Characterization of caspases from Lepidoptera and their role in apoptosis. Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) Vorgelegtdem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Juliette Courtiade (M. Sc.) geboren am 18. Juni 1980 in Longjumeau; Frankreich Gutachter: Prof. Dr. David G. Heckel, Max Planck for Chemical Ecology, Jena, Germany Prof. Dr. Günter Theiβen, Friedrich Schiller University, Jena, Germany Prof. Dr. Rollie J. Clem, Kansas State University, Kansas, USA Tag des öffentlichen Verteidigung: 15. July 2011 Table of Contents Table of Contents Acknowledgments ............................................................................................ 7 Summary ........................................................................................................... 9 Zusammenfassung .......................................................................................... 11 General Introduction ...................................................................................... 15 Apoptosis versus Necrosis ........................................................................................... 15 Apoptotic pathways ..................................................................................................... 16 Consequences of apoptosis.......................................................................................... 18 Apoptosis and development ....................................................................................... 18 Apoptosis and homeostasis ........................................................................................ 19 Apoptosis and immunity ............................................................................................ 19 Importance of apoptosis in holometabolous insects ................................................... 20 Aims of this work ......................................................................................................... 21 Chapter 1. Characterization of the Caspase gene family in Lepidoptera ..... 23 Introduction ................................................................................................................. 23 Metacaspases and Paracaspases ................................................................................ 23 The True caspases .................................................................................................... 24 Inflammatory caspases .............................................................................................. 25 Apoptotic caspases .................................................................................................... 26 Caspases in Lepidoptera ............................................................................................ 26 Methods ....................................................................................................................... 27 Preparation of cDNA libraries and EST sequencing ...................................................... 27 Database mining for putative caspase sequences ........................................................ 28 Caspase gene amplification and sequencing ................................................................ 28 Sequence alignment and phylogenetic analysis ........................................................... 29 BAC library screening and sequencing ........................................................................ 30 Intron-Exon structure ................................................................................................ 31 Results and discussion ................................................................................................. 31 Lepidopteran caspases cluster into six distinct clades .................................................. 31 Classification of the lepidopteran caspases .................................................................. 34 Alternative splicing of Caspase-3 and -5 in B. mori ...................................................... 36 A noctuid-specific caspase gene arose from duplication of Caspase-1 ........................... 37 Ms-Caspase-4 subfamily has evolved through duplication events .................................. 39 Future directions and conclusions ............................................................................... 40 Supplementary materials ............................................................................................. 41 Chapter 2. Functional analyses of Caspases in Helicoverpa armigera .......... 49 Introduction ................................................................................................................. 49 Materials and methods ................................................................................................ 50 Apoptosis induction ................................................................................................... 50 Feeding experiments, tissue and developmental stages expression .............................. 50 Page3 Table of Contents Real time quantitative RT-PCR ................................................................................... 51 Results ......................................................................................................................... 52 Caspase expression patterns in H. armigera ............................................................... 52 Caspase expression after immune challenge .............................................................. 53 Caspase expression upon induction of apoptosis ........................................................ 55 Discussion .................................................................................................................... 56 Caspase-1 and -2, shared functions? ......................................................................... 56 Caspase-5, the development manager? ..................................................................... 56 Caspase-3 and -6, to serve and protect? .................................................................... 57 Caspase-4, an outcast? ............................................................................................. 59 Conclusion and further directions ............................................................................... 59 Chapter 3. Comparative proteomic analysis of Helicoverpa armigera cells undergoing apoptosis ..................................................................................... 61 Introduction................................................................................................................. 61 Material and Methods .................................................................................................. 62 Cell culture, apoptosis induction and extraction of protein samples. ............................. 62 Two-dimensional Differential Fluorescence Gel Electrophoresis (DIGE)......................... 63 Protein identification by mass spectrometry. .............................................................. 64 Heterologous expression of Ha-Caspase-1.................................................................. 65 Caspase activity assays. ............................................................................................ 65 RNA isolation and quantitative real-time PCR. ............................................................ 66 Results and Discussion ................................................................................................ 67 Comparative analysis and protein identification. ......................................................... 67 Functional characterization of Ha-Caspase-1. ............................................................. 70 Actin. ....................................................................................................................... 73 Voltage dependent anion channel. ............................................................................. 73 Glyceraldehyde 3-phosphate dehydrogenase. ............................................................. 74 Annexin IX. .............................................................................................................. 74 Aldo-keto reductase. ................................................................................................. 75 Receptor for activated protein kinase C 1. .................................................................. 75 Chaperone proteins. ................................................................................................. 76 Seryl tRNA synthetase (SerRS). ................................................................................. 77 Conclusion ............................................................................................................... 78 Supplementary materials ............................................................................................ 79 General discussion .......................................................................................... 82 References ...................................................................................................... 85 Selbständigkeitserklärung ............................................................................. 96 Curriculum vitae ............................................................................................. 97 Page4 Acknowledgments Acknowledgments First of all I would like to thank all the teachers, professors, supervisors,and scientists