Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

Les protéines à cuivre jouent un rôle crucial dans de nombreux processus biologiques essentiels à la vie tels que la respiration. De nombreuses études ont été menées afin de décrypter le lien entre la structure de leur centre actif, les propriétés électroniques qui en découlent et la fonction de ces protéines.Les travaux réalisés au sein du laboratoire sur l’étude de la chaîne respiratoire d’un organisme acidophile, A. ferrooxidans, ont permis de mettre en évidence une protéine à cuivre (AcoP), appartement à la vaste famille des cuprédoxines, indispensable au fonctionnement de cette voie. Une approche pluridisciplinaire mêlant des méthodes de spectroscopies, d’électrochimie, de cristallographie aux rayons X combinée à des expériences de mutagénèse dirigée, a permis de dévoiler la présence d’un centre cuivre atypique associé à des propriétés électroniques et d’oxydoréduction rarement retrouvées au sein de cette vaste famille. Le rôle d’une telle protéine au sein de la chaîne respiratoire d’A. ferroxidans a par la suite fait l’objet de notre attention. AcoP interagit avec le cytochrome c et l’enzyme terminale de la chaîne respiratoire, la cytochrome c oxydase. L’étude du complexe cytochrome c – AcoPcytochrome c oxydase nous a permis de proposer un rôle d’AcoP dans le recrutement du cytochrome c au sein de ce complexe, ainsi que dans le transfert d’électron entre ces deux partenaires. Ces travaux de recherche démontrent que l’étude de la biodiversité permet non seulement la découverte de nouveaux systèmes permettant la vie dans des environnements extrêmes, mais également la découverte de nouvelles protéines aux propriétés remarquables. / Copper proteins play key roles in many biological processes, such as in respiratory chains. Although many studies have been carried out to decipher the relationship between their active site structure, electronic properties and function, these features are still not fully understood. Previous studies on the respiratory chain of an acidophilic organism, Acidithiobacillus ferrooxidans, have revealed the presence of a new copper-binding protein: AcoP. This cupredoxin is critical for the correct functioning of this respiratory pathway. Using site-directed mutagenesis and a wide-range of biophysical approaches, electrochemistry and X-ray crystallography, we can show that an unconventional copper-active site in AcoP might underlie its rare electronic and redox features. The function of such a protein in the respiratory chain of A. ferrooxidans has subsequently raised our curiosity. It was shown that AcoP interacts with cytochrome c and the cytochrome c oxidase. We showed that AcoP could act as a linker between the cytochrome c and the cytochrome c oxidase, by recruiting the former, and could also participate in the electron transfer between these two partners. This work shows how exploring biodiversity leads to the discovery of new systems that allow life in extreme environments, as well as of new proteins with remarkable features.

Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function. So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival. In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.:List of Figures and Tables Abbreviations Abstract 1 Indroduction 1.1 Mitochondria and the respriratory chain 1.2 The human mitochondrial genome 1.3 Homoplasmy and heteroplasmy 1.4 Mitochondrial disorders 1.4.1 Mutations in mitochondrial DNA 1.4.2 Mutations in nuclear DNA 1.5 Cytochrome c oxidase 1.6 Cytochrome c oxidase assembly 1.7 Copper and its trafficking in the cell 1.8 Mitochondrial copper metabolism 1.9 Cox17 1.10 Aims of the thesis 2 Materials and Methods 2.1 Materials 2.1.1 Chemicals and reagents 2.1.2 Antibodies 2.1.3 Plasmid 2.1.4 Kits 2.1.5 Marker 2.1.6 Enzymes 2.1.7 Primers 2.1.8 siRNAs 2.2 Methods 2.2.1 Cell culture 2.2.1.1 Cell culture: HeLa cells 2.2.1.2 Cell culture: HeLa cells transfected with pTurboRFP-mito 2.2.1.3 Subcultivation 2.2.1.4 Determination of cell number 2.2.1.5 Cell storage and thawing 2.2.2 Transient transfection of HeLa cells 2.2.3 Transfection of HeLa cells with pTurboRFP-mito 2.2.4 Immunocytochemistry 2.2.5 RNA extraction and quantitative real-time PCR 2.2.6 Isolation of mitochondria 2.2.6.1 Isolation of mitochondria for BN-PAGE Analysis 2.2.6.2 Isolation of mitochondria for localization studies 2.2.6.3 Isolation of bovine heart mitochondria 2.2.7 Proteinase K treatment of mitochondria and mitoplasts 2.2.8 Photometric activity assay 2.2.8.1 Citrate synthase activity 2.2.8.2 Cytochrome c oxidase activity 2.2.9 Blue native polyacrylamide gel electrophoresis (BN-PAGE) 2.2.9.1 In gel activity assay 2.2.9.2 2D-BN/SDS-PAGE 2.2.10 SDS-PAGE and Western blot analysis 2.2.11 Direct stochastic optical reconstruction microscopy (dSTORM) 2.2.12 Flow cytometric phenotyping 2.2.12.1 Determination of cell cyle phase 2.2.12.2 Identification of apoptotic cells 2.2.12.3 Detection of ROS 2.2.13 Oxygen measurement 2.2.14 Cu–His supplementation 3 Results 3.1 Subcellular localization of Cox17 3.2 Transient knockdown of COX17 in HeLa cells 3.2.1 Knockdown of COX17 mRNA 3.2.2 Knockdown of Cox17 protein 3.2.3 Effect of COX17 knockdown on the steady-state levels of OXPHOS subunits 3.2.4 Effect of COX17 knockdown on the steady-state levels of copperbearing COX subunits 3.2.5 Subdiffraction-resolution fluorescence imaging 3.3 Phenotypical characterization 3.3.1 Growth analyis 3.3.2 Cell cycle analysis 3.3.3 Apoptosis assay 3.3.4 Detection of ROS 3.3.5 Oxygen measurement 3.4 Cytochrome c oxidase activity 3.5 Characterization of mt OXPHOS complexes 3.5.1 BN-PAGE/in gel activity assays 3.5.2 Supramolecular organization of COX 3.5.3 Molecular organization of Cox17 3.5.4 Molecular organisation of copper-bearing COX subunits Cox1 and Cox2 3.5.5 Supramolecular organization of RC complexes 3.5.6 dSTORM of supercomplexes 3.6 Copper supplementation 4 Discussion 4.1 Dual localization of human Cox17 4.2 COX17 knockdown affects steady-state levels of copper-bearing COX subunits Cox1 and Cox2 4.3 Supramolecular organization of RC is affected as an early response to COX17 knockdown 4.4 Cox17 is primarily engaged in copper delivery to Sco1/Sco2 4.5 Copper supplementation alone cannot rescue the COX17 phenotype 4.6 Outlook 5 Appendix 6 PhD publication record 7 References

Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...

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Maintaining energy organismal homeostasis under changing physiological and environmental conditions is vital, and requires constant adjustments of the energy metabolism. Central to meeting energy demands is the regulation of mitochondrial oxidative capacity. When demands increase, animals can increase mitochondrial content/enzymes, known as mitochondrial biogenesis. Central to mammalian mitochondrial biogenesis is the transcriptional master regulator PPARγ (peroxisome proliferator-activated receptor γ) coactivator-1α (PGC-1α), and the network of DNA-binding proteins it coactivates (e.g. nuclear respiratory factor 1 and 2 [NRF-1, NRF-2], estrogen-related receptor α [ERRα], thyroid receptor α [TRα-1], retinoid X receptor α [RXRα]). However, the mechanisms by which mitochondrial content in lower vertebrates such as fish is controlled are less studied. In my study I investigate underlying mechanisms of the phenomenon that many fish species alter mitochondrial enzyme activities, such as cytochrome c oxidase (COX) in response to low temperatures. In particular, I investigated (i) if the phenomenon of mitochondrial biogenesis during cold-acclimation is related to fish phylogeny, (ii) what role PGC-1α and other transcription factors play in mitochondrial biogenesis in fish, and (iii) if mRNA decay rates are important in the transcriptional control of a multimeric protein like COX. This study shows that mitochondrial biogenesis does not follow a phylogenetic pattern: while distantly related species displayed the same response to low temperatures, closely related species showed opposite responses. In species exhibiting mitochondrial biogenesis, little evidence was found for PGC-1α as a master regulator, whereas NRF-1 is supported to be an important regulator in mitochondrial biogenesis in fish. Further, there was little support for other transcription factors (NRF-2, ERRα, TRα-1, RXRα) to be part of the regulatory network. Lastly, results on the post-transcriptional control mechanism of mRNA decay indicate that this mechanism is important in the regulation of COX under mitochondrial biogenesis: it accounts for up to 30% of the change in subunit transcript levels. In summary, there is no simple temperature-dependent mitochondrial response ubiquitous in fish. Further, the pathways controlling mitochondrial content in fish differ from mammals in the important master regulator PGC-1α, however, NRF-1 is important in regulating cold-induced mitochondrial biogenesis in fish. Lastly, COX subunit mRNA decay rates seem to have a part in controlling COX amounts during mitochondrial biogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-10-21 09:53:59.46

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Cardiac injury is increased following ischemia-reperfusion. Mitochondria are the “effector organelles” that are damaged during ischemia (ISC) when there is no blood flow. Resumption of metabolism by damaged mitochondria during reperfusion (REP) results in increased cell injury. Current therapeutic interventions to pre-condition and post-condition the heart during ISC are ineffective during certain conditions like aging and diabetes due to defects in the signaling cascades. In contrast, mitochondrial-based strategies are effective in protecting the heart during ISC-REP. Hence direct therapeutic targeting of dysfunctional mitochondria will provide the potential to bypass the upstream signaling defects and intervene directly upon the effector organelle. Novel mitochondrial-targeted therapy relies on understanding the sites in the electron transport chain (ETC) that are damaged by ISC and produce cell-injury during REP. This project identifies a novel pathological role of cytochrome c in depleting cardiolipin during ischemia after which the mitochondria are in a defective condition that leads to additional cell death during reperfusion. During ischemia oxidants from complex III oxidize cytochrome c, forming a peroxidase, which causes oxidative damage and depletion of cardiolipin. Depletion of cardiolipin disrupts normal physiology and augments cell death. Identification of the innovative pathobiology during ISC-REP recognizes a novel therapeutic target, cytochrome c peroxidase, which can be a focal point for new therapeutic interventions to decrease cardiac injury. In order to maintain homeostatis, living organisms have the methionine sulfoxide reductase system, which reduce both free and protein bound Met(O) back to methionine (Met) in the presence of thioredoxin. Oxidized Trx is inactive and unable to bind to ASK1 thereby activating ASK1 and causing cell death via p38/JNK pathways thereby contributing to the pathogenesis of myocardial ISC-REP injury. In this study we have shown that inhibition of ASK1 protects the heart during REP via the modulation of mitochondria that sustained damage during ISC. The mitochondrial-based mechanism of cardioprotection with ASK1 inhibition enhanced the functional integrity of the inner mitochondrial membrane retaining cytochrome c thereby decreasing cell death. This therapeutic intervention is a key step to achieve the ultimate goal to improve clinical outcomes in patients that suffer an acute myocardial infarction.

AAbbssttrraacctt:: The mammalian organism is entirely dependent on ATP production by oxidative phosphorylation system (OXPHOS) on the inner mitochondrial membrane. OXPHOS is composed of respiratory chain complexes I-IV, ATP synthase and also include two electron transporters cytochrome c and coenzyme Q. Disorders of mitochondrial energy metabolism caused by OXPHOS defects are characterized by extreme heterogeneity of clinical symptoms, variability of tissues affected and the severity of the defect at the level of individual tissues. The mitochondrial disorders are not always clearly expressed at the level of available tissue or most easily available cultured fibroblasts and/or currently available methods are not capable to detect the defects on the fibroblasts level. The aim of this master thesis was to identify by biochemical methods, especially by high sensitive polarography, OXPHOS disorders in cultured fibroblasts. Cell lines from 10 patients with isolated (SURF21, SCO1 ND1, ND5) or combined defects of OXPHOS complexes whose biochemical defect was confirmed in muscle tissue as well as 14 patients with non- mitochondrial diseases (8 patients with Huntington disease, 6 patients with disorder of sulphur amino acids metabolism) were analysed. Furthermore impact of various cultivation conditions on OXPHOS...