Title Page Ubiquitin E3 Ligases in Lung Disease by Travis Bradley Lear BA, Saint Mary’s College of Maryland, 2012 Submitted to the Graduate Faculty of the Department of Environmental and Occupational Health Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2020 Committee Page UNIVERSITY OF PITTSBURGH GRADUATE SCHOOL OF PUBLIC HEALTH This dissertation was presented by Travis Bradley Lear It was defended on March 6, 2020 and approved by Aaron Barchowsky, PhD, Professor, Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Bruce R. Pitt, PhD, Professor, Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Toren Finkel, MD, PhD, Professor of Medicine, Department of Medicine, School of Medicine, University of Pittsburgh Dissertation Advisor: Bill B. Chen, PhD, Associate Professor of Medicine, Department of Medicine, School of Medicine, University of Pittsburgh ii Copyright © by Travis Bradley Lear 2020 iii Abstract Bill B. Chen, PhD Ubiquitin E3 Ligases in Lung Disease Travis Bradley Lear, PhD University of Pittsburgh, 2020 Abstract Diseases of the lung form the one of the largest causes of death globally. Inflammatory diseases such as Acute Respiratory Distress Syndrome, and fibrosis such as interstitial lung disease, in particular have high mortality rates with limited therapy. Thus, there is an unmet public health need for new avenues of intervention. Inflammatory, fibrotic, and nutrient lung diseases are driven by cellular signaling pathways, leading to pathological cell responses. Effector lung cells naturally dampen these deleterious signaling pathways; dysfunction of these dampening mechanisms play causal roles in lung disease, notably through excessive destruction of critical signal transduction proteins. Modulation of signal transduction protein degradation may have therapeutic effect by controlling deleterious signaling. The ubiquitin-proteasome system is the major cellular mechanism controlling protein degradation. Ubiquitin E3 ligase proteins are a critical part of ubiquitination, specifically targeting substrates for degradation. Research shows the importance of protein degradation in lung disease, however the potential to identify and inhibit specific E3-ligase-substrate interactions remains unexplored. Through both candidate-based and unbiased high-throughput screening techniques, we probed the importance of E3 ligases in lung disease through their targeted degradation of signal transduction proteins, and the therapeutic potential of E3 ligase inhibition. We investigated three aspects of lung disease – 1) inflammation and innate immunity, 2) fibrosis and interstitial lung disease, and 3) regulation of nutrient sensing mechanisms. Here we report multiple E3 ligase-substrate axes, including those associated with iv fibrosis: FIEL1-PIAS4; KLHL42-PPP2R5e, with innate immunity: PPP1R11-TLR2, RNF113A- CXCR4, KIAA0317-SOCS2, RNFT2-IL3Ra, and with nutrient sensing: RNF186-SESN2. We observed that E3 ligases potently control inflammatory signaling through control of cytokine receptors and signal modulators during acute inflammation and bacterial infection. We uncovered that E3 ligases are significantly associated with fibrotic signaling in interstitial lung fibrosis, and can be targeted by small molecules. Finally, we detected new mechanisms of nutrient sensor control leading to manipulation of anabolism. These results show the criticality of ubiquitin e3 ligases in the biology of lung inflammation, fibrosis, and nutrient sensing. Further, these studies validate ubiquitin E3 ligases as potential targets for therapeutic intervention to provide new tools to combat lung diseases. v Table of Contents Preface ......................................................................................................................................... xxi 1.0 Introduction ............................................................................................................................. 1 1.1 Diseases of Lung ............................................................................................................. 1 1.1.1 Global Public Health Significance of Lung Disease ..........................................2 1.1.2 Fibrosis in the Lung .............................................................................................3 Idiopathic Pulmonary Fibrosis ............................................................... 3 Determinants of IPF Pathology .............................................................. 4 Therapeutic Options for IPF .................................................................. 4 1.1.3 Inflammation in the Lung ...................................................................................7 1.1.4 Nutrient Sensing in the Lung ..............................................................................9 1.2 Protein Degradation ..................................................................................................... 10 1.2.1 Protein Lifespan .................................................................................................10 1.2.2 The Ubiquitin Proteasome System ...................................................................11 1.2.3 Ubiquitin E3 Ligases ..........................................................................................12 Ubiquitin E3 Ligases in disease ............................................................ 14 1.3 Specific Aims ................................................................................................................. 16 2.0 Ubiquitination in Fibrosis .................................................................................................... 18 2.1 Ubiquitin E3 Ligase FIEL1 Regulates Fibrotic Lung Injury through SUMO-E3 Ligase PIAS4 ....................................................................................................................... 18 2.1.1 Study Overview ..................................................................................................18 2.1.2 Introduction ........................................................................................................19 vi PIAS proteins and SMAD signaling ..................................................... 20 2.1.3 FIEL1-PIAS4 pathway in pulmonary fibrosis ................................................21 2.1.4 FIEL1 promotes TGF signaling ......................................................................25 2.1.5 PIAS4 phosphorylation by PKCζ is required for FIEL1 binding .................27 2.1.6 GSK3 phosphorylation of FIEL1 is required for PIAS4 targeting .............29 2.1.7 Gene transfer of FIEL1 exacerbates bleomycin induced lung injury in vivo .......................................................................................................................................33 2.1.8 FIEL1 knockdown ameliorates bleomycin-induced lung injury in vivo .......35 2.1.9 Anti-fibrotic activity of a FIEL1 small molecule inhibitor.............................37 In vitro .................................................................................................... 37 In vivo ..................................................................................................... 38 2.1.10 Discussion ..........................................................................................................42 2.1.11 Acknowledgements ...........................................................................................45 2.1.12 Materials and Methods ....................................................................................45 Materials ............................................................................................... 45 Human Samples ................................................................................... 46 Cell culture ........................................................................................... 46 In vitro protein binding assays ........................................................... 47 In vitro peptide binding assays ........................................................... 47 In vitro drug binding assays ............................................................... 47 In vitro ubiquitin conjugation assays ................................................. 48 In vitro kinase assays ........................................................................... 48 Hydroxyproline Assay ......................................................................... 48 vii SMAD reporter assay ........................................................................ 49 Immunostaining ................................................................................. 49 Molecular docking studies and compound design .......................... 49 BC-1485 synthesis .............................................................................. 50 RT–qPCR, cloning, and mutagenesis............................................... 50 Lentivirus construction ..................................................................... 51 Animal studies .................................................................................... 51 Tissue Staining ................................................................................... 52 Statistical Analysis ............................................................................. 52 2.2 Kelch-like Protein 42 is a Pro-Fibrotic Ubiquitin E3 Ligase in Systemic