Defining the Mechanisms of Neutrophil Exocytosis Using Proteomic Techniques
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University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 8-2005 Defining the mechanisms of neutrophil exocytosis using proteomic techniques. George Lominadze 1978- University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Recommended Citation Lominadze, George 1978-, "Defining the mechanisms of neutrophil exocytosis using proteomic techniques." (2005). Electronic Theses and Dissertations. Paper 852. https://doi.org/10.18297/etd/852 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. DEFINING THE MECHANISMS OF NEUTROPHIL EXOCYTOSIS USING PROTEOMIC TECHNIQUES By George Lominadze B.S. University of Louisville, 2000 A Dissertation Submitted to the Faculty of the Graduate School of the University of Louisville in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Biology University of Louisville Louisville, Kentucky August 2005 DEFINING THE MECHANISMS OF NEUTROPHIL EXOCYTOSIS USING PROTEOMIC TECHNIQUES By George Lominadze B.S., University of Louisville, 20000 A Dissertation Approved on June 1, 2005 by the following Thesis Committee Thesis Director ii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Kenneth McLeish for his guidance, trust, support, and patience. I would also like to thank Dr. Richard Ward, who has acted as a co-mentor on the granule proteome project. I am very grateful to other members of my dissertation committee, Dr. Jon Klein, Dr. Robert Gray, and Dr. William Dean. I also wish to express my gratitude to Dr. Madhavi Rane for her insightful suggestions, encouragement and advice. I thank Dr. David Powell for carrying out the ESI-MSIMS analysis at Vanderbilt University. Finally, I would like to thank my parents and my brother for being a perfect family. 111 ABSTRACT DEFINING THE MECHANISMS OF NEUTROPHIL EXOCYTOSIS USING PROTEOMIC TECHNIQUES George Lominadze June 1,2005 Exocytosis of intracellular granules is critical for conversion of inactive, circulating neutrophils to fully activated cells. The p38 MAPK pathway plays a central role in neutrophil exocytosis, although its mechanism of action is unknown. We used several proteomic approaches to identify granule proteins and find targets of the p38 MAPK and its downstream kinase, MK2, on granules. Our analysis identified 286 proteins on neutrophil granules, four of which were known MK2 substrates. Known p38 substrates were not detected. However, MRP-14 was identified as a novel p38 MAPK substrate. MRP-14 was phosphorylated by p38 MAPK in neutrophils upon cell stimulation and translocated to plasma membranes and gelatinase granules, as well as to Triton X-IOO-insoluble structures at the base of lamellipodia. Phosphorylation of the MRP-14 by p38 MAPK increased binding to actin in vitro. These results suggest that MRP-14 is a potential mediator of p38 MAPK-dependent exocytosis in human neutrophils. IV TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS ....................................................................... .iii ABSTRACT ......................................................................................... .iv LIST OF TABLES ................................................................................. vii LIST OF FIGURES ................................................................................ .ix CHAPTER I. GENERAL INTRODUCTION Role ofNeutrophils in Host Defense and Tissue Damage ........................... 1 Neutrophil Granules and Their Role in Neutrophil Function ....................... .2 Neutrophil Granule Exocytosis ......................................................... 8 Application ofProteomics to Protein and Phosphoprotein Identification ........ 27 II. PROTEOMIC ANALYSIS OF HUMAN NEUTROPHIL GRANULES Foreword .............. ".................................................................. 37 Introduction .............................................................................. 38 Methods ...................................................................................40 Results .................................................................................... 44 Discussion ................................................................................ 72 III. MYELOID-RELATED PROTEIN-14 IS A P38 MAPK SUBSTRATE IN HUMAN NEUTROPHILS Foreword ................................................................................. 80 Introduction .............................................................................. 83 Materials and Methods ..................................................................40 Results .................................................................................... 95 Discussion .............................................................................. 117 IV. SUMMARY AND FINAL CONCLUSIONS ....................................... 126 v REFERENCES .................................................................................. .131 APPENDIX ....................................................................................... 161 CURRICULUM VITAE ........................................................................ 193 VI LIST OF TABLES PAGE TABLE 1. Known Proteins of Neutrophil Granules ................................................. 13 2. Proteins Identified From 2D Gels of Neutrophil Granules ............................ 56 3. A Complete List of Proteins Identified on Neutrophil Granules ..................... 59 4. Supplementary Table 1: Peptides Used for Identification of Proteins From More Than One 2D-HPLC ESI-MS/MS Experiment. ....................................... 162 5. Supplementary Table 2: Peptides Used for Identification of Proteins From a Single 2D-HPLC ESI-MS/MS Experiment. ............................................. 176 6. Proteins Rejected as Granule Components ............................................ 181 Vll LIST OF FIGURES PAGE FIGURE 1. A diagram of granule biogenesis during stages of neutrophil maturation ............ 5 2. Schematic representation of the steps leading to secretary granule exocytosis .... .1 0 3. Assessment of purity of granule fractions .............................................. .45 4. 2D gels of whole granules ................................................................ .48 5. 2D gels of gelatinase granule proteins fractionated by carbonate lysis and by ammonium sulfate precipitation of Triton X-100 solubilized proteins .............. 52 6. 2D gels of specific granule proteins fractionated by carbonate lysis and by ammonium sulfate precipitation of Triton X-I00 solubilized proteins .............. 54 7. Assessment of actin content on neutrophil granules by western blotting ........... 69 8. Phosphorylation of urea-solubilized gelatinase granule proteins by MAPKAPK-2 and p38 MAPK in the presence of [y32p]ATP ............................................ 81 9. Identification ofp38 MAPK substrates in human neutrophil lysate .................... 97 10. In vitro phosphorylation ofMRP14 by p38 MAPK ................................... 99 11. Phosphorylation of MRP 14 by p38 MAPK in intact neutrophils ................... 102 12. P38 MAPKphosphorylates MRP14 on Thr-I13 ...................................... 105 13. P38 MAPK phosphorylates wild type hrMRP14, but not the TI13A mutant. .. 107 14. Phosphorylation-induced redistribution ofMRP-14 in intact neutrophils ........ .110 15. Association ofMRP-14 with neutrophil granule and plasma membrane/secretory vesicle fractions ........................................................................... .113 Vlll 16. Translocation ofMRP-14 to neutrophil gelatinase granule and plasma membrane/secretory vesicle fractions in tMLP-stimulated neutrophils ............ 115 17. Phosphorylation by p38 MAPK enhanced actin binding ofMRP-14/MRP-8 .... 118 IX CHAPTER I GENERAL INTRODUCTION Role of Neutrophils in Host Defense and Tissue Damage Neutrophils (also called polymorphonuclear leukocytes or PMNs) are phagocytic granulocytes that are crucial to host defense. These cells provide the initial cellular response at sites of infection and inflammation. Upon exposure to pro-inflammatory chemo attractants , chemokines, and cytokines, neutrophils are transformed from benign circulating cells to cells capable of adhesion to vascular endothelium, migration along chemoattractant gradients to sites of infection or trauma, phagocytosis of opsonized pathogens, and the release of antimicrobial agents [1]. In all of the above-mentioned processes, exocytosis of mobilizable membrane-bound compartments plays a central role. Although the infiltration of neutrophils into injured tissue protects it from invading pathogens, substantial evidence exists showing that neutrophil-induced host tissue injury contributes to inflammatory diseases, ischemia-reperfusion injury, and impaired wound healing [2-5]. Upon neutrophil activation the secretion of granule proteinases, peroxidases and cationic peptides acts in concert with the release of reactive oxygen intermediates to promote chemotaxis by digestion of extracellular matrix or to directly damage host