interacting protein serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 TIMELESS interacting protein origin recognition complex, subunit 6 like (yeast) CD44 molecule (Indian blood group) ezrin plasminogen activator, tissue TTK protein kinase perilipin 2  Fold Change -2.049 -2.045 -2.037 -2.033 -2.024 -2.024 -2.020 -2.016 -2.012 -1.988 -1.965 -1.961 -1.957 -1.949 -1.949 -1.946 -1.942 -1.923 -1.919 -1.919 -1.908 -1.894 -1.890 -1.887 -1.883 -1.883 -1.883 -1.880 -1.873 -1.873 -1.869 -1.869 -1.862 -1.855 -1.848 -1.848 -1.845 -1.842 -1.825 -1.821 -1.818 -1.818 -1.818 -1.815 -1.808 -1.799 -1.799 -1.799 -1.792  178    Gene Symbol KIF11 CCND3 NFRSF10B DUSP5 PCNA COL6A3 PRKCE USP10 ANLN SUV39H1 SMC3 ACVR1B BCL6 CDK14 CDC25C KIF15 ITGB5 EZH2 MCM5 CDCA4 SFRP1 CEBPB HMMR CAST PPAP2C POLA1 BIRC5 MCM2 TGFBR2 WWTR1 TIMELESS MCM7 SLC22A18 GSTM1 GPER SPARC COL6A1 ACTG1 VAV3 IFITM1 MGP RARRES3 POSTN MAGED2 SOX4 CDKN2C LAMA5 FOLR1 MXD4 TMEFF2 NR3C1  Gene Name kinesin family member 11 cyclin D3 tumor necrosis factor receptor superfamily, member 10b dual specificity phosphatase 5 proliferating cell nuclear antigen collagen, type VI, alpha 3 protein kinase C, epsilon ubiquitin specific peptidase 10 anillin, actin binding protein suppressor of variegation 3-9 homolog 1 (Drosophila) structural maintenance of chromosomes 3 activin A receptor, type IB B-cell CLL/lymphoma 6 cyclin-dependent kinase 14 cell division cycle 25 homolog C (S. pombe) kinesin family member 15 integrin, beta 5 enhancer of zeste homolog 2 (Drosophila) minichromosome maintenance complex component 5 cell division cycle associated 4 secreted frizzled-related protein 1 CCAAT/enhancer binding protein (C/EBP), beta hyaluronan-mediated motility receptor (RHAMM) calpastatin phosphatidic acid phosphatase type 2C polymerase (DNA directed), alpha 1, catalytic subunit baculoviral IAP repeat-containing 5 minichromosome maintenance complex component 2 transforming growth factor, beta receptor II (70/80kDa) WW domain containing transcription regulator 1 timeless homolog (Drosophila) minichromosome maintenance complex component 7 solute carrier family 22, member 18 glutathione S-transferase mu 1 G protein-coupled estrogen receptor 1 secreted protein, acidic, cysteine-rich (osteonectin) collagen, type VI, alpha 1 actin, gamma 1 vav 3 guanine nucleotide exchange factor interferon induced transmembrane protein 1 (9-27) matrix Gla protein retinoic acid receptor responder (tazarotene induced) 3 periostin, osteoblast specific factor melanoma antigen family D, 2 SRY (sex determining region Y)-box 4 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) laminin, alpha 5 folate receptor 1 (adult) MAX dimerization protein 4 transmembrane protein with EGF-like and two follistatin-like domains 2 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)  Fold Change -1.779 -1.776 -1.773 -1.767 -1.767 -1.764 -1.764 -1.764 -1.761 -1.757 -1.748 -1.745 -1.745 -1.742 -1.739 -1.739 -1.727 -1.724 -1.724 -1.721 -1.721 -1.718 -1.718 -1.712 -1.712 -1.709 -1.706 -1.706 -1.706 -1.706 -1.704 -1.701 1.716 1.719 1.721 1.737 1.738 1.758 1.763 1.783 1.785 1.814 1.850 1.854 1.862 1.911 1.916 1.930 1.951 1.973 2.007  179    Gene Symbol NFE2 MUC1 CD24 S100A4 CDKN1C SOCS2 BMF ALDH3A1 SFRP4 GUCY1A3 GPNMB CD36 LUM ALDH1A1 DCN  Gene Name nuclear factor (erythroid-derived 2), 45kDa mucin 1, cell surface associated CD24 molecule S100 calcium binding protein A4 cyclin-dependent kinase inhibitor 1C (p57, Kip2) suppressor of cytokine signaling 2 Bcl2 modifying factor aldehyde dehydrogenase 3 family, memberA1 secreted frizzled-related protein 4 guanylate cyclase 1, soluble, alpha 3 glycoprotein (transmembrane) nmb CD36 molecule (thrombospondin receptor) lumican aldehyde dehydrogenase 1 family, member A1 decorin  Fold Change 2.141 2.150 2.283 2.364 2.578 2.643 2.762 2.892 2.997 3.054 3.122 3.329 3.379 4.080 4.215  180     Supplemental Table A5.4. Genes associated with cell movement as annotated by Ingenuity Pathway Analysis molecular functions in response to Spn4A WT-S. Gene symbols, gene names, and fold changes are listed.  181    Gene Symbol IL8 ITGA2 ETV5 TGFA GDF15 SPP1 ERRFI1 AJAP1 (includes EG:55966) ITGA6 RGS1 F3 CCND1 VEGFA TFAP2C JAG1 SDC1 STX16 ETV4 DCBLD2 TGM2 PRNP FHL2 MMP3 SPRY2 PTPN12 PBK HMGA1 HEY1 KIF23 TFPI2 KIF20B PLAUR ECT2 IL11 AURKA NUSAP1 EFNB2 TGFBR2 BCAR3 CCL26 (includes EG:10344) PTPRF  Gene Name interleukin 8 integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) ets variant 5 transforming growth factor, alpha growth differentiation factor 15 secreted phosphoprotein 1 ERBB receptor feedback inhibitor 1  Fold Change -5.319 -4.695 -4.348 -3.846 -3.236 -2.959 -2.933  adherens junctions associated protein 1 integrin, alpha 6 regulator of G-protein signaling 1 coagulation factor III (thromboplastin, tissue factor) cyclin D1 vascular endothelial growth factor A transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) jagged 1 (Alagille syndrome) syndecan 1 syntaxin 16 ets variant 4 discoidin, CUB and LCCL domain containing 2 transglutaminase 2 (C polypeptide, protein-glutamine-gammaglutamyltransferase) prion protein four and a half LIM domains 2 matrix metallopeptidase 3 (stromelysin 1, progelatinase) sprouty homolog 2 (Drosophila) protein tyrosine phosphatase, non-receptor type 12 PDZ binding kinase high mobility group AT-hook 1 hairy/enhancer-of-split related with YRPW motif 1 kinesin family member 23 tissue factor pathway inhibitor 2 kinesin family member 20B plasminogen activator, urokinase receptor epithelial cell transforming sequence 2 oncogene interleukin 11 aurora kinase A nucleolar and spindle associated protein 1 ephrin-B2 transforming growth factor, beta receptor II (70/80kDa) breast cancer anti-estrogen resistance 3  -2.924 -2.857 -2.710 -2.545 -2.519 -2.500  -2.278 -2.262 -2.252 -2.232 -2.193 -2.188 -2.155 -2.128 -2.110 -2.110 -2.083 -2.075 -2.070 -2.062 -2.049 -2.045 -2.024 -2.012 -1.996 -1.988  chemokine (C-C motif) ligand 26 protein tyrosine phosphatase, receptor type, F  -1.965 -1.961  -2.481 -2.469 -2.445 -2.392 -2.387 -2.288  182    Gene Symbol DIAPH3 MYC BMP7 TFPI CDK1 KIF14 PPAP2B CCNB1 TGFB2 FOXM1 EPHB2 NUP85 TRIB1 KIF4A SERPINE2 CD44 EZR PLAT GNG12 DEK YARS PRKCE ANLN ITGB5 EZH2 SFRP1 HMMR CAST KIFC1 WWTR1 NDRG1 SPARC ACTG1 VAV3 CITED2 MGP MYLK POSTN LAMA5 AMOT MUC1 KAL1 C5ORF13 ALPP  Gene Name diaphanous homolog 3 (Drosophila) v-myc myelocytomatosis viral oncogene homolog (avian) bone morphogenetic protein 7 tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) cyclin-dependent kinase 1 kinesin family member 14 phosphatidic acid phosphatase type 2B cyclin B1 transforming growth factor, beta 2 forkhead box M1 EPH receptor B2 nucleoporin 85kDa tribbles homolog 1 (Drosophila) kinesin family member 4A serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2 CD44 molecule (Indian blood group) ezrin plasminogen activator, tissue guanine nucleotide binding protein (G protein), gamma 12 DEK oncogene tyrosyl-tRNA synthetase protein kinase C, epsilon anillin, actin binding protein integrin, beta 5 enhancer of zeste homolog 2 (Drosophila) secreted frizzled-related protein 1 hyaluronan-mediated motility receptor (RHAMM) calpastatin kinesin family member C1 WW domain containing transcription regulator 1 N-myc downstream regulated 1 secreted protein, acidic, cysteine-rich (osteonectin) actin, gamma 1 vav 3 guanine nucleotide exchange factor Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxyterminal domain, 2 matrix Gla protein myosin light chain kinase periostin, osteoblast specific factor laminin, alpha 5 angiomotin mucin 1, cell surface associated Kallmann syndrome 1 sequence chromosome 5 open reading frame 13 alkaline phosphatase, placental (Regan isozyme)  Fold Change -1.957 -1.957 -1.949 -1.949 -1.946 -1.934 -1.934 -1.919 -1.919 -1.908 -1.848 -1.848 -1.845 -1.818 -1.818 -1.808 -1.799 -1.799 -1.786 -1.776 -1.767 -1.764 -1.761 -1.727 -1.724 -1.721 -1.718 -1.712 -1.706 -1.706 1.730 1.737 1.758 1.763 1.764 1.785 1.785 1.850 1.916 1.984 2.150 2.182 2.203 2.206  183    Gene Symbol CD24 S100A4 SOCS2 PPAP2A GUCY1A3 CD36 OLR1 DCN  Gene Name CD24 molecule S100 calcium binding protein A4 suppressor of cytokine signaling 2 phosphatidic acid phosphatase type 2A guanylate cyclase 1, soluble, alpha 3 CD36 molecule (thrombospondin receptor) oxidized low density lipoprotein (lectin-like) receptor 1 decorin  Fold Change 2.283 2.364 2.643 2.717 3.054 3.329 3.630 4.215  184     Supplemental Table A5.5. Genes associated with significant canonical pathways identified by Ingenuity in response to Spn4A WT-S. Fisher’s exact test was used to calculate the p-value. Ratio represents the ratio of focus molecules to total molecules in the canonical pathway. Ingenuity Canonical Pathways Role of CHK Proteins in Cell Cycle Checkpoint Control Aryl Hydrocarbon Receptor Signaling  -log(p-value) 6.67  Ratio 0.29  4.54  0.11  Cell Cycle: G1/S Checkpoint Regulation Role of BRCA1 in DNA Damage Response Mitotic Roles of Polo-Like Kinase ATM Signaling  3.79  0.16  3.73  0.15  3.66  0.16  3.24  0.15  Hereditary Breast Cancer Signaling  2.52  0.09  Pancreatic Adenocarcinoma Signaling  2.31  0.09  Molecules PCNA, CDC25C, RFC4, RFC2, RFC5, BRCA1, CDK1, E2F2, CDC25A, RFC3 HSPB3, GSTM1, TFDP1, POLA1, CCND1, MYC, TGM2, CCNE1, NCOA7, GSTM2, ALDH1A1, CCND3, TGFB2, ALDH3B1, ALDH3A1, MCM7 MYC, CCNE1, CCND3, TFDP1, SUV39H1, TGFB2, CCND1, E2F2, CDC25A RFC4, FANCG, BARD1, RFC2, RBBP8, RFC5, BRCA1, E2F2, RFC3 KIF23, CDC25C, PLK4, CCNB1, PPP2R2B, PKMYT1, CDK1, KIF11, CDC25A SMC3, CDC25C, CCNB1, SMC2, ATF4, BRCA1, CDK1, CDC25A CDC25C, CCNB1, RFC4, FANCG, BARD1, RFC2, RFC5, BRCA1, CCND1, CDK1, RFC3 VEGFA, TGFBR2, CCNE1, TFDP1, SUV39H1, TGFA, TGFB2, CCND1, E2F2, BIRC5  185  

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