THE STUDY OF GRIM-19 FUNCTION IN MITOCHONDRIA Lu Hao A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 i ACKNOWLEDGEMENTS In the last years, I have learnt and grown, with great appreciation to many peoples. First and foremost, I would like to express my deep and sincere gratitude to my supervisor, Dr. Xinmin Cao, for giving me the opportunity to pursue my Ph.D. research work in her laboratory. Her understanding, encouragement and personal guidance provided a very good basis for my thesis. I am really grateful to all my labmates and friends in IMCB, past and present, for the stimulating and extensive discussions, sharing of reagents, technical assistances and friendship. I owe my graduate supervisory committee, Prof. Hong Wanjin and Dr. Cai Mingjie my sincere gratitude, for their constructive suggestions and critical comments. I also wish to express my thanks to Prof. Christopher J Leaver for sharing the Blue-Native PAGE protocol, which is a really useful and important method for me to study the GRIM-19 functions in this thesis. I am deeply grateful to Dr. Lim Cheh Peng for her critical comments on my thesis writing. Finally, I am forever indebted to my parents and my wife for their understanding, endless patience and encouragement when it was most required. ii LIST OF PUBLICATIONS Hao Lu, and Xinmin Cao. GRIM-19 is essential for maintenance of mitochondrial membrane potential. Mol, Biol, Cell. (Submitted to MBC after revision). Huang G, Lu H, Hao A, Ng DC, Ponniah S, Guo K, Lufei C, Zeng Q, Cao X. GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I. Mol. Cell. Biol. 2004. 24, 8447-8456. Huang G., Chen Y., Lu H., and Cao X. (2007). Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death. Cell Death Differ. 2007. 14, 327-337. Chen Y, Yuen W., Fu J., Huang G., Melendez A. J., Ibrahim F.B., Lu H., and Cao X. Mitochondrial respiratory chain controls intracellular calcium signaling and NFAT activity essential for heart formation in Xenopus. Mol. Cell. Biol. 2007. 27, 6420-6432. iii TABLE OF CONTENTS Acknowledgements……………………………………………………………………….II List of publications …………………………………………………………………… .III Table of contents…………………………………………………………………………IV Abbreviations…………………………………………………………………………….IX List of figures………………………………………………………………………… XIV List of tables…………………………………………………………………………….XV Summary……………………………………………………………………………….XVI Chapter Introduction…………………………………………………………… 1.1 Mitochondrion and mitochondrial respiratory chain………………………… .2 1.1.1 Mitochondrion………………………………………………………… .2 1.1.2 Mitochondrial structure………………………………………………….3 1.1.3 Mitochondrial oxidative phosphorylation……………………………….5 1.1.4 Mitochondrial respiratory chain and membrane potential………………6 1.1.5 Mitochondrial dysfunction and disease……………………………… 13 1.2 Mitochondrial Complex I………………………………………………… .18 1.2.1 The subunits of mitochondrial Complex I………………………… 18 1.2.2 The structure of mitochondrial Complex I…………………………… 20 1.2.3 the import of Complex I subunits…………………………………… .21 1.2.4 The assembly of mitochondrial ComplexI…………………………… 25 1.3 GRIM-19…………………………………………………………………… 30 iv 1.4 Objective and significance of this study…………………………………….31 Chapter Material and Methods……………………………………………… 34 2.1 Chemicals and reagents …………………………………………………… .35 2.2 Cells culture………………………………………………………………….35 2.3 Generation and culture of ρ0 cells………………………………………… .35 2.4 Plasmid constructions……………………………………………………… 36 2.5 Preparation of DH5α Escherichia coli competent cells…………………… .38 2.6 DNA transformation…………………………………………………………38 2.7 DNA transfection byLipofectamine 2000……………………………………39 2.8 QuikChange™ Site-Directed Mutagenesis………………………………… 39 2.9 Western blot analysis……………………………………………………… .40 2.10 Immunoprecipitation……………………………………………………… 40 2.11 Immunofluorescence ……………………………………………………….41 2.12 Generation of a mouse antibody against human GRIM-19 …………41 2.13 In vitro transcription and translation……………………………………… 42 2.14 Measurement of ∆ψm………………………………………………………42 2.15 Mitochondrial isolation…………………………………………………… 42 2.16 Cytochrome c release assay……………………………………………… .43 2.17 Blue-Native PAGE and in gel activity assay……………………………….43 2.18 Complex I spectrophotometric enzyme assay………………………………44 2.19 Apoptosis assay (sub-G1 assay)…………………………………………….44 2.20 Isolation and culture of blastocysts in vitro………………………….45 v 2.21 Preparation of RNA……………………………………………………… .45 Chapter GRIM-19 is a subunit of mitochondrial complex I………… .47 3.1 GRIM-19 interacts with various subunits of mitochondrial complex I……………………………………………………………………….48 3.2 GRIM-19 is a component of mitochondrial complex I……………….52 3.3 GRIM-19 is essential for mitochondrial complex I assembly and enzymatic activity…………………………………………………….52 Chapter GRIM-19 functional domains…………………………………… 56 4.1 Residues 20-30 and 40-60 Are Required for Mitochondrial Localization of GRIM-19…………………………………………………………………….57 4.2 Residues 134-144 Affects GRIM-19 Insertion to Complex I………………62 4.3 Aa 70-80 and 90-100 Are Required for Maintenance of ΔΨm…………….65 Chapter Dominant negative mutant of GRIM-19 impairs mitochondrial membrane potential…………………………….71 5.1 Generation of a Dominant Negative GRIM-19 Mutant Which Disrupts ΔΨm…………………………………………………………………… ….72 5.2 DN-GRIM-19 Compromises Complex I Activity without Affecting Its Assembly………………………………………………………………… .72 5.3 Loss of ΔΨm Caused by DN-GRIM-19 does not Induce Cytochrome c vi Release and Apoptosis………………………………………………… … 79 5.4 Loss of ΔΨm Caused by DN-GRIM-19 Sensitizes Cells to Undergo Apoptosis……………………………………………………………………82 Chapter Discussion .88 6.1 GRIM-19 Is Localized in Mitochondria…………………………………… .89 6.2 GRIM-19 Is a Critical Subunit of RC Complex I……………………… .90 6.2.1 GRIM-19 is essential for mitochondrial complex I assembly and activity……………………………………………………………… 90 6.2.2 GRIM-19 is a functional subunit in mitochondrial complex I… .90. 6.3 The Position of GRIM-19 in Mitochondrial Complex I………………………92 6.4 The Important Function of Accessory Subunits in Mitochondrial Complex I……………………………………………………………… 93 6.5 DN-GRIM-19 as a Novel Tool for Functional Study of ΔΨm in Apoptosis………………………………………………………………….…94 6.6 RC/Complex I Regulates Cell Death via Different Mechanisms…………….95 6.7 GRIM-19 could be a potential therapy candidate in diseases…………………96 6.7.1 GRIM-19 and embryonic development disorders……………………… 96 vii 6.7.2 GRIM-19 and cancer…………………………………………………….97 6.7.3 GRIM-19 and bacterium/virus infection…………………………… .…98 References .100 viii ABBREVIATIONS ∆ψm mitochondrial membrane potential ACP acyl-carrier protein ATP adenosine triphosphate Β-gal β-galactosidase BSA bovine serum albumin CIA complex I intermediate-associated Cyt cytochrome DMEM Dulbecco’s modified Eagle’s medium DMSO dimethyl sulfoxide DMF dimethylformamide DNA deoxyribonucleic acid DTT dithiothreitol ECL enhanced chemiluminescence EDTA ethylenediamine tetra-acetic acid ER endoplasmic reticulum ETC electron transport chain FAD flavin adenine nucleotide; oxidized state FADH2 flavin adenine nucleotide; reduced state FBS fetal bovine serum FMN Flavin MonoNucleotide FP flavoprotein ix GRIM-19 genes associated with retinoid IFN-induced Mortality-19 GST glutathione S-transferase HA hemagglutinin HEPES N-(2-hydroxyethyl) piperazine-N’-(2ethanesulfonic acid) HGNC HUGO Gene Nomenclature Committee HP hydrophobic protein Hsp60 heat shock protein 60 kDa IFN-β interferon- β IP iron-sulfur protein IPTG isopropylthio-β-D-galactoside MIB mitochondrial isolation buffer MIM mitochondrial inner membrane MOM mitochondrial outer membreane MRC mitochondrial respiratory chain mtRNA mitochondrial DNA NaCl sodium chloride NAD nicotinamide adenine dinucleotide; oxidized state NADH nicotinamide adenine dinucleotide; reduced state NDUFA NADH dehydrogenase (ubiquinone) alpha subcomplex NDUFS NADH dehydrogenase (ubiquinone) Fe-S protein x Hedderich , R. 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Acta.1241, 139-176. 122 123 [...]... (2) Mitochondrial intermembrane space The mitochondrial intermembrane space is the space between the outer membrane and the inner membrane It contains about 5% of mitochondrial proteins Some portion of cytochrome c (Cyt c), an extrinsic protein of the respiratory electron transport chain involved in transferring electrons from Complex III to Complex IV, is located here Most of the dehydrogenases of the. .. There are three major types of enzyme complexes in the mitochondrial inner membrane including all of the complexes of the electron transport system, the ATP synthetase complex, and transport proteins (Alberts et al., 199 4) (4) Mitochondrial cristae space Mitochondrial cristae are the internal compartments which are formed by the convoluted inner membrane Many types of cytochromes are localized in the. .. the mitochondrial critstae space (5) Matrix The majority of mitochondrial proteins are located in the mitochondrial matrix The matrix contains the enzymes responsible for the citric acid cycle reactions and fatty acid oxidation (Alberts et al., 199 4) In addition, the matrix also contains the ribosomes and other enzyme systems responsible for the synthesis of mitochondrial DNA (mtDNA), RNA and proteins... as: succinate + Q fumarate + QH2 Complex II is the simplest complex in RC, containing only 4 nuclearencoded subunits (Cecchini, 2003) The two largest subunits form the peripheral arm of Complex II which acts as the succinate dehydrogenase in the citric acid cycle The remaining two subunits (anchor proteins) are integrated into the mitochondrial inner membrane (Horsefield et al., 2004) The peripheral... al., 199 6: Green et al., 199 8 and Zoratti et al., 199 5) Subsequently, the mitochondrial membrane potential will collapse and the inner membrane will swell All of these will finally lead to the release of cell death-promoting factors into the cytosol The release of cytochrome c will activate the cytosolic caspase cascade, which will finally destroy the cellular structures The release of AIF from mitochondria. .. electrons to ubiquinone which then pass the electrons to Complex III Ubiquinone is the only non-protein electron carrier of the mitochondrial respiratory chain Its highly hydrophobic property makes ubiquione dissolved only within the membrane The quinine ring of ubiquinone accepts 2 9 electrons and is reduced to ubiquinol (QH2) Ubiquinone can also accept a single electron to generate ubisemiquinone radicals... and the last 10 amino acid at C-terminus enhanced the assembly ability of GRIM- 19 to xvi complex I Based on the domain-mapping information, a dominant-negative GRIM- 19, which can specifically decrease mitochondrial membrane potential, was generated In summary, I have demonstrated that GRIM- 19 was a crucial subunit in mitochondrial complex I xvii xviii Chapter 1 Introduction 1 1.1 Mitochondrion and mitochondrial... (mtDNA), RNA and proteins Because of the folds of the cristae, no part of the matrix is far from the inner membrane Dissolved oxygen, water, carbon dioxide and the recyclable intermediates can diffuse into the matrix rapidly 1.1.3 Mitochondrial oxidative phosphorylation 5 Why is the mitochondrion the “powerhouse” of the cell? This is because of the special capability of mitochondria to carry out oxidative... retinoic acid (RA) induced apoptosis and was also reported to copurify with mitochondrial complex I However, the relationship between GRIM- 19 and complex I was not clear This study clearly demonstrates that GRIM- 19 is a subunit of mitochondrial complex I by showing: (1) GRIM- 19 interacted with various mitochondrial complex I subunits and was physically present in mitochondrial complex I (2) In GRIM1 9... as exercise intolerance Medically, Complex I is also important in a lot of aging related diseases For example, the mitochondrial complex I has reduced activity in Parkinson’s disease Moreover, the mitochondrial complex I is a major source of reactive oxygen species in cells, one of the major causes of aging (Turrens, 199 7; St-Pierre et al., 2002 and Li et al., 2003) Therefore the study of complex I . THE STUDY OF GRIM- 19 FUNCTION IN MITOCHONDRIA Lu Hao A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE INSTITUTE OF MOLECULAR AND CELL. sequences were defined at the N-terminus, the electron transfer activity domain was found in the middle and the last 10 amino acid at C-terminus enhanced the assembly ability of GRIM- 19 to xvi complex. functional subunit in mitochondrial complex I… 90. 6.3 The Position of GRIM- 19 in Mitochondrial Complex I………………………92 6.4 The Important Function of Accessory Subunits in Mitochondrial Complex