Protein phosphorylation is one of the major post-translational modifications to allow for signal transmission and fine tuning of metabolism on the cellular proteomic level. As such it is “one of the last instances” to modulate the activity of enzymes and hence to impact the cellular life irrespective of the basic conditions provided by the genome – and epigenome– controlled gene expression. The evolutionary increase in cellular complexity is reflected by highly sophisticated regulatory networks in multicellular eukaryotes based on the transfer of phosphate mostly onto the side chains of serine, threonine and tyrosine residues. Nature has chosen phosphate for inter- and intracellular communication, which is also an integral component of nucleic acids and can be regarded as the molecule of choice for life. Currently, life science is interested to unravel the network of reversible protein phosphorylation that is catalyzed by two antagonistic enzyme classes: the protein kinases and protein phosphatases. We are currently in the era of proteomics and enormously benefit from the progress of mass-spectrometry methods. This is documented by a huge number of “proteomic studies” that mostly provide a simple inventory of the existence of proteins – and/or their phosphorylated forms – under more or less defined conditions. So far, the physiological correlations could be established only in a few cases, e.g. by comparing two physiological conditions. Another strategy, which was addressed in this work, is the systematic screening of mutants defective in genes encoding either protein kinases or protein phosphatases. This approach benefits from the ease to predict these enzymes due to the presence of characteristic protein motifs. In combination with the major goal of this work – to shed light on the impact of protein phosphorylation in the mitochondrial (mt) compartment – the yeast Saccharomyces cerevisiae was chosen as a model system because of its respiro-fermentative metabolism, that allows for the maintenance of respiratory defective mutants. Indeed, this reverse genetic approach successfully revealed two kinases (Pkp1p, Pkp2p) and two phosphatases (Ppp1p, Ppp2p) as the key components regulating the pyruvate dehydrogenase complex by phosphorylation of serine 313 of its α- subunit Pda1p. In addition, evidence is provided that Pkp1p has an additional role in the assembly process of the PDH complex. Also, the effect of the deletion of the COQ8 gene (gene engaged in coenzyme Q synthesis; originally named ABC1) leading to respiratory deficiency, could be correlated with the phosphorylation of subunit Coq3p of the mitochondrial ubiquinone biosynthesis complex. Finally, in the case of the kinase Sat4p (protein involved in salt tolerance), overexpression of the enzyme was used as an alternative approach to unravel the molecular basis of the originally observed salt sensitivity of sat4 mutants. The data suggest that Sat4p has a direct or indirect role in the late steps of iron-sulfur (Fe/S) cluster assembly of the so-called “aconitase-type” enzymes in mitochondria, accompanied by a strongly reduced steady state concentration of the Fe/S-cluster protein aconitase. Interestingly, a secondary phenotype became apparent upon overexpression of Sat4p: the abundance of the lipoic acid containing mitochondrial proteome was markedly reduced. Most likely this phenotype is due to the fact that the synthesis and/or attachment of lipoic acid depend on a Fe/S-cluster bearing enzyme. In the course of the work it became clear that the regulatory (mt) protein phosphorylation network of yeast evolved to meet the criteria of a life adapted to the ecological niche on temporarily available sugar rich sources. Clearly, the transfer of the respective data to higher eukaryotes is limited. However, it shows that yeast is primarily an excellent model system for the principal molecular reactions shared with higher eukaryotes.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 82 / Phosphorylierungen von Aminosäuren ist eine der verbreitetsten post-translationalen Modifikationen für zelluläre Signalübertragungswege und zur Regulation des Metabolismus auf Proteom-Ebene. Mit der reversiblen Protein-Phosphorylierung eng verbunden ist die unabhängige Modulation der Aktivität von Enzymen ungeachtet der Genom- und Epigenom-basierten Genexpression. Die evolutionäre Zunahme der zellularen Komplexität äußert sich in zunehmend komplexeren Regulations-Netzwerken in mehrzelligen eukaryotischen Organismen basierend auf dem Transfer von Phosphatgruppen vorzugsweise auf die Aminosäuren Serin, Threonin und Tyrosin. Die Natur hat evolutionär als Baustein der inter- und intrazellulären Kommunikation Phosphat gewählt, welches auch ein integraler Bestandteil der Nukleinsäuren ist und somit als das „Molekül der Wahl“ für das Leben bezeichnet werden darf. Die Lebenswissenschaften sind gegenwärtig daran interessiert das Netzwerk der Proteinphosphorylierung aufzuklären, welches durch zwei antagonistisch wirkende Enzymklassen, die Proteinkinasen und Proteinphosphatasen charakterisiert ist. Dabei profitieren wir gegenwärtig von den Fortschritten der „Proteomics-Ära“ auf dem Gebiet der massenspektrometrischen Proteinidentifizierung. Ausdruck dessen ist eine Vielzahl von Proteom-Studien, die jedoch meist nur eine einfache Inventarisierung der unter mehr oder weniger gut definierten zellulären Bedingungen existierenden Proteine in ihrer Phosphat-modifizierten oder unphosphorylierten Form darstellen. Die beteiligten Enzyme werden dabei kaum berücksichtigt. Insbesondere gilt dies für extra-cytoplasmatische Ereignisse. Bisher gelang es nur in wenigen Fällen eine Korrelation der physiologischen Rolle dieser Proteinmodifikation, z.B. durch den Vergleich der Phospho-Proteome unter zwei unterschiedlichen physiologischen Bedingungen, herzustellen. Eine andere Strategie, die auch Gegenstand dieser Arbeit ist, sieht ein Screening von Mutanten vor, die durch Deletionen von Genen, die für Proteinkinasen bzw. –phosphatasen kodieren, gekennzeichnet sind. Dieser Ansatz profitiert von der Existenz und leichten bioinformatischen Vorhersagbarkeit charakteristischer Kinase- bzw. Phosphatase- Sequenzmotive. In Kombination mit dem Hauptziel der Arbeit – Licht ins Dunkel der Proteinphosphorylierung im mitochondrialen Kompartiment zu bringen – wurde die Hefe Saccharomyces cerevisiae als Modellsystem gewählt, insbesondere vor dem Hintergrund ihres fermentativen Metabolismus. Als Beleg der prinzipiellen Funktionalität des vorgeschlagenen Ansatzes konnten zwei Kinasen (Pkp1p, Pkp2p) und zwei Phosphatasen (Ppp1p, Ppp2p) als Schlüsselkomponenten der Regulation des Pyruvatdehydrogenase (PDH) Komplexes identifiziert und charakterisiert werden. Darüber hinaus konnte sowohl das Zielprotein der Phosphorylierung, Pda1p, die α-Untereinheit des Komplexes, als auch die modifizierte Aminosäure (Serin 313) experimentell bestätigt werden. Ferner konnte der Atmungsdefekt von Stämmen mit einer nicht-funktionellen Abc1p-Kinase mit dem Phosphorylierungszustand der Untereinheit Coq3p des Ubiquinon-Biosynthese Komplexes und dem Ausfall der Ubiquinonsynthese korreliert werden. Eine alternative Herangehensweise, die Überexpression einer Kinase, führte zur Identifizierung möglicher Zielproteine von Sat4p. Vergleichende Analysen des 2D-gelelektrophoretisch separierten mitochondrialen Genoms mit dem des Wildtyps legen die Vermutung nahe, dass Sat4p eine direkte oder indirekte Rolle bei der Regulation der „Aconitase-Typ“ Eisen-Schwefel (Fe/S) Proteine besitzt. Der darüber hinaus beobachtete Effekt einer Abnahme von Liponsäure-tragenden mitochondrialen Enzymen, ist wahrscheinlich sekundärer Natur und kann durch die Zugehörigkeit der Liponsäure-Synthase zur oben erwähnten Gruppe der „Aconitase-Typ“ -Fe/S-Proteine erklärt werden. Im Verlauf der Arbeit wurde deutlich, dass das regulatorische Netzwerk der Proteinphosphorylierung der Hefe eher den Kriterien einer evolutionären Adaptation an eine spezifische ökologische Nische – der temporären Verfügbarkeit zuckerreicher Substanzen – entsprechen. Das schränkt die Übertragbarkeit der gewonnen Einsichten in die Regulation des mitochondrialen Metabolismus auf höhere Eukaryonten ein. Es zeigt jedoch, dass Hefe in erster Linie ein exzellentes Modellsystem für die prinzipiellen molekulare Mechanismen ist, die sie mit den höheren Eukaryonten teilt.:1 SUMMARY 1 ZUSAMMENFASSUNG 3 2 INTRODUCTION 5 2.1 Why phosphate? 5 2.2 Protein phosphorylation in prokaryotes 6 2.3 Protein phosphorylation in mitochondria 7 2.4 Regulation of mammalian pyruvate dehydrogenase complex (PDH) by phosphorylation 9 2.5 Mammalian cytochrome c oxidase (COX) 10 2.6 Protein phosphorylation in yeast mitochondria 11 3 AIM OF THIS WORK 13 4 PROLOG 14 4.1 Critical evaluation of tools for phosphoproteomics 14 4.2 Introducing a new method for in gel profiling of phospho-proteins 17 4.3 In-gel screening of phosphorus of yeast mitochondrial proteins by LA-ICP-MS 20 4.4 Detection of phosphorylated subunits of ATPase 22 5 RESULTS 23 5.1 YIL042c and YOR090c encode the kinase and phosphatase of the Saccharomyces cerevisiae pyruvate dehydrogenase complex 28 5.2 Yeast Pyruvate Dehydrogenase Complex Is Regulated by a Concerted Activity of Two Kinases and Two Phosphatases 29 5.3 Proteomic analysis reveals a novel function of the kinase Sat4p in yeast mitochondria 30 5.4 Ubiquinone biosynthesis in Saccharomyces cerevisiae: the molecular organization of O –methylase Coq3p depends on Abc1p/Coq8p 53 6 DISCUSSION AND PERSPECTIVES 54 6.1 Mitochondrial phosphorylation in yeast 54 6.1.1 An evolutionary view 54 6.1.2 The yeast ABC1-kinase family 55 6.1.3 Regulation of yeast pyruvate dehydrogenase (PDH) complex 57 6.1.4 Regulation of iron sulfur cluster biogenesis 60 6.2 Challenges to investigate the network of (mt) protein phosphorylation 62 6.2.1 When is a kinase a mitochondrial kinase? 64 6.2.2 Epilog 66 7 LITERATURE 69 8 APPENDIX 78 8.1 Related publications 78 8.2 List of publications 80 8.3 ERKLÄRUNGEN 82

No description available.

Menschliche Mitochondrien enthalten etwa 1500 bis 2000 Proteine. Die meisten dieser Proteine werden im Zellkern kodiert und im Zytoplasma synthetisiert, und müssen daher über eine spezielle Maschinerie in die Mitochondrien transportiert werden. Obwohl mittlerweile viele Details über die Wirkungsweise dieser Proteinschleusen bekannt sind, wurden einige wichtige Aspekte des Proteinimports noch nicht ausreichend untersucht. Zum einen ist nicht bekannt, ob die einzelnen Importkomplexe einen Einfluss auf die mitochondrienvermittelte Apoptose haben. Weiterhin ist offen, welche genaue Rolle der Mitochondrienimport in der Pathogenese von Neisseria gonorrhoeae spielt. Außerdem ist unklar, ob Faktoren des Importapparates für die Aufrechterhaltung der mitochondrialen Morphologie notwendig sind. Um diese Fragestellungen zu untersuchen, wurden im Rahmen der vorliegenden Arbeit permanente Zelllinien hergestellt, in denen die Expression einzelner am Mitochondrienimport beteiligter Proteine mittels RNA-Interferenz (RNAi) inhibiert werden kann. Mithilfe dieser Zelllinien wurde getestet, ob die proapoptotischen Proteine Bax und Bak die Importmaschinerie benötigen, um in die äußere Mitochondrienmembran zu gelangen. Die Präsenz der beiden proapoptotischen Proteine in Mitochondrien während der Apoptose ist sehr entscheidend, da Bax und Bak in den Mitochondrien oligomerisieren und damit weitere Schritte der Apoptose einleiten. Im Widerspruch zu früheren Publikationen konnte hier gezeigt werden, dass die Translokation von Bax und Bak in die äußere Mitochondrienmembran unabhängig von Proteinimportfaktoren erfolgt. Der zweite Teil dieser Arbeit beschäftigt sich mit dem Einfluss mitochondrialer Importproteine auf die Pathogenese von Neisseria gonorrhoeae. Das Neisserienprotein PorB transloziert während der Infektion in die Mitochondrien der Wirtszelle und induziert Apoptose. Aufgrund der strukturellen Ähnlichkeit von PorB zu bestimmten Proteinen der äußeren Mitochondrienmembran wurde bisher angenommen, dass PorB diesen endogenen Proteinen auf ihrem Importweg in die äußere Mitochondrienmembran folgt. Überraschenderweise wurde im Rahmen dieser Arbeit entdeckt, dass PorB nicht von allen Komplexen der Importmaschinerie in den Mitochondrien erkannt wird. Infolgedessen transloziert es in die innere Mitochondrienmembran und wirkt dadurch toxisch auf die Wirtszelle. In einem weiteren Projekt wurde untersucht, welche Rolle die Proteinimportkomplexe der äußeren mitochondrialen Membran in der Aufrechterhaltung der Mitochondrienmorphologie spielen. Unter Verwendung der beschriebenen Zelllinien wurde entdeckt, dass in Abwesenheit des SAM (sorting and assembly) Importkomplexes die Struktur der inneren Mitochondrienmembran derangiert ist. Es wurden zudem Hinweise darauf gefunden, dass die Ursache für diesen Befund in einer Unterbrechung von Kontaktstellen zwischen den beiden Mitochondrienmembranen liegen könnte, für deren Aufrechterhaltung möglicherweise der SAM-Komplex verantwortlich ist. Die in dieser Arbeit vorgestellten Ergebnisse erlauben neue Einblicke in verschiedene Aspekte des Proteinimports in Mitochondrien. Zudem wurde mit der Entwicklung der stabilen Zelllinien ein neues Model geschaffen, anhand dessen in Zukunft weitere Detail des mitochondrialen Proteinimports erforscht werden können. / Human mitochondria comprise about 1500 to 2000 proteins. While only 13 proteins are encoded by the mitochondrial DNA the vast majority of mitochondrial proteins is encoded in the nucleus, synthesized in the cytosol, and translocated into mitochondria by a special protein import machinery. Although many details are now known about its function several important aspects of protein import in mitochondria were not unraveled yet. To begin with, the influence of the different mitochondrial import complexes on apoptosis is not known. Further, the exact role of the protein import machineries in mitochondria in the pathogenesis of Neisseria gonorrhoeae has not been clarified yet. Moreover, the question whether factors involved in protein import are required for the maintenance of the mitochondrial morphology is still unsolved. In order to address these open issues, permanent cell lines were generated within the frame of the present thesis in which the expression of single proteins implicated in mitochondrial import can be inhibited via RNA interference (RNAi). Using these cell lines, it was investigated whether the proapoptotic proteins Bax and Bak require the import machinery in order to gain access to the outer mitochondrial membrane. The presence of both proapoptotic proteins in mitochondria is essential during apoptosis as Bax and Bak oligomerize in the outer mitochondrial membrane leading to the execution of apoptosis. In contrast to earlier publications, results presented here prove that the translocation of Bax and Bak into the outer mitochondrial membrane occurs independent of its import machineries. The second part of this thesis explores the influence of mitochondrial import proteins on the pathogenesis of Neisseria gonorrhoeae. The neisserial protein PorB translocates into the mitochondria of host cells during infection and induces apoptosis. Because of structural similarities of PorB to a certain class of proteins in the outer mitochondrial membrane, it was assumed that PorB would follow the import pathway of these endogenous proteins into the outer mitochondrial membrane. Surprisingly, it was found within the present study that PorB is not recognized by all complexes implicated in this import pathway. As a consequence, it translocates into the inner mitochondrial membrane to exert its toxic effect on the host cell. In a further project, the role of import complexes of the outer mitochondrial membrane in the maintenance of the mitochondrial morphology was investigated. Using the described cell lines, it was found that in the absence of the SAM (sorting and assembly) import device, the structure of the inner mitochondrial membrane was disrupted. Further, evidence was found that the reason for this phenotype could be an interruption of contact sites between the two mitochondrial membranes, whose preservation possibly requires the SAM complex. The results presented here allow new insights into different aspects of mitochondrial protein import. Further, with the development of the stable cell lines a new model was generated that will allow future investigations on details about mitochondrial protein import.

Aufgrund der vielfältigen metabolischen Prozesse und Funktionen von Mitochondrien finden durch beide mitochondriale Membranen zahlreiche Transportprozesse statt. Es wird weitgehend angenommen, dass der Transfer von metabolischen Intermediaten durch die äußere Membran von den zahlreichen Porinporen gewährleistet wird. Im Gegensatz dazu sind in der inneren Membran spezifische Transportproteine für die Translokationsprozesse verantwortlich. Neben dem gut untersuchten Porinmolekül (Por1p) gibt es in der Hefe S. cerevisiae unter respiratorischen Bedingungen zwei weitere abundante, aber funktionell unbekannte Proteine in der äußeren Membran von Mitochondrien - Om14p und Om45p -, deren molekular-biologische Charakterisierung Gegenstand dieser Arbeit war. Mit drei unabhängigen Methoden (2D BN - SDS-PAGE, Co-IP und TAP) konnte gezeigt werden, dass die beiden Proteine Om14p und Om45p zusammen mit Por1p einen Proteinkomplex in der äußeren Membran ausbilden, wobei Por1p eine von Om14p und Om45p unabhängige Porenstruktur ausbildet. Bei Bedarf, möglicherweise über Phosphorylierungen signalisiert, binden Om14p und Om45p an diese Struktur, wobei Om45p dabei der direkten Interaktion von Om14p mit Por1p bedarf. Die Identifikation von Interaktionspartnern des Fusionsproteins Om14p-TAP durch Einsatz einer präparativen TAP mit anschließender massenspektrometrischer Analyse sowie die Untersuchungen der Effekte von OM14- und/oder OM45- Gendeletionen auf das mitochondriale Proteom mit einem 2D DIGE-Verfahren führten zur Aufstellung von funktionalen Zusammenhängen des Proteinpaares Om14p/Om45p. Mit Wachstumsuntersuchungen von Deletionsmutanten in Gegenwart von in den Mitochondrien toxisch wirkenden Substanzen sowie durch ein in dieser Arbeit entwickeltes Testverfahren zur Bestimmung des mitochondrialen ATP-Flusses, konnten die funktionalen Hypothesen für die Proteine Om14p und Om45p initial verifiziert werden. Zusammengefasst unterstützen die Daten dieser Arbeit die Idee von einem hochgradig flexiblen System der Mitochondrien, zur Gewährleistung von effizienten Transportvorgängen durch beide Membranen. Eine koordinierte Bindung der Porinpore an die spezifischen Transporter der inneren Membran wird wahrscheinlich durch die Aktivität des Proteinpaares Om14p/Om45p vermittelt. In diesem Zusammenhang könnten beide Proteine als eine Art Lizenzierungsfaktor fungieren und die Positionierung der Porinpore an die entsprechenden Proteine der inneren Membran erzeugen. Dadurch würde ein effektives System für den Austausch von metabolischen Intermediaten und Substraten der mitochondrialen Atmungskette entstehen. Ebenfalls durch diese Arbeit nicht auszuschließen ist die Vorstellung, dass die Proteine Om14p und Om45p einen Einfluss auf die spezifischen Transportproteine der inneren Membran oder die Porinpore der äußeren Membran ausüben. Phosphatrest-Übertragungen, die zu Konformationsänderungen oder Porenöffnungen führen könnten, sind beispielsweise vorstellbar. Die Stoffwechseladaption einer Zelle bei einem diauxic shift ist durch einen verstärkten mitochondrialen Import von Metaboliten, Co-Faktoren und Proteinen sowie häufigerer mitochondrialer Teilungsprozesse charakterisiert. Om14p und Om45p sind bei einem Wechsel zu nicht-fermentativen Bedingungen verstärkt präsent. Diese beiden Proteine könnten der Hefe einen entscheidenden Vorteil bei der Synchronisierung der genannten Prozesse liefern, indem sie eine verbesserte Erreichbarkeit bzw. eine Veränderung der Selektivität von bereitgestellten Kanälen bzw. Transportproteinen in beiden mitochondrialen Membranen bewirken.

COX deficiency in human presents a plethora of phenotypes which is not surprising given the complexity of the enzyme structure and the multiple factors and many steps required for its assembly. A functional COX requires three mitochondrially encoded subunits (Cox1p, Cox2p and Cox3p), at least 10 nuclearly encoded subunits, some of which are tissue specific, and a yet unknown number of assembly factors. Mutations in four of these factors, hSco1p, hSco2p, hCox10p and hSurf1p, have been associated with lethal COX deficiency in patients. Sco proteins, conserved from prokaryotes to eukaryotes, are probably involved in the insertion of copper in COX. The role of hSco1p and hSco2p in this process was investigated in this work. Moreover the importance of some hSco mutations found in patients was analysed. Both in vitro and in vivo analyses show that the hSco proteins are localised in the mitochondria. Both proteins are per se unable to substitute for ySco1p. However, a chimeric construct consisting of the N-terminal portion, the TM and a part of the C-terminal portion of ySco1p and the remaining C-terminal part derived from hSco1p was able to complement a ysco1 null mutant strain. This construct was used to define the role of a point mutation (P174L) found in the hSCO1 gene of infants suffering from ketoacidotic coma. These mutation was shown to affect the COX activity and the levels of Cox1p and Cox2p. The fact that copper was able to suppress this mutation, strongly outlined the importance of Sco proteins in the copper insertion in COX. The C-terminal portions of recombinant hSco1p and hSco2p were purified from E. coli by affinity chromatography. The purified proteins were subjected to atomic emission and absorption analyses and were shown to specifically bind copper. A stoichiometry of 1:1 for hSco2p and of 0,6:1 for hSco1p was determined. To identify the Aa residues involved in copper binding, in vitro mutagenesis was performed. hSco1p and hSco2p, lacking the cysteines of the predicted metal binding site CxxxC, show a dramatic decrease in the ability to bind copper. A model for the structure of the metal binding site in hSco proteins is proposed. hSco proteins could bind copper with trigonal coordination, involving the two cysteines of the CxxxC motif and a conserved histidine. The purified recombinant proteins were also used in an enzymatic assay to test their ability to reduce disulfide bridges, similar to thioredoxin-like proteins involved in the assembly of bacterial COX. Both hSco proteins were not able to act as thioredoxins suggesting a role for the hSco proteins as copper chaperones. To define the pathway of the copper transfer to COX, hSco proteins were tested for their ability to interact with hCox17p, a mitochondrial copper chaperone, and with Cox2p, which contains two copper ions. An interaction between hSco1p and Cox2p was detected. Both hSco proteins were shown to homomerise and to form heterodimers one with each other. Two mutations found in hSCO2 patients suffering from hypertrophic cardiomyopathy, (E140K and S225F) were shown not to affect the copper binding properties, the intracellular localisation and the ability to form homomers. In accordance to these data, a model is proposed in which hSco2p dimers transfer copper to hSco1p dimers. hSco1p dimers interact with COX and insert copper in the binuclear centre of Cox2p.

Mitochondria are an attractive target for the design of cancer therapy. One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis through extrinsic or intrinsic apoptotic pathways. Extrinsic pathways target cell surface receptors whilst intrinsic pathways target mitochondria. Several studies have shown cancer cell destruction through the extrinsic pathways, which target cancer-specific overexpressed growth factor receptors on the cell membrane. Although the mitochondria dependent apoptotic process is well understood, its application in cancer therapy is still not well developed. Therefore, to design an effective cancer therapy targeting mitochondria, a good understanding in mitochondria dependent apoptotic process is required. Recent developments in nanotechnology have enabled live cell investigations and non-destructive methods to obtain cellular information. The availability of such information would assist to design methods of targeted apoptosis induction. In view of this, I report on studies towards development of cancer therapy where nanoparticles (NPs) were targeted to human cell mitochondria for two purposes: (a) development of cell-imaging tools to investigate the fundamental cell biological pathways inside cells and (b) induction of apoptosis by targeting nanoparticles to mitochondria. Current medical and biological fluorescent imaging methods are mainly based on dye markers, which are limited in light emission per molecule, as well as photostability. Consequently, NPs are gaining prominence for molecular imaging because of their strong and stable fluorescence. Additionally, in order to get insight of mitochondrial molecular information, I investigated the use of optical properties of gold nanoparticles (Au NPs) for surface enhanced Raman spectroscopy (SERS). In this study, two types of Au NPs - nanospheres (Au NS) and nanorods (Au NR) were investigated. Results from this study showed the enhancement effect of Au NPs in Raman spectra of mitochondria, especially in the region from 1500 to 1600 cm-1. In this region, normal Raman spectra of mitochondria showed the presence of some understated Raman peaks probably due to the excitation wavelength dependence. Au NRs showed a larger enhancement effect than Au NS with respect to the penetration depth of the plasmonic nearfield enhancement effect. Although, the details of the enhancement mechanism are beyond the current studies, Au NPs could be enhancing vibrations of aromatic residues in proteins. This study therefore showed that Au NPs could enhance Raman spectra of mitochondria and in addition the shape of the nanoparticles had a significant effect on SERS spectra. In living cells, I investigated some transfection methods and targeting of NPs to mitochondria or cytosolic actin subunits. I tested the performance of three transfection reagents to deliver nanodiamonds (NDs) into living cells. Antibody functionalized NDs were targeted to mitochondria or cytosolic actin subunits. Three transfection reagents were used: cationic liposomes PULSin™, the cell penetrating peptide protamine, and oligosaccharide modified polypropylene imine (PPI) dendrimers. Fluorescence imaging results revealed that dendrimers were the most efficient in delivering ND conjugates to targeted organelles. Protamine-mediated transfections appeared to target ND conjugates to intended organelles, although there was a tendency of unfunctionalized NDs to be directed to the nucleus. PULSin™-mediated transfection formed ND aggregates regardless of the functionalization moiety. This reflected the unsuitability of the cationic liposome to mediate ND transfections. Further, I investigated the potential use of Au NPs for cell imaging and photothermal lysis of mitochondria inside cells. Just as above, I also tested the performance of the three-transfection reagents mentioned above on transfection capacity of Au NPs into living cells. Using transmission electron microscopy (TEM), oligosaccharide modified dendrimers showed the best transfection of functionalized Au NPs. Further experiments explored the use of the nearfield enhancement effect of Au NPs in combination with low-level laser irradiation (LLLI) to induce apoptosis in living cells. Analysis of the apoptotic process using cytochrome c release showed that Au NPs induced apoptosis most probably through mechanical disruption of the outer mitochondrial membrane. However, apoptosis was significantly accelerated in cells with mitochondrially targeted Au NRs than in cells without Au NRs. This study showed successful targeting of Au NPs to mitochondria in living cells, and demonstrated the potential of using Au NPs in combination with laser irradiation to induce the mitochondria dependent apoptotic pathway. In conclusion, the potential use of Au NPs in SERS of mitochondria and the application of NDs for cell imaging of intracellular organelles were demonstrated. Lastly, Au NPs were targeted to mitochondria in living cells and could induce apoptosis due to mechanical disruption of the outer mitochondrial membrane. Consequently, application of low-level laser irradiation to Au NP transfected cells accelerated the apoptotic process.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

No description available.

Mitochondria are an attractive target for the design of cancer therapy. One of the mechanisms by which chemotherapeutics destroy cancer cells is by inducing apoptosis through extrinsic or intrinsic apoptotic pathways. Extrinsic pathways target cell surface receptors whilst intrinsic pathways target mitochondria. Several studies have shown cancer cell destruction through the extrinsic pathways, which target cancer-specific overexpressed growth factor receptors on the cell membrane. Although the mitochondria dependent apoptotic process is well understood, its application in cancer therapy is still not well developed. Therefore, to design an effective cancer therapy targeting mitochondria, a good understanding in mitochondria dependent apoptotic process is required. Recent developments in nanotechnology have enabled live cell investigations and non-destructive methods to obtain cellular information. The availability of such information would assist to design methods of targeted apoptosis induction. In view of this, I report on studies towards development of cancer therapy where nanoparticles (NPs) were targeted to human cell mitochondria for two purposes: (a) development of cell-imaging tools to investigate the fundamental cell biological pathways inside cells and (b) induction of apoptosis by targeting nanoparticles to mitochondria. Current medical and biological fluorescent imaging methods are mainly based on dye markers, which are limited in light emission per molecule, as well as photostability. Consequently, NPs are gaining prominence for molecular imaging because of their strong and stable fluorescence. Additionally, in order to get insight of mitochondrial molecular information, I investigated the use of optical properties of gold nanoparticles (Au NPs) for surface enhanced Raman spectroscopy (SERS). In this study, two types of Au NPs - nanospheres (Au NS) and nanorods (Au NR) were investigated. Results from this study showed the enhancement effect of Au NPs in Raman spectra of mitochondria, especially in the region from 1500 to 1600 cm-1. In this region, normal Raman spectra of mitochondria showed the presence of some understated Raman peaks probably due to the excitation wavelength dependence. Au NRs showed a larger enhancement effect than Au NS with respect to the penetration depth of the plasmonic nearfield enhancement effect. Although, the details of the enhancement mechanism are beyond the current studies, Au NPs could be enhancing vibrations of aromatic residues in proteins. This study therefore showed that Au NPs could enhance Raman spectra of mitochondria and in addition the shape of the nanoparticles had a significant effect on SERS spectra. In living cells, I investigated some transfection methods and targeting of NPs to mitochondria or cytosolic actin subunits. I tested the performance of three transfection reagents to deliver nanodiamonds (NDs) into living cells. Antibody functionalized NDs were targeted to mitochondria or cytosolic actin subunits. Three transfection reagents were used: cationic liposomes PULSin™, the cell penetrating peptide protamine, and oligosaccharide modified polypropylene imine (PPI) dendrimers. Fluorescence imaging results revealed that dendrimers were the most efficient in delivering ND conjugates to targeted organelles. Protamine-mediated transfections appeared to target ND conjugates to intended organelles, although there was a tendency of unfunctionalized NDs to be directed to the nucleus. PULSin™-mediated transfection formed ND aggregates regardless of the functionalization moiety. This reflected the unsuitability of the cationic liposome to mediate ND transfections. Further, I investigated the potential use of Au NPs for cell imaging and photothermal lysis of mitochondria inside cells. Just as above, I also tested the performance of the three-transfection reagents mentioned above on transfection capacity of Au NPs into living cells. Using transmission electron microscopy (TEM), oligosaccharide modified dendrimers showed the best transfection of functionalized Au NPs. Further experiments explored the use of the nearfield enhancement effect of Au NPs in combination with low-level laser irradiation (LLLI) to induce apoptosis in living cells. Analysis of the apoptotic process using cytochrome c release showed that Au NPs induced apoptosis most probably through mechanical disruption of the outer mitochondrial membrane. However, apoptosis was significantly accelerated in cells with mitochondrially targeted Au NRs than in cells without Au NRs. This study showed successful targeting of Au NPs to mitochondria in living cells, and demonstrated the potential of using Au NPs in combination with laser irradiation to induce the mitochondria dependent apoptotic pathway. In conclusion, the potential use of Au NPs in SERS of mitochondria and the application of NDs for cell imaging of intracellular organelles were demonstrated. Lastly, Au NPs were targeted to mitochondria in living cells and could induce apoptosis due to mechanical disruption of the outer mitochondrial membrane. Consequently, application of low-level laser irradiation to Au NP transfected cells accelerated the apoptotic process.

Direkte Zellreprogrammierung basiert auf Transkriptionsfaktoren (TFs), die die Identität bestimmter Zelltypen induzieren.Diese TF vermittelte Zellreprogrammierung ist in den meisten Zelltypen häufig durch hemmende Mechanismen limitiert.Um solche Barrieren in C. elegans zu identifizieren, verwendeten wir den zuvor charakterisierten Zinkfinger-TF CHE-1, der erforderlich ist, um glutamaterge ASE-Neuronen zu induzieren.In dieser Studie haben wir eine mögliche Barriere für die Reprogrammierung von Keimzellen zu Neuronen identifiziert und charakterisiert:die NAD+ abhängige mitochondriale Isocitratdehydrogenase 3 (IDH3).Der RNAi-Knockdown ergab in Kombination mit ektopischer Expression von CHE-1 einen konsistenten Phänotyp hinsichtlich der Expression des neuronalen ASE- Reporters in der Keimbahn. Wir konnten feststellen,dass IDH3-Knockdowns zu globalen Veränderungen der repressiven Histonmodifikationen führen.Mittels genetischer Untersuchungen identifizierten wir Mitglieder der Jumonji-Proteine sowie die a-KG-abhängigen Histon-Demethylasen, die an diesem Reprogammierungsphänotyp beteiligt sind. Durch Massenspektrometrie und weiterführenden genetische Untersuchungen haben wir festgestellt, dass Zellen bei IDH3-Mangel eine sogenannte Glutamin-Anaplerose verwenden, um den a-KG-Spiegel wieder aufzufüllen und somit einen teilweise aktiven Zitronensäurezyklus beizubehalten.Des Weiteren sind diese Prozesse erforderlich, damit die TF vermittelte Zellreprogrammierung stattfinden kann.Wir haben außerdem festgestellt, dass Signale von Zellen der somatischen Gonade diesen durch IDH3-Mangel vermittelten Zellreprogrammierungsprozess von Keimzellen ermöglichen.Daher ist anzunehmen, dass der Reprogrammierungsphänotyp in der Keimbahn nicht gewebsautonom ist. Zusammengefasst identifiziert diese Studie die Rolle der evolutionär konservierten Isocitrat-Dehydrogenase 3 (IDH3) bei der Aufrechterhaltung der Zellidentität und damit auch als Barriere für die Zellreprogrammierung. / Direct reprogramming makes use of transcription factors (TFs) that induce the identity of specific cell types. These TFs often are restricted in most cell types by inhibitory mechanisms. In order to identify these barriers in C. elegans, we used the previously described zinc-finger TF CHE-1 that is required to induce the glutamatergic ASE neuron fate. In this study, we identified and characterized a candidate barrier for reprogramming germ cells into neurons, the NAD+ dependent mitochondrial isocitrate dehydrogenase 3 (IDH3). RNAi knockdown of alpha (IDHA-1) or gamma (IDHG-1) subunit of this complex gave a consistent and reliable phenotype of the expression of ASE reporter in the germ line upon ectopic expression of CHE- 1. This study shows that idha-1 depletion-mediated reprogramming of germ cells to neurons is partially repressed in animals that lack the hypoxia-induced factor, TF HIF-1. It has been shown that mitochondrial dynamics change during differentiation. This suggests that disturbing mitochondrial function may feed-back to chromatin thus altering gene expression and allowing reprogramming. We were able to identify that knock down of idha-1 leads to global histone modification changes; and by performing a genetic screen we identified members of the Jumonji proteins, the a-KG dependent histone demethylases, involved in this conversion phenotype. By performing Mass Spectrometry and genetic screens, we have identified that cells utilize glutamine anaplerosis to replenish a-KG levels and display a partially active citric acid cycle upon IDH3 depletion; and these processes are required for the TF- mediated reprogramming to occur. Furthermore, the IDH3 depletion-mediated germ cell reprogramming is not tissue autonomous. We identified that signals from the somatic gonad enable the reprogramming process. Overall, this study identifies the role of the conserved Isocitrate Dehydrogenase 3 in cell fate safeguarding and thus as a barrier to reprogramming.