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

Stefan Löfving

Description

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.

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2010
• Nombre de lectures: 58
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

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

A CKNOWLEDGEMENTS

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.

 v

22 22 24 24 25 25 25 26 26 28 28 28 28

2 4 6 6 7 8 9 11 13 15 19 21

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

2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.1.8 2.2 2.2.1 2.2.1.1 2.2.1.2

i

2

Cell culture: HeLa cells transfected with pTurboRFP-mito

2

Mitochondrial copper metabolism Cox17 Aims of the thesis

1.1 1.2 1.3 1.4 1.4.1 1.4.2 1.5 1.6 1.7 1.8 1.9 1.10

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

C ONTENTS

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

2.2.1.3 2.2.1.4 2.2.1.5 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6 2.2.6.1 2.2.6.2 2.2.6.3 2.2.7 2.2.8 2.2.8.1 2.2.8.2 2.2.9 2.2.9.1 2.2.9.2 2.2.10 2.2.11 2.2.12 2.2.12.1 2.2.12.2 2.2.12.3 2.2.13 2.2.14

3

3.1 3.2 3.2.1 3.2.2 3.2.3

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

29 29 29 30 30 31 31 32 32 33 33 34 34 34 35 36 37 37 37 38 39 39 40 41 42 43

4 4

Subcellular localization of Cox17 44 Transient knockdown ofCOX1746in HeLa cells Knockdown ofCOX17mRNA 47 Knockdown of Cox17 protein 49 Effect ofCOX17knockdown on the steady-state levels of OXPHOS subunits 50 Effect ofCOX17knockdown on the steady-state levels of copper- bearing COX subunits 51

ii

CONTENTS

3.2.5 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.4 3.5 3.5.1 3.5.2 3.5.3 3.5.4

3.5.5 3.5.6 3.6

4

4.1 4.2

4.3

4.4 4.5

4.6

5

6

7

Subdiffraction-resolution fluorescence imaging 51 Phenotypical characterization 56 Growth analyis 57 Cell cycle analysis 57 Apoptosis assay 59 Detection of ROS 61 Oxygen measurement 63 Cytochromecoxidase activity 64 Characterization of mt OXPHOS complexes 65 BN-PAGE/in gelactivity assays 65 Supramolecular organization of COX 67 Molecular organization of Cox17 68 Molecular organisation of copper-bearing COX subunits Cox1 and Cox2 69 Supramolecular organization of RC complexes 70 d72STORM of supercomplexes Copper supplementation 74

D iscu ssion

7 5

Dual localization of human Cox17 75 COX17knockdown affects steady-state levels of copper-bearing COX subunits Cox1 and Cox2 77 Supramolecular organization of RC is affected as an early response toCOX17knockdown 79 Cox17 is primarily engaged in copper delivery to Sco1/Sco2 82 Copper 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

84 85

8 8

9 6

9 7

LIST OF FIGURES AND TABLES

L IST OF FIGURES AND TABLES

Figure 1.10.3Oxidative phosphorylation. Figure 1.20.Model of mammalian I 1 III 2 IV 1 supercomplex. 4 Figure 1.30.Molecular organization of COX. 10 Figure 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 Cu 1 -Cox17 2S-S . 20 Figure 2.10.Respective positions of the siRNA sequence onCOX17mRNA. 27 Figure 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. 42 Figure 3.10.45Localization of human Cox17. Figure 3.20.siRNA transfection efficiency in HeLa cells. 47 Figure 3.30.Transient knockdown ofCOX17mRNA. 48 Figure 3.40.Effect of transient knockdown ofCOX1749in HeLa cells. Figure 3.50.The principle ofdSTORM image analysis. 52 Figure 3.60.Subdiffraction-resolution imaging of immunolabeled pTurboRFP  mito HeLa cells transfected withCOX17siRNAs. 55 Figure 3.70.Growth ofCOX17knockdown cells. 57 Figure 3.80.58Cell cycle analysis. Figure 3.90.Identification of apoptotic cells by flow cytometry. 60 Figure 3.10.62ROS production in HeLa cells. Figure 3.11.Respiration rate inCOX1763knockdown cells. Figure 3.12.COX activity ofCOX17knockdown cells. 64 Figure 3.13.BN-PAGE/in gel66activity of digitonin solubilized mitochondria. Figure 3.14.Supramolecular organization of RC complexes. 68 Figure 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

25 26

ABBREVIATIONS

A BBREVIATIONS

AEBSF adPEO APS BHM BN

BSA CHAPS

COX CPEO CS Cu-His DCFH-DA Ddp DTNB

ddH 2 O 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 Cx 9 C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is

A BSTRACT

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

I NDRODUCTION

M ITOCHONDRIA 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) (Gilkerson et 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 (F 1 F 0 -ATP synthase) (Figure 1.1).

2