University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2010-03-29 A View of the IMD Pathway from the RHIM Kamna Aggarwal University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Animal Experimentation and Research Commons, Cells Commons, Genetic Phenomena Commons, Hemic and Immune Systems Commons, and the Immunity Commons Repository Citation Aggarwal K. (2010). A View of the IMD Pathway from the RHIM. GSBS Dissertations and Theses. https://doi.org/10.13028/69q1-d386. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/463 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. A VIEW OF THE IMD PATHWAY FROM THE RHIM A Dissertation Presented By Kamna Aggarwal Submitted to the Faculty of the University of Massachusetts Medical School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MARCH 29, 2010 INTERDISCIPLINARY GRADUATE PROGRAM ii A Dissertation Presented By Kamna Aggarwal Approved as to style and content by: Neal Silverman, Ph.D., Thesis Advisor Michael Brodsky, Ph.D., Member of Committee Katherine Fitzgerald, Ph.D., Member of Committee Scot Wolfe, M.D., Member of Committee François Leulier, Ph.D., Member of Committee Michelle Kelliher, Ph.D., Chair of Committee Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program March 29, 2010 iii Acknowledgments There are many people I would like to thank for making my graduate school journey so memorable. Foremost I would like to thank my advisor Dr. Neal Silverman who is not only a gifted scientist, but also a great mentor and teacher. I have learnt from him that there is no substitute for a well planned and structured approach for testing a hypothesis. He also taught me to think critically and to plan every experiment and to think about all the controls as every control is important for proper analysis of the data. I am particularly grateful to him for being extremely patient with me and allowing me the freedom to work independently. I can say with confidence that Neal is one of the best mentors I will have ever worked with. It was an honor to have Drs, Michelle Kelliher, Katherine Fitzgerald, Michael Brodsky and Scot Wolfe on my thesis committee. Their comments and suggestions were highly valuable and I thank them for their time. I am also grateful to Dr. François Leulier for taking the time to evaluate my dissertation. I was incredibly lucky in training in an environment so well stocked in scientists with diverse interest and expertise in their field. I would especially like to thanks the past and present members of the Silverman. In particular I would like to thank Takashi Kaneko, Charles Sweet, Nicholas Paquette and Deniz Hasdemir for teaching me various techniques in the lab and also for providing a great working environment. iv My graduate school journey would have been tough without the love and good will of the following people. So a big thank you to all my friends and family members for being a part of my life and in particular, to listening to my woes (Kayla Morlock, Florentina Rus and my loving sister Aparna Aggarwal) and for providing me with a home away from home (Sujan Rafi-ul Kabir, Hema Bashyam, the Jain family and the WCBA family). Although I have had a great time at UMass one of the most difficult thing about this journey has been to be away from my family. They are my pillars of strength and responsible for all the success that has come my way. I am indebted to them for supporting me in all my endeavors and having faith in times when even I lacked it. I am also thankful to my parent in laws for loving me like a daughter and letting me be a part of their family. Last but definitely not the least I would like thanks my husband Tathagat Dutta Ray. I lack the words to express my love for him. Without him this journey would have been impossible. I thank him for giving me endless encouragement, help and advice and for always being there besides me. I am very lucky to share my life with him. v TABLE OF CONTENTS Title i Signature Page ii Acknowledgments iii Table of Contents v List of Figures ix List of Publications xii ABSTRACT xiii Preface to CHAPTER I 1 CHAPTER I: INTRODUCTION 2 Overview of the Drosophila Immune Response 4 Microbial Recognition -- the Peptidoglycan Recognition Proteins 6 Peptidoglycan 8 The Toll signaling 12 The IMD signaling 20 Down-regulation of Immune Signaling Pathways by PGN Digesting Enzymes 28 Negative Regulation in the Toll Pathway 30 Negative Regulation in the IMD Pathway 32 Transcriptional regulation of the Drosophila immune response 33 Tissue specific immune regulation 35 vi Mammalian NF-κB signaling 40 Negative Regulation in the TLR Pathway 47 Extracellular regulation 48 Transmembrane Protein Regulators 48 Intracellular Protein Regulators 49 Intracellular Proteins that negatively regulate TLR signaling by degradation 51 Intracellular Proteins that negatively regulate TLR signaling by deubiquitnation 52 Thesis objective 52 Preface to CHAPTER II 56 CHAPTER II: PGRP-LC and PGRP-LE play essential yet distinct roles in the drosophila immune response to monomeric DAP-type peptidoglycan. 57 Abstract 58 Introduction 59 Results PGRP-LE and PGRP-LC recognize TCT 61 PGRP-LE, an intracellular TCT receptor 65 CD14-like activity of PGRP-LEpg 70 PGRP signaling through a RHIM-like motif 72 IMD-PGRP-LC interaction is dispensable for signaling 76 Discussion 79 vii Materials and methods 83 CHAPTER III: Yeast two hybrid screen 89 Abstract 90 Introduction 91 Results and Discussion 92 Materials and methods 97 Preface for CHAPTER IV 99 CHAPTER IV: Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. 100 Abstract 101 Introduction 102 Results Isolation of Rudra 104 Induction of rudra expression 109 Rudra is a negative regulator of IMD signaling 113 Rudra inhibits signaling at the receptor 118 Discussion 121 Materials and Methods 124 CHAPTER V: RYBP: a new regulator of the IMD pathway 128 Abstract 129 Introduction 130 Results viii PGRP-LCx RHIM mutants fail to activate any downstream event 134 A receptor proximal complex is assembled when components of the IMD pathway are over- expressed 137 RYBP interacts with the receptor PGRP–LC and it is a new component of the IMD pathway 140 Discussion 142 Materials and Methods 145 CHAPTER VI: DISCUSSION: Speculations on the regulation of IMD pathway via the RHIM-like motif and the connection with ubiquitination 147 Overview 149 Ubiquitination 150 Ubiquitination Regulates Innate Immune Signaling 152 Regulation of IMD signaling via the RHIM-like motif 156 RYBP is a new component of the IMD pathway 160 BIBLIOGRAPHY 165 ix LIST OF FIGURES Figure 1.1: Peptidoglycan structure 9 Figure 1.2: Toll signaling pathway 14 Figure 1.3: IMD signaling pathway 23 Figure 1.4: Gut immunity 39 Figure 1.5 Mammalian NF-κB signaling 44 Figure 1.6: Negative Regulators of Toll-like receptor 53 Figure 2.1: PGRP-LE is a second receptor for monomeric DAP-type peptidoglycan. 63 Figure 2.2 TCT-induced immune activation is reduced but not eliminated in PGRP-LC mutant flies. 64 Figure 2.3: PGRP-LE is not expressed in cultured S2* cells. 66 Figure 2.4: PGRP-LE is an intracellular receptor for TCT 69 Figure 2.5: Intracellular localization of PGRP-LE 71 Figure 2.6: CD14-like activity of the PGRP domain of PGRP-LE 73 Figure 2.7: PGRP-LC and PGRP-LE signal via a conserved motif 75 Figure 2.8: RHIM-like features of the conserved PGRP signaling motif. 77 Figure 2.9: The interaction between PGRP-LC and IMD is not essential for signal transduction 78 x Figure 2.10: Model of PGRP-LC and PGRP-LE-mediated recognition of monomeric and polymeric DAP-type peptidoglycan. 80 Figure 3.1: X gal screening 93 Table 3.2: Interaction analysis to identify clones that interact with the RHIM-like motif of PGRP-LC 95 Figure 3.3: RfaBg, Scylla and CG12935 are not regulators of IMD signaling in cells 96 Table 4.1: Rudra interacts with cytoplasmic domain of PGRP-LC by yeast two-hybrid 105 Figure 4.2: Rudra interacts with the receptors PGRP-LE and PGRP-LC 106 Figure 4.3: PGRP-LE and Rudra interact in vitro 107 Figure 4.4: Rudra interacts with all the deletion mutants spanning the cytoplasmic domain of PGRP-LCx 108 Figure 4.5 rudra, a negative feedback regulator of IMD signaling in cells 110 Figure 4.6 Over-expression of rudra blocks IMD signaling in both cells and flies 111 Figure 4.7: Rudra inhibits IMD signaling but not the Toll pathway 112 Figure 4.8: Characterization of rudra mutant flies 114 Figure 4.9: Rudra functions upstream of IMD, Dredd and Relish 117 Figure 4.10: Rudra disrupts the interaction between PGRP-LCx and IMD 119 Figure 5.1: PGRP-LCx RHIM mutants fail to activate any downstream event 138 Figure 5.2: A receptor proximal complex is assembled when components of the IMD pathway are over- expressed 139 Figure 5.3: RYBP interacts with the receptor PGRP-LC and it is a new component of the IMD pathway 141 Figure 6.1: Comprehensive IMD signaling pathway model 149 xi Figure 6.2: Sequence alignment of NZF domains containing proteins 162 Figure 6.3: Proposed model of IMD signaling 164 xii LIST OF PUBLICATIONS Aggrawal K, Silverman N.