INTERACTIONS OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) WITH ADVANCED GLYCATION END PRODUCTS AND S100B A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Venkata Shravan Kumar Indurthi In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Pharmaceutical Sciences October 2015 Fargo, North Dakota North Dakota State University Graduate School Title INTERACTIONS OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) WITH ADVANCED GLYCATION END PRODUCTS (AGEs) AND S100B By Venkata Shravan Kumar Indurthi The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Stefan Vetter Chair Dr. Stephen O’Rourke Dr. Bin Guo Dr. Christopher Colbert Approved: 11/20/2015 Dr. Jagdish Singh Date Department Chair ABSTRACT RAGE is a multi-ligand pattern recognition receptor. RAGE can bind several damage associated molecular pattern proteins. RAGE- ligand interaction is pathophysiologically relevant to several major diseases including diabetes and certain cancers. RAGE inhibition has been reported to reduce morbidity in these disease states. However, to design better RAGE inhibitors it is necessary to understand the structural basis behind the RAGE-ligand interaction and currently this is not well understood. This thesis focuses on understanding the interaction of RAGE with two of its ligands; AGEs and S100B. AGEs are highly heterogeneous and are formed as a result of non-enzymatic glycation. A panel of AGEs were characterized in terms of their side chain modifications, thermal stability, secondary structure, aggregation and surface charge. These glycation induced changes were then correlated to RAGE binding. Building on these results the role of AGE-RAGE interaction in pancreatic cancer cell proliferation and migration was determined. Ribose modified BSA induced ROS formation, which then triggered NF-κB upregulation via RAGE induced ROS signaling. Ribose BSA increased pancreatic cell proliferation and migration. Anti-RAGE antibodies and RAGE inhibitors prevented AGE induced cellular effects. The role of ribose modified BSA was also determined in macrophage activation and pro-inflammatory cytokine release. Rapid internalization was observed of the ribose-BSA and confocal imaging revealed the internalization of the AGE compound into the lysosomes which lead to the ROS production, NF- κB activation and pro-inflammatory cytokine release in a RAGE independent signaling mechanism. Finally, the role of tryptophan residues of the V domain in domain stability and S100B binding was determined. We have generated single, double and triple tryptophan mutants of the iii V domain by site directed mutagenesis. The effect of Trp residues in the domain stability could not elucidated as no change was observed in the secondary structure of the mutants when compared to the wild type suggesting the plasticity of the V-domain. The fluorescence emission and life time properties of each Trp residue was determined. Our binding assays of the Trp Ala mutants indicate tighter binding of the S100B to the mutants. The S100-RAGE peptide structures suggest multi modal interaction of S100B-RAGE interaction. iv ACKNOWLEDGEMENTS I am bereft of all words to describe the contribution of my advisor, mentor and guru Dr. Stefan Vetter. I joined the Ph.D. program at 21, I needed a guru to be the guiding light in my scientific endeavors and I am grateful to Dr. Vetter for accepting me into his lab and being the guiding light. The role he has played in my growth as a scientist is indescribable. He will always be an inspiration. I consider myself extremely fortunate to have been advised by Dr. Vetter and I am proud to be his first student. His contribution extends way beyond honing my scientific skills and has been instrumental in the overall growth of me as an individual. In the same breath, I would also like to express my deepest gratitude to Dr. Estelle Leclerc for all her contributions. To be successful it is very important to have a strong family. I am extremely fortunate to have a very supporting family. I would like to acknowledge my family; my parents, my grandparents, my sister and my brother-in-law for the unconditional support. My cousin Abhishek has been my pillar of strength during my graduate studies and would like to thank him for all his support. The last year of my graduate studies have been the toughest and the most frustrating. I cannot thank my lab mate Priyanka enough for bearing with me through this phase and making sure that I had a smile on my face every single day. My special thanks to Jean; the nicest person I have ever met and Clyde. Thank you for making me feel a part of your family and making sure I did not miss home. I have made some fabulous friendships during my graduate studies in the college of health professions, these are the people who believed in me and always motivated me to take that extra step. I would like to express my sincere gratitude to Dana, Sara, Lori and Kelly for all their support and making me feel at home here at North Dakota State University. They have an v immense contribution in making me a better person. I would like to acknowledge my friends Rhishi, Mayura, Anil, Prajakta and Gaurav for taking care of me and being there for me whenever I needed. I would like to express my heartfelt gratitude to my advisory committee, Prof. Guo, Prof. O’Rourke, and Prof. Colbert for your valuable suggestions and expert advice. Their input was instrumental in shaping me as a scientist and honing my critical reasoning skills. Finally, I would like to express my sincere gratitude to ND-EPSCoR and the graduate school for the doctoral dissertation assistantship, I am grateful to the financial support provided to me through this assistantship. vi DEDICATION Dedicated to my parents Krishnaveni and Ramesh Kumar Indurthi And my sister, Swetha Mula vii TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................................ v DEDICATION .............................................................................................................................. vii LIST OF TABLES ........................................................................................................................ xv LIST OF FIGURES .................................................................................................................... xvii LIST OF ABBREVIATIONS ..................................................................................................... xxii CHAPTER 1: INTRODUCTION ................................................................................................... 1 Receptor for advanced glycation end products ........................................................................... 1 RAGE-mediated NF- κB signaling ............................................................................................. 3 RAGE expression and physiological function ............................................................................ 5 RAGE and disease ....................................................................................................................... 6 CHAPTER 2: GLYCATION, ADVANCED GLYCATION END PRODUCTS (AGES) AND THEIR INTERACTION WITH RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) .......................................................................................................... 10 Introduction ............................................................................................................................... 10 Non-Enzymatic glycation and AGE compound formation ................................................... 10 AGE compounds and disease ................................................................................................ 12 AGE-specific cell surface receptors ...................................................................................... 14 Serum albumin, drug binding and glycation ......................................................................... 15 Problems associated with AGE compound research ............................................................. 17 Materials and Methods .............................................................................................................. 18 Preparation of glycated serum albumin ................................................................................. 19 UV/Vis and fluorescence spectroscopy ................................................................................. 21 Amino acid analysis .............................................................................................................. 22 Lysine side chain modification .............................................................................................. 22 viii Arginine side chain modification .......................................................................................... 22 Fructosamine content estimation ........................................................................................... 23 Carboxymethyl lysine estimation .........................................................................................