The interplay between pro-death and pro-survival signaling pathways converging at mitochondria in myocardial ischemia, reperfusion [Elektronische Ressource] : apoptosis meets autophagy / vorgelegt von Anne Hamacher

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2006
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

THE INTERPLAY BETWEEN PRO-DEATH AND PRO-SURVIVAL
SIGNALING PATHWAYS CONVERGING AT MITOCHONDRIA IN
MYOCARDIAL ISCHEMIA/REPERFUSION:
APOPTOSIS MEETS AUTOPHAGY





Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation



vorgelegt von

Diplom-Biologin

Anne Hamacher

aus Aachen









Berichter: Universitätsprofessor Dr. Fritz Kreuzaler
Privatdozent Dr. Christoph Peterhänsel


Tag der mündlichen Prüfung: 15. September 2006




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
























































FOR MY DAUGHTER ZOË












































The research described in this thesis was carried out at the Department of Molecular and Experimental
Medicine of The Scripps Research Institute, California, USA under the auspices of the RWTH
Aachen, Germany. ABBREVIATIONS
.
Abbreviations

ADP adenosine diphosphate
AIF apoptosis-inducing factor
ANT adenine nucleotide translocator
Apaf-1 apoptotic protease activating factor 1
ATP adenosine triphosphate
AV(s) autophagic vacuole(s)
Baf A bafilomycin A 1 1
Bax Bcl-2 associated X protein
Bcl-2 B-cell leukemia/lymphoma 2
BD binding domain
BH3 Bcl-2 homology domain 3
Bid Bcl-2 interacting domain
Bnip3 Bcl-2/adenovirus E1B 19 kDa interacting protein
CFP cyan fluorescent protein
2+
Ca calcium
CsA cyclosporin A
DCF 2',7'-dichlorofluorescein
CM-DCFH -DA chloromethyl dichlorodihydrofluorescein diacetate 2
DNA deoxyribonucleic acid
ΔΨ mitochondrial transmembrane potential m
EDTA ethylene diamine tetraacetic acid
ETC electron transport chain
FADD Fas-associated death domain
FADH 1,5-dihydroflavin adenine dinucleotide 2
4D four-dimensional (X/Y/Z/time)
FRET fluorescence resonance energy transfer
GFP green fluorescent protein
GDP guanosine diphosphate
GTP guanosine triphosphate
+
H proton
IAPs inhibitor of apoptosis proteins
I/R ischemia/reperfusion
kDa kilo Dalton
KH Krebs-Henseleit
Lamp2 lysosomal-associated membrane protein 2
LC3 microtubule-associated protein light chain 3
LSCM laser scanning confocal microscopy
LTR LysoTracker Red
MAPK mitogen-activated protein kinase
MCS multiple cloning site
μM micro-molar (μmol/L)
mM mill-molar (mmol/L)
MPT(P) mitochondrial permeability transition (pore)
i ABBREVIATIONS
.
mRNA messenger ribonucleic acid
mTOR mammalian target of rapamycin
N.A. numerical apertur
NADH nicotinamide adenine dinucleotide
ORF open reading frame
PBS phosphate buffered saline
PCD programmed cell death
PE phosphatidylethanolamine
PI3-K phosphatidylinositol 3-kinase
PI3-P phosphatidylinositol 3-phosphate
RFP red fluorescent protein
RIRR ROS-induced ROS release
RNA ribonucleic acid
RNAi RNA interference
ROS reactive oxygen species
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
S/ER sarco/endoplasmic reticulum
SERCA sarco/endoplasmic reticulum calcium ATPase
sI/R simulated ischemia/reperfusion
tBid truncated Bid
TM transmembrane
TMRM tetramethylrhodamine methyl ester
TNF-α tumor necrosis factor-α
TRADD TNF-α-associated death domain
3D 3-dimensional (X/Y/Z)
3-MA 3-methyladenine
v-ATPase vacuolar ATPase
VDAC voltage dependent anion channel
YFP yellow fluorescent protein

ii TABLE OF CONTENTS .

Table of contents


Abbreviations……………………………………………………………………………..i
Table of contents…………………………………………………………………………iii

I Introduction ……...………………………………...……...………………………….1

I-1 Heart disease ....................................................................................................... 3
I-2 The heart and its working units........................................................................... 4
I-3 Myocardial cell death during I/R injury.............................................................. 5
I-3.1 Pathways and executers of apoptosis (PCD type I) ........................................ 6
I-3.1.1 Receptor-mediated death pathway.......................................................... 7
I-3.1.2 Mitochondrial death pathway................................................................. 7
I-3.2 Death signaling at the mitochondria ............................................................... 9
I-3.2.1 Mitochondrial permeability transition.................................................. 10
I-3.2.2 Mitochondrial ROS generation............................................................. 10
I-3.2.3 Mitochondrial calcium handling........................................................... 11
I-3.3 Regulation of mitochondrial integrity by Bcl-2 family members................. 12
I-3.3.1 Anti-apoptotic Bcl-2 family members ................................................... 13
I-3.3.2 Pro-apoptotic Bcl-2 family members .................................................... 14
I-4 Autophagy – at the interface between death and survival ................................ 15
I-4.1 Molecular mechanism of autophagy............................................................. 15
I-4.1.1 Autophagosome formation.................................................................... 16
I-4.1.2 Autophagosome maturation.................................................................. 19
I-4.1.3 Degradation within the autophagolysosome ........................................ 19
I-4.2 Regulation of autophagy............................................................................... 20
I-4.2.1 Negative regulation of autophagy......................................................... 20
I-4.2.2 Positive regulation of autophagy.......................................................... 20
I-4.3 Cross-talk of autophagy and apoptosis: Bcl-2 family members ................... 21
I-4.3.1 Anti-apoptotic Bcl-2 family members ................................................... 22
I-4.3.2 Pro-apoptotic Bcl-2 family members .................................................... 22
I-4.4 Autophagy in the heart.................................................................................. 23
I-5 Thesis objectives............................................................................................... 24
I-5.1 Cell culture model of I/R injury to the heart................................................. 24
I-5.2 Elucidation of the regulation of Beclin1-mediated autophagy ..................... 24
I-5.3 Role of Beclin1-mediated autophagy in myocardial I/R injury.................... 25
I-5.4 Connection between mitochondrial dysfunction and autophagy.................. 25

I Materials and Methods ……...……………………..……...……………………….27

II-1 Materials ........................................................................................................... 29
II-1.1 Antibodies................................................................................................. 29
II-1.1.1 Primary antibodies............................................................................ 29
II-1.1.2 Horseradish peroxidase-conjugated secondary antibodies.............. 30
II-2 Methods............................................................................................................. 30
iii TABLE OF CONTENTS .


II-2.1 Cloning and plasmid construction ............................................................ 30
II-2.1.1 pmCherry-C1 .................................................................................... 31
II-2.1.2 mCherry-Bax..................................................................................... 31
II-2.1.3 Mito-ECFP........................................................................................ 31