PROTEOME ANALYSIS OF HUMAN LUNG Ph EPITHELIAL CELLS INTERACTING WITH . D ASPERGILLUS TERREUS CONIDIA AND . STRUCTURE-FUNCTION RELATION OF nsSNPs IN INNATE IMMUNE RECEPTORS Thesis submitted in fulfillment of the requirements for the Degree of RAMAN RAMAN DOCTOR OF PHILOSOPHY By RAMAN JUIT, WAKNAGHAT Department of Biotechnology & Bioinformatics JAYPEE UNIVERSITY OF INFORMATION TECHNOLOGY WAKNAGHAT, DISTRICT SOLAN, H.P., INDIA January 2018 2018 PROTEOME ANALYSIS OF HUMAN LUNG EPITHELIAL CELLS INTERACTING WITH ASPERGILLUS TERREUS CONIDIA AND STRUCTURE-FUNCTION RELATION OF nsSNPs IN INNATE IMMUNE RECEPTORS Thesis submitted in fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY By RAMAN Department of Biotechnology & Bioinformatics JAYPEE UNIVERSITY OF INFORMATION TECHNOLOGY WAKNAGHAT, DISTRICT SOLAN, H.P., INDIA January 2018 I Copyright @ JAYPEE UNIVERSITY OF INFORMATION TECHNOLOGY WAKNAGHAT JANUARY 2018 ALL RIGHTS RESERVED II TABLE OF CONTENTS CONTENT PAGE NO. INNER FIRST PAGE I DECLARATION VIII SUPERVISOR’S CERTIFICATE IX ACKNOWLEDGEMENT X-XI ABSTRACT XII LIST OF ABBREVIATIONS XIII-XV LIST OF FIGURES XVI-XVII LIST OF TABLES XVIII-XIX CHAPTER - 1 THESIS INTRODUCTION 1-3 CHAPTER - 2 REVIEW OF LITERATURE 4-25 2.1 ASPERGILLI 4 2.2 TAXONOMY OF ASPERGILLUS TERREUS 5 2.3 DISTRIBUTION AND MORPHOLOGY OF A. TERREUS 5 2.4 DISEASES OR MANIFESTATIONS CAUSED BY A. 6-8 TERREUS 2.5 DIAGNOSIS 8-9 2.6 IMMUNE RESPONSE TO ASPERGILLI 9-12 2.7 AIRWAY EPITHELIAL CELLS 12-13 III 2.8 THERAPIES FOR INFECTIONS CAUSED BY A. 14-17 TERREUS 2.9 SINGLE NUCLEOTIDE POLYMORPHISMS AND 18-21 PATHOGEN RECOGNITION RECEPTORS 2.11 PROTEOMICS OF ASPERGILLI AND THE 21-25 SIGNIFICANCE CHAPTER - 3 OBJECTIVE 1: TO ELUCIDATE THE PROTEIN/ENZYMES OR 26-43 BIOCHEMICAL PATHWAY OF ASPERGILLUS TERREUS CONIDIA DURING THE GERMINATION 3.1 INTRODUCTION 26-27 3.2 MATERIAL AND METHODS 27-31 3.2.1 ASPERGILLUS TERREUS STRAIN AND CULTURE 27 CONDITIONS 3.2.2 ASPERGILLUS TERREUS MORPHOTYPES 28 IDENTIFICATION 3.2.3 LARGE SCALE CULTURE FOR GERMINATING 28 CONIDIA 3.2.4 TOTAL PROTEIN EXTRACTION 28 3.2.5 SDS -PAGE ELECTROPHORESIS OF EXTRACTED 29 PROTEINS 3.2.6 PROTEIN DIGESTION 29 IV 3.2.7 QUADRUPOLE TIME OF FLIGHT-LC-MS/MS AND 29-30 DATA ANALYSIS 3.2.8 GENE ONTOLOGY 30 3.2.9 PROTEIN INTERACTION NETWORK FOR PROTEINS 30 FROM MAJOR BIOSYNTHETIC PATHWAYS 3.2.10 TOTAL RNA EXTRACTION FROM ASPERGILLUS 30 TERREUS CONIDIA AND GERMINATING CONIDIA 3.2.11 SYNTHESIS OF CDNA FROM TOTAL RNA 31 3.2.12 COMPARATIVE EXPRESSION ANALYSIS OF 31 SELECTED GENES USING QRT-PCR 3.3 RESULTS 31-39 3.4 DISCUSSION 40-43 CHAPTER - 4 OBJECTIVE 2: GLOBAL PROTEOME ANALYSIS OF HUMAN 44-64 LUNG EPITHELIAL CELLS (A549) IN RESPONSE TO A. TERREUS 4.1 INTRODUCTION 44-45 4.2 MATERIAL AND METHODS 46-48 4.2.1 ASPERGILLUS TERREUS STRAIN AND CULTURE 46 CONDITIONS 4.2.2 CULTURE OF A549 CELLS 46 V 4.2.3 A495 CELLS INTERACTION WITH ASPERGILLUS 46 TERREUS FOR PROTEOME STUDY 4.2.4 TOTAL PROTEIN EXTRACTION 47 4.2.5 PROTEIN DIGESTION 47 4.2.6 QUADRUPOLE TIME OF FLIGHT-LC-MS/MS AND 48 DATA ANALYSIS 4.2.7 GENE ONTOLOGY 48 4.2.8 PROTEIN INTERACTION NETWORK OF PROTEINS 48 INVOLVED IN VARIOUS BIOLOGICAL FUNCTIONS DURING INTERACTION OF A549 CELLS 4.3 RESULTS 49-60 4.4 DISCUSSION 61-64 CHAPTER - 5 OBJECTIVE 3: STRUCTURAL-FUNCTIONAL RELATION OF 65-86 DELETERIOUS NSSNPS IN PENTRAXIN-3 (A SOLUBLE RECEPTOR) AND IN DECTIN-1 (A CELL BOUND RECEPTOR) THAT RECOGNIZE ASPERGILLI CONIDIA DURING THEIR PHENOTYPIC TRANSITION TO GERMINATING CONIDIA 5.1 INTRODUCTION 65-68 5.2 MATERIAL AND METHODS 68-71 5.2.1 SNP DATA RETRIEVAL FOR DECTIN-1 AND 68 PENTRAXIN-3 VI 5.2.2 FUNCTIONAL CONSEQUENCES PREDICTION OF 68 NON-SYNONYMOUS-SNPS USING DIFFERENT ALGORITHMS 5.2.3 CONSERVATION PROFILE OF DECTIN-1 AND 69 PENTRAXIN-3 PROTEIN RECEPTORS 5.2.4 NON-SYNONYMOUS SNPS EFFECT ON STABILITY 69 OF DECTIN-1 AND PENTRAXIN-3 PROTEINS 5.2.5 IDENTIFICATION OF NON-SYNONYMOUS SNPS 70 LOCATIONS IN DOMAINS OF DECTIN-1 AND PENTRAXIN-3 PROTEINS 5.2.6 HOMOLOGY MODELING OF DOMAINS OF DECTIN- 70 1 AND PENTRAXIN-3 PROTEINS AND STRUCTURE STABILITY DETERMINATION 5.2.7 PROTEIN -PROTEIN INTERACTION ANALYSIS FOR 71 DECTIN-1 AND PENTRAXIN-3 PROTEINS 5.2.8 MOLECULAR DOCKING OF NATIVE CTLD-DOMAIN 71 AND ITS VARIANT PROTEIN STRUCTURES WITH Β- GLUCAN 5.3 RESULTS 71-82 5.4 DISCUSSION 82-86 6. SUMMARY AND FUTURE PROSPECTS 87-88 7. REFERENCES 89-112 8. APPENDIX-I 113-118 9. APPENDIX-II 119-129 10.PUBLICATIONS 129-131 VII DECLARATION I hereby declare that the work reported in the Ph.D. thesis entitled “Proteome analysis of human lung epithelial cells interacting with Aspergillus terreus conidia and structure-function relation of nsSNPs in innate immune receptors” submitted at Jaypee University of Information Technology, Waknaghat, India, is an authentic record of my work carried out under the supervision of Dr. Jata Shankar. I have not submitted this work elsewhere for any other degree or diploma. I am fully responsible for the contents of my Ph.D. Thesis. Raman Date: Enrollment No. 136558 Department of Biotechnology & Bioinformatics Jaypee University of Information Technology Waknaghat, India - 173234 VIII CERTIFICATE This is to certify that the work reported in the Ph.D. thesis entitled “Proteome analysis of human lung epithelial cells interacting with Aspergillus terreus conidia and structure-function relation of nsSNPs in innate immune receptors” submitted by Mr. Raman at Jaypee University of Information Technology, Waknaghat, India, is a bonafide record of his original work carried out under my supervision. This work has not been submitted elsewhere for any other degree or diploma. Supervisor: Dr. Jata Shankar Date: Associate Professor Department of Biotechnology & Bioinformatics Jaypee University of Information Technology Waknaghat, India - 173234 IX ACKNOWLEDGEMENT This thesis is the end of my Ph.D. journey and my thesis would not be able to complete without the efforts, well wishes, support and encouragement of many peoples. At the end of this journey, I would like to thank all those who helped me to reach this destination. First and foremost I would like to thank the omnipresent ‘GOD’ for bestowing upon me, his choicest blessing without which this work would have not been accomplished. The worlds “one who direct the path of progress is angelic” are inadequate to express my deep sense of indebtedness and gratitude to my supervisor Dr. Jata Shankar for his solemn efforts, resolute guidance, steadfast encouragement, constructive criticism, meticulous supervision and his kind cooperation throughout the course of the work has enabled me to complete this work. He was always there for me with positive attitude to solve any problem. I especially thank him for his kind support during though time and helping me to learn how to manage the pressure during work. Along with his generous guidance, he gave me space to learn independently, so that I can grow as an ingenious individual. Next, I am thankful to Dr. Sudhir Kumar, Head of the Department of Biotechnology & Bioinformatics, for providing me space and all facilities in the department to carried out this work. Also thanks for his positive attitude and encouragement throughout work period. I also owe my sincere thanks to JUIT administration, Vice Chancellor, Prof. (Dr.) Vinod Kumar, Director and Academic Head, Prof. (Dr.) Samir Dev Gupta and Registrar Maj. Gen. (Retd.) Rakesh Bassi for providing all the facilities and infrastructure to accomplish this work. I would like to express my gratitude to my doctoral progress committee members and other faculty members of the department of biotechnology and Bioinformatics for their cooperation and fruitful suggestions throughout my research work. Among laboratory staff, I would like to thank Mr. Baleshwar for his generous help during tough time of my work. Also thanks to other lab staff, Mrs. Mamta Mishra, X Mrs. Somlata Sharma, Mrs. Sonika, Mr. Ismail and Mr. Ravikant for their kind help during study period. Friends are the important part of life and without their support this journey would not be completed. Special thanks to my dear friends Mr. Anil Sharma and Mr. Rakesh Sharma for their constant support during up and downs for last nine years. I always remember the cheerful time that we have spent together. This acknowledgement would not be complete without thanking my lab mates for their generous help and support during entire work period. Thanks to Mrs. Shraddha Tiwari, Mrs. Sonia and junior lab mates Mr. Deepak and others. I extend my heartfelt thanks to all my seniors, Dr. Varun Gupta, Dr. Swapinl Jain, Dr. shivani Sood, Dr. Archit Sood, Mr. Jibesh, Mr. Arun Dhiman and others for their help and support. I own my thanks to fellow mates Ritika, Poonam mam, and other research scholars for their kind help during study period. A special thanks to my Junior Mr. Avinash Jaswal (Research Scholar, National Dairy Research Institute, Karnal) for his constant motivation to carry out this work. Family members are the important part of this journey and without their support and love; I would not be able to end this journey. A special thank to my younger sister Sarkia Thakur and my cousins Anu Di, Geetanjli, Arti and Vishal Thakur for all their love, affection and support. Thanks and gratitude are the small words for what I owe to my beloved parents, Mr. Prem Singh Thakur and Mrs. Pratima Thakur, whose moral support and great inspiration made me to complete this work. Their unconditional love moral teachings, guidance and motivations will always inspire me throughout my life. At last, It was pleasant task to express my heartfelt gratitude to all those who have contributed directly or indirectly towards completion of this work.