Characterization of caspases from Lepidoptera and their role in apoptosis [Elektronische Ressource] / Juliette Courtiade. Gutachter: David G. Heckel ; Günter Theißen ; Rollie J. Clem
99 Pages
English

Characterization of caspases from Lepidoptera and their role in apoptosis [Elektronische Ressource] / Juliette Courtiade. Gutachter: David G. Heckel ; Günter Theißen ; Rollie J. Clem

-

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Description

Characterization of caspases from Lepidoptera and their role in apoptosis. Dissertation zur Erlangung des akademischen Gra desdoctor rerum naturalium (Dr. rer. nat .) Vorgelegtd em Rat der Biologisc-hPharmazeutischen Fakul tätder Friedrich-Schiller-Universität Jen a von Juliette Courtiade (M. Sc. ) geboren am 18. Juni 1980 in Longjum;eau Frankreich Gutachter: Prof. Dr. David G. Hecke , Ml ax Planck for Chemical Ecology, Jena, German y Prof. Dr. Günter Th eiβen, Friedrich Schiller University, Jena, German y Prof. Dr. Rollie J. Clem, Kansas State University, Kansas, U SA Tag des öffentlichen Verteidigun 15. Julg: y 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 .......................................................

Subjects

Informations

Published by
Published 01 January 2011
Reads 12
Language English
Document size 4 MB





Characterization of caspases from
Lepidoptera and their role in apoptosis.




Dissertation
zur Erlangung des akademischen Gra des
doctor rerum naturalium (Dr. rer. nat .)


Vorgelegtd em Rat der Biologisc-hPharmazeutischen Fakul tät
der Friedrich-Schiller-Universität Jen a



von Juliette Courtiade (M. Sc. )

geboren am 18. Juni 1980 in Longjum;eau Frankreich
































Gutachter:

Prof. Dr. David G. Hecke , Ml ax Planck for Chemical Ecology, Jena, German y

Prof. Dr. Günter Th eiβen, Friedrich Schiller University, Jena, German y

Prof. Dr. Rollie J. Clem, Kansas State University, Kansas, U SA




Tag des öffentlichen Verteidigun 15. Julg: y 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

Page 3 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


Page 4



























Acknowledgments

Acknowledgments
First of all I would like to thank all the teachers, professors, supervisors, and scientisst of all kind
who, during the past 10 years, showed me how wonderful it is to study the steries om f Nature .

I would like to thank Professor David G. Heckel for giving me the opportunity to do my PhD in
his department.
I‟m incredibly grateful to Yannick Pauchet for helping and supervising me even when it was not
his role.
Many thanks to Heiko Vogel for his advice in molecular biology and his help duringque thest
for caspase sequenc . es
I‟m also grateful to Dr. Dalial Freitak and Dr. Hanna Heidel-Fischer for being such good PhD
companion s and for teaching me some new technique s.
I also would like to thank Bianca Ulitzch for her technical assistance and for making my life in
the lab easier, Domenica Schnabelrauch hforer assistance in the sequencing facility and
HenrietteR ingys-Beckstein for technical advice .
Of course, his t whole enterprise wouldn‟t have been possible without the financial support of the
Max Planck Gese lclhaft, but also :
Antje Dudda and Olaf Kniemeye,r from the Hans Knöll Institute, who helped me with the DIGE
experiments.
The MS group who analyzed a lot of samples for mnd e aespecially Alexander Muck who spet n
quite some time explaining me the arcnaeness of mass spectrometr y.
The members of the IT department, who we re always there when computers turned rebellious
and started a lifeof their own.
Regina and her army of Hiwi s, for rearing the insects.
All my colleagues in the Entomology department, for the good atmosp ahend rethe , scientific
and non scientific discussions .
At last but n ot least, I would like to than: k

Page 7 Acknowledgments

Dalial and Paulina who welcomed me he tfirst day like anold friend, thanks for the countless
and priceless adventures. See you in Paris!
Sammy the Snail who made it to Krakow with us ;
Verena, probably the only German girl speaking French with n aAfrican accent, who made me
feel at home in Jen ; a
Frau Doctor Jana Bechern, ot only an accomplished chemist who succeeded in safely heating up
rocket fuel in a microwave but also an accomplished translator who helped me dealing with the
“Bürokratie”, many thanks for all the concerts too ;
David Schlepp for the cookes suppli ies and his help with my c ar;
Hanna, first for being such a model of apparent calm and steadiness when storms came, but also
for sharing with meso me very stressful mome, ntsand luckily, a lot of normal ones;
Yannick, aka The Björn, for all the f un in the lab, and so much mo re;
Roy, with whom I ran the final straight;
Steffi and Nicole, for being such nice office mates ;
Kathrin, who introduced me to the city of Jena when I arrive; d
Katharina (and the president Paul), for the lovely dinners a scnd diussions;
Brent, for sharing the “short lived flatmate” curse with me;
JuL. who showed me the way to the “handicraft” path;
All my friends from “La Garde du Neuvième Cercle”, “Le Conseil” et “Les Archanges” for the
countless hours we spe t tongethe;r
Every person who tried and apparently managed to understand Gemrmay n;
My father and my stepmother, for their unfailing support;
Thierry, for his love and his patienc e.


Page 8 Summary

Summary
Multicellular organisms need a tightly controlled regulation of cell prolife ion raat nd of
cell specialization together with a controlled cell death to maintain their in tegrity. Apoptosis as a
part of the programmed cell death mechanism is required to clear out undesirable cells which are
supernumerary, malfunctioning or infected pabyt hogens. The importance of such a cell suicide
pathway has been demonstrated numerous times since its first description. In holometabolous
insects, and more specifically in Lepidoptera, which undergo a complete metamorphosis
between their larval and ultad stages, the importance of apoptotic events during pupation has
been demonstrated as early as in the 1960‟s. More recently, it has been shown that apoptosis of
cells infected with baculovirus can significantly reduce viral spread throughout the body.
However, the molecular pathway regulating apoptosis in Lepidoptera is still poorly understood.
A family of evolutionarily conserved proteases, callcead spase, splays a central role in regulating
apoptosis in anima. ls Reception of death tismuli triggers th e activation of initiator caspases,
which in turn activate the effector caspa, se ressponsible for the cleavage of many other
intracellular components. The discovery o fp35, a baculovirus protein inhibiting caspase
activity, has led to the characterization of the first lepidopteran caspase, -CSafspase-1, from
Spodoptera frugiperda. Studies on the Sf-Caspas-e1 mode of activation suggested that apoptosis
in Lepidopteraa lso requires a cascade of caspase activation, as demonstrated in many other
species.
In order to obtain an overview of thi sgene family in Lepidoptera, wepe rformed an
extensive urves y of lepidopteran-derived EST datasets. We identified 66 sequence ens coding
putative caspases, distributed amon27 gsp ecies of butterflies and moths . Phylogenetic analyses
showed that Lepidopt era possess at least 5 caspases, for which we propose a unified
nomenclature . According to their homology to their Drosophila melanogaster counterparts and
their primary structure, we propo sed that Lep-Caspase-1, -2 and -3 are effector caspas, e
whereas Le-pCaspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4
remains un clear. Lep-Caspase-2 is absent from thesil kworm genome and appears to be noctui- d
specific, and to have arisen from tandea m duplication of the Caspase-1 gene. In the tobacco
hawkmoth , 3 distinct transcripts encoding putative Caspa-4 swee re identified, suggesting at least
2 duplication events in this species. The basic repertoire of five major types of caspases shared
among Lepido ptera seems to be smaller than for most other groups studied to date, but gene

Page 9 Summary

duplication still plays an essential role in linea -spege cific increases in diversity, similar to what
has been described in Diptera and mamm als.
Despite an overall decrease in RmNA and protein biosynthesis, apoptosis requires the
selective transcription and translation of several genes involved in regulation and execution. In
an attempt to characterize caspase expression profilHees liin coverpa armigera, we used real
time quanti tative RT-PCR to assess their resting levels of expression in different larval tissues
and during development. Furthermore, we analyzed the effect of immune challenge and
induction of apoptosis on their expression. We found that Ca-spa1 and se-2 have complementary
expression profiles during larval development, suggesting a differential regulation. C-3 aspase
and -6 are upregulated upon immune challenge, suggesting a roleimd in the pathway. Caspa-s5 e
is upregulated during pupation and upon induction with -hy20droxyecdysone, supporting the
hypothesis of Caspa-se5 playing a similar role as Dronc in developmental apoptosis in
Drosophila.
In mammalian systems, almost 300 potential targets of caspases have been described and
the expression of more than 120 protens ci an be altered during apoptosis. In an attempt to obtain
further insights into the molecular pathway(s) underlying apoptosis in lepidopteran insects, we
used 2D differential electrophoresis to identif3y prote1 ins, for which relative abundance was
altered during apoptosis of H. armigera derived cells, induced by actinomy cD. in Among these
13 proteins, we identified the putative effector Caspa-1,se 3 chaperone proteins and several - pro
apoptotic proteins, such as actin and a voltage dependant anion el. cha W nne also identified two
anti-apoptotic proteins, a member of the aldo/keto reductase family and anne xin IX.
In conclusion, the molecular pathway(s) underlying apoptosis in Lepidoptera seems to be
similar to what has been described in other organism Ho s. we ver, a better understanding of the
apoptotic pathway(s) in lepidopteran insects would contribute to improve our knowledge in
fields such as interaction between the insect and its pathogens, or between the insect and its host
plant. This could lead to he t discovery of deterrents that could be used as potential
“environmentally-friendly” insecticides.



Page 10