Mitochondrial copper homeostasis in mammalian cells [Elektronische Ressource] / von Corina Oswald

Mitochondrial copper homeostasis in mammalian cells [Elektronische Ressource] / von Corina Oswald

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Mitochondrial copper homeostasis in mammalian cells Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Corina Oswald (Diplom-Biochemikerin) geboren am 10.04.1981 in Dohna, Deutschland Gutachter: Prof. Dr. Gerhard Rödel Prof. Dr. Alexander Storch Eingereicht am 30. April 2010 Verteidigt am 13. August 2010 ACKNOWLEDGEMENTS I sincerely thank my supervisor Prof. Dr. Gerhard Rödel for giving me the opportunity to do my PhD in his group and to join the Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB). He introduced me to the world of mitochondria, supported and provided me with all resources and comprehension necessary to conduct my research. I thank Dr. Udo Krause-Buchholz for his scientific advice and for helping writing the paper by giving constructive comments on the manuscript. I honestly thank my TAC members Dr. Frank Buchholz and Prof. Dr.

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Mitochondrial copper homeostasis in
Gutachter:
mammalian cells
Dissertation
zur Erlangung des akademischen Grades Doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt der Fakultät Mathematik und Naturwissenschaften
der Technischen Universität Dresden von
Corina Oswald
(Diplom-Biochemikerin) geboren am 10.04.1981 in Dohna, Deutschland
Prof. Dr. Gerhard Rödel Prof. Dr. Alexander Storch
Eingereicht am 30. April 2010
Verteidigt am 13. August 2010
ACKNOWLEDGEMENTS
I sincerely thank my supervisor Prof. Dr. Gerhard Rödel for giving me the
opportunity to do my PhD in his group and to join the Dresden International
Graduate School for
Biomedicine and Bioengineering (DIGS-BB). He
introduced me to the world of mitochondria, supported and provided me with
all resources and comprehension necessary to conduct my research.
I thank Dr. Udo Krause-Buchholz for his scientific advice and for helping
writing the paper by giving constructive comments on the manuscript.
I honestly thank my TAC members Dr. Frank Buchholz and Prof. Dr.
Alexander Storch for their interest in this work, for guiding me scientifically,
and for stimulating discussions in the TAC meeting. Especially, Dr. Frank
Bucholz for giving insightful suggestions as RNAi specialist, and Prof. Dr.
Alexander Storch for acting as reviewer of this thesis.
ThedSTORM images would not have been possible without the very friendly
collaboration with Prof. Dr. Markus Sauer and Sebastian van de Linde,
Institute for Applied Laser Physics and Laser Spectroscopy of the University of
Bielefeld. Thank you!
I am furthermore grateful to all former and present lab members for the
friendly working atmosphere, for fruitful discussions, for providing advice and
assistance in many situations. In particular, I thank the “girls” in the lab – 2 Anja, Kirsten, Susi , Simone and Uta for struggling together through the ups
and downs.
I am grateful to Uta Gey, Dr. Kristof Zarschler and Dr. Kai Ostermann for
critical proof reading of the thesis.
Special thanks are dedicated to my friends for all our unforgettable moments
together.
I sincerely thank my parents, who helped, supported and encouraged me all
the time.
Above all, I want to thank Stefan for always being there, motivating and
believing in me.
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Cytochromecoxidase assembly
Copper and its trafficking in the cell
Cell culture: HeLa cells
Ab st r a ct
Primers siRNAs Methods
The human mitochondrial genome
Mitochondria and the respriratory chain
Mitochondrial disorders
Homoplasmy and heteroplasmy
2.12.1.12.1.22.1.32.1.42.1.52.1.62.1.72.1.82.22.2.12.2.1.12.2.1.2
i
2
Cell culture: HeLa cells transfected with pTurboRFP-mito
2
Mitochondrial copper metabolism Cox17 Aims of the thesis
1.11.21.31.41.4.11.4.21.51.61.71.81.91.10
Materials Chemicals and reagents Antibodies Plasmid
List of Fig u r e s a n d Ta b le s
Ab b r e v ia t ion s
Mutations in mitochondrial DNA Mutations in nuclear DNA Cytochromecoxidase
CONTENTS
2 2
 1
 iv
1
CONTENTS
Kits Marker Enzymes
Cell culture
M a t e r ia ls a n d M e t h od s
I n d r od u ct ion
CONTENTS
2.2.1.32.2.1.42.2.1.52.2.22.2.32.2.42.2.52.2.62.2.6.12.2.6.22.2.6.32.2.72.2.82.2.8.12.2.8.22.2.92.2.9.12.2.9.22.2.102.2.112.2.122.2.12.12.2.12.22.2.12.32.2.132.2.14
3
3.13.23.2.13.2.23.2.3
3.2.4
Subcultivation
Determination of cell number
Cell storage and thawing
Transient transfection of HeLa cells
Transfection of HeLa cells with pTurboRFP-mito
Immunocytochemistry
RNA extraction and quantitative real-time PCR
Isolation of mitochondria
Isolation of mitochondria for BN-PAGE Analysis
Isolation of mitochondria for localization studies
Isolation of bovine heart mitochondria
Proteinase K treatment of mitochondria and mitoplasts
Photometric activity assay
Citrate synthase activity
Cytochromecoxidase activity
Blue native polyacrylamide gel electrophoresis (BN-PAGE)
In gelactivity assay
2D-BN/SDS-PAGE
SDS-PAGE and Western blot analysis
Directstochastic optical reconstruction microscopy (dSTORM)
Flow cytometric phenotyping
Determination of cell cyle phase
Identification of apoptotic cells
Detection of ROS
Oxygen measurement
Cu–His supplementation
Re su lt s
2929293030313132323333343434353637373738393940414243
4 4
Subcellular localization of Cox17 44Transient knockdown ofCOX1746in HeLa cells Knockdown ofCOX17mRNA 47Knockdown of Cox17 protein 49Effect ofCOX17knockdown on the steady-state levels of OXPHOS subunits 50Effect ofCOX17knockdown on the steady-state levels of copper-bearing COX subunits 51
ii
CONTENTS
3.2.53.33.3.13.3.23.3.33.3.43.3.53.43.53.5.13.5.23.5.33.5.4
3.5.53.5.63.6
4
4.14.2
4.3
4.44.5
4.6
5
6
7
Subdiffraction-resolution fluorescence imaging 51Phenotypical characterization 56Growth analyis 57Cell cycle analysis 57Apoptosis assay 59Detection of ROS 61Oxygen measurement 63Cytochromecoxidase activity 64Characterization of mt OXPHOS complexes 65BN-PAGE/in gelactivity assays 65Supramolecular organization of COX 67Molecular organization of Cox17 68Molecular organisation of copper-bearing COX subunits Cox1 and Cox2 69Supramolecular organization of RC complexes 70d72STORM of supercomplexes Copper supplementation 74
D iscu ssion
7 5
Dual localization of human Cox17 75COX17knockdown affects steady-state levels of copper-bearing COX subunits Cox1 and Cox2 77Supramolecular organization of RC is affected as an early response toCOX17knockdown 79Cox17 is primarily engaged in copper delivery to Sco1/Sco2 82Copper supplementation alone cannot rescue theCOX17
phenotype
Outlook
Ap p e n d ix
Ph D p u b lica t ion r e cor d
Re f e r e n ce s
iii
8485
8 8
9 6
9 7
LIST OF FIGURES AND TABLES
LIST OF FIGURES AND TABLES
Figure 1.10.3Oxidative phosphorylation. Figure 1.20.Model of mammalian I1III2IV1supercomplex. 4Figure 1.30.Molecular organization of COX. 10Figure 1.40.11Illustration of the electron flow through the COX. Figure 1.50.12Model of the assembly pathway of human COX. Figure 1.60.14Pathways of copper trafficking within a mammalian cell. Figure 1.70.NMR solution structures of apo- and Cu1-Cox172S-S. 20Figure 2.10.Respective positions of the siRNA sequence onCOX17mRNA. 27Figure 2.20.40Quantification of cell cycle distribution of HeLa cells. Figure 2.30.41Sample data using Annexin V-FITC Apoptosis Detection Kit. Figure 2.40.DCF fluorescence in HeLa cells. 42Figure 3.10.45Localization of human Cox17. Figure 3.20.siRNA transfection efficiency in HeLa cells. 47Figure 3.30.Transient knockdown ofCOX17mRNA. 48Figure 3.40.Effect of transient knockdown ofCOX1749in HeLa cells. Figure 3.50.The principle ofdSTORM image analysis. 52Figure 3.60.Subdiffraction-resolution imaging of immunolabeled pTurboRFP  mito HeLa cells transfected withCOX17siRNAs. 55Figure 3.70.Growth ofCOX17knockdown cells. 57Figure 3.80.58Cell cycle analysis. Figure 3.90.Identification of apoptotic cells by flow cytometry. 60Figure 3.10.62ROS production in HeLa cells. Figure 3.11.Respiration rate inCOX1763knockdown cells. Figure 3.12.COX activity ofCOX17knockdown cells. 64Figure 3.13.BN-PAGE/in gel66activity of digitonin solubilized mitochondria. Figure 3.14.Supramolecular organization of RC complexes. 68Figure 3.15.702D-BN/SDS-PAGE of OXPHOS complexes. Figure 3.16.72Molecular organization of RC complexes. Figure 3.17.dSTORM of supercomplexes. 73
Table 2.1. List of gel electrophoresis markers.
Table 2.2. List of siRNAs.
iv
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ABBREVIATIONS
ABBREVIATIONS
AEBSF adPEO APS BHM BN
BSA CHAPS
COX CPEO CS Cu-His DCFH-DA Ddp DTNB
ddH2O DMEM DOX dNTP dSTORM EDTA et al. FBS FMN FITC FL FRET GAPDH HMW Hsp60 HRP
IEF IgG IMM
4-(2-Aminoethyl)benzenesulfonyl fluoride
Autosomal dominant progressive external ophthalmoplegia
Ammonium persulphate
Bovine heart mitochondria
Blue native
Bovine serum albumine
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
Cytochromecoxidase
Chronic progressive external ophthalmoplegia
Citrate synthase
Copper-histidine
2,7-Dichlorodihydrofluorescein diacetate
Deafness dystonia protein 5,5’-dithio-bis(2-nitrobenzoate)
Double distilled water
Dulbecco’s Modified Eagle Medium
Doxycycline
Deoxynucleoside triphosphate
Directstochastic optical reconstruction microscopy
Ethylendiamin-tetraacetic acid
et alii, and others
Fetal bovine serum
Flavin mononucleotide
Fluorescein isothiocyanate
Fluorescence Fluorescence resonance energy transfer Glyceraldehyde 3-phosphate dehydrogenase
High molecular weight
Heat shock protein 60
Horse radish peroxidase
Isoelectric focusing
Immunoglobulin G
Inner mitochondrial membrane
v
ABBREVIATIONS
IMS KSS LDH MELAS
MERRF MIDD MOPS mRNA
mt mtDNA MW
+ NAD NADH NARP
NDUFB9 nDNA NMR nt NTB OMM OXPHOS PAA PBS PDH PS
PMS PI PIC PVDF RC RFP RITOLS
RNAi ROS rRNA RT
Intermembrane space
Kearns-Sayre syndrome
Lactate dehydrogenase
Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes
Myoclonic epilepsy associated with ragged-red fibres Maternally inherited diabetes with deafness 3-[N-Morpholino]propanesulfonic acid
Messenger RNA
Mitochondrial
Mitochondrial DNA
Molecular weight
β-nicotinamide adenine dinucleotide
Reducedβ-nicotinamide adenine dinucleotide
Neurogenic muscle weakness, ataxia, and retinitis pigmentosa NADH dehydrogenase (ubiquinone) subunit 9 Nuclear DNA
Nuclear magnetic resonance Nucleotide Nitro tetrazolium blue
Outer mitochondrial membrane
Oxidative phosphorylation
Polyacrylamide
Phosphate buffered saline
Pyruvate dehydrogenase
Phosphatidylserine
Phenazine methosulfate
Propidium iodide
Protease inhibitor cocktail
Polyvinylidene fluoride
Respiratory chain
Red fluorescent protein
RNA incorporation throughout the lagging strand
RNA interference Reactive oxygen species Ribosomal RNA
Room temperature
vi
ABBREVIATIONS
RT-PCR S. cerevisiae SCs SDM SDS SEM shRNA siRNA SOD1 SOD2 STED TBS
TCEP TEMED Tet
TOM Tris Triton X-100
tRNA Tween-20 ultra ddH2O v/v VDAC w/v
Reverse transcription – polymerase chain reaction
Saccharomyces cerevisiae
Supercomplexes
Strand-displacement mechanism
Sodium dodecyl sulphate
Standard error of the mean
Short hairpin RNA
Short interfering RNA
Cu/Zn-superoxide dismutase
Mn-superoxide dismuatse
Stimulated Emission Depletion microscopy
Tris buffered saline Tris (2-carboxyethyl) phosphine hydrochloride N,N,N',N'-tetramethylethylenediamine
Tetracycline
Translocase outer membrane
Tris (hydroxymethyl) aminomethane
T-octylpenoxpolyethoxethanol
Transfer RNA
Polyoxyethylene (20) sorbitan monolaurate
Ultra pure double distilled water
Volume per volume
Voltage-dependent anion channel, porin
Weight per volume
vii
structures, so called
formation is not simply due to assembly of completely assembled complexes.
may indicate that the absence of Cox17 interferes with copper delivery to
In this thesis, the role ofCOX17 in the biogenesis of the respiratory chain in
knowledge concerning mitochondrial copper homeostasis and insertion of
has not been well elucidated. Homozygous disruption of the mouseCOX17
indispensable role for Cox17 in cell survival.
a reduced steady-state concentration of the copper-bearing subunits of COX
Assembly of cytochromec oxidase (COX), the terminal enzyme of the
formation
of
this pathway. It is a low molecular weight protein containing highly conserved
chaperones and factors for the correct insertion of subunits, accessory
mitochondrial respiratory chain, requires a concerted activity of a number of
supercomplexes as an early response. Accumulation of a novel ~150 kDa
ABSTRACT
blue native gel electrophoresis reveals the disappearance of COX-containing
supercomplexes is proposed that requires the coordinated synthesis and
the
Cox2, but not to Cox1. Data presented here suggest that supercomplex
an
association of individual complexes.
complex containing Cox1, but not Cox2 could be observed. This observation
as
well accepted that the multienzyme complexes of the respiratory chain are
HeLa cells was explored by use of siRNA. The knockdown ofCOX17results in
assembly
1
supramolecular
functional
scenario
for
and apoptotic cells. Furthermore, in accordance with its predicted function as
cerevisiae.this organism, Cox17 was the first identified factor involved in In
organizedin vivo
proteins, cofactors and prosthetic groups. Most of the fundamental biological
So far, the role of Cox17 in the mammalian mitochondrial copper metabolism
copper into COX derives from investigations in the yeast Saccharomyces
essential for its function.
twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is
ABSTRACT
interdependent
gene leads to COX deficiency followed by embryonic death, which implies an
and affects growth of HeLa cells accompagnied by an accumulation of ROS
COX,COX17knockdown affects COX-activity and -assembly. It is now siRNA
supercomplexes. While the abundance of COX dimers seems to be unaffected,
Instead
a copper chaperone and its role in formation of the binuclear copper center of
INTRODUCTION
1
1.1
INDRODUCTION
MITOCHONDRIA AND THE RESPRIRATORY CHAIN
Mitochondria are complex subcellular organelles which perform a wide range
of necessary functions, including ATP production, citric acid cycle, fatty acid
oxidation, calcium homeostasis, and the production of heme and iron-sulfur
clusters (Rizzuto et al., 1998; Scheffler, 2001; McBride et al., 2006). Finally,
they play an important role in the regulation of apoptosis (Green and Reed,
1998).
Structurally, mitochondria contain two membranes that separate four distinct
compartments: the outer membrane (OMM), intermembrane space (IMS),
inner membrane (IMM), and the matrix. The two membranes are themselves
very different in structure and in function. The OMM contains numerous
integral porin proteins, resulting in a membrane that is permable to water,
ions, and small proteins (< 5000 Da) (Mannella, 1992; Vander Heiden et al.,
2000). The IMM is
impermeable, enabling the maintenance of the
transmembrane potential (ΔΨ). It is highly folded into cristae, which house
the megadalton complexes of oxidative phosphorylation (OXPHOS) (Gilkersonet al., 2003).As the site of OXPHOS, mitochondria provide a highly efficient route to
generate ATP from energy-rich molecules. Respiration consists of the
sequential transfer of electrons extracted from nutrient compounds through
the chain of oxidoreductase reactions, leading to reduction of molecular
oxygen to water. Briefly, electrons are transported in the respiratory chain
(RC) from NADH or succinate to complex I (NADH ubiquinone oxidoreductase)
or complex II (succinate ubiquinone oxidoreductase), respectively, and further
via(ubiquinol cytochrome complex III cIVand complex  oxidoreductase)
(cytochromec oxidase, COX) to the terminal acceptor molecular oxygen + (Hatefi, 1985). In the process, protons (H ) are pumped from the matrix
across the IMM into the IMS through respiratory complexes I, III, and IV
(Babcock and Wikstrom, 1992). The resulting electrochemical proton gradient
(ΔΨ) finally leads to the production of ATP by phosphorylation of ADPvia
complex V (F1F0-ATP synthase) (Figure 1.1).
2