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.

No description available.

Im ersten Teil dieser Dissertation wurde der Einfluss des mitochondrialen Proteins TCAIM (T cell activation inhibitor, mitochondrial) auf die T-Zellaktivierung untersucht. Hierzu wurde eine transgene Mauslinie mit einem T-zellspezifischen knock-in (KI) von Tcaim in den Rosa26 Lokus generiert. Die Tcaim-Überexpression beeinflusste die Fission und Umverteilung von Mitochondrien und reduzierte die T-Zellrezeptor (TZR)-induzierte Bildung mitochondrialer, radikaler Sauerstoffspezies. In vitro stimulierte CD4+ Tcaim KI T-Zellen zeigten eine geringere Aktivierung, Proliferation und IL-2 Sekretion als Kontrollzellen. T-Zellen aus Tcaim KI Mäusen, die in Rag-1 knock-out Mäuse transferiert wurden, waren nicht fähig ein allogenes Haut-Transplantat abzustoßen und behielten einen naiven Phänotyp. Diese Ergebnisse zeigen, dass TCAIM als mitochondriales Protein wichtige Schritte in der Zellaktivierung und der Bildung von Gedächtnis-T-Zellen beeinflusst. Der zweite Teil der Dissertation beschäftigte sich mit dem Einfluss der CD44-Oberflächenexpression auf die Differenzierung von T-Helfer (TH)-Zellen. Eine hohe CD44-Expression unterscheidet Effektor- von naiven T-Zellen. Durch die allogene Stimulation von CD4+ T-Zellen bildeten sich drei verschiedene Populationen: CD44+, CD44++ und CD44+++. Sowohl in vitro als auch in vivo generierte alloreaktive TH17-Zellen wurden in der CD44+++ Population, TH1-Zellen hingegen in der CD44++ Population, detektiert. Es wurde beschrieben, dass sowohl eine geringe TZR- als auch eine geringe CD28-Stimulation eher die Bildung von TH17- als TH1-Zellen unterstützen. Unter genau diesen Bedingungen kann CD44 als kostimulatorisches Molekül die Signaltransduktion verstärken. Tatsächlich zeigten allogenreaktive CD44+++ TH-Zellen eine höhere ZAP-70-Phosphorylierung als CD44++ TH-Zellen. Diese Ergebnisse unterstützen die Annahme, dass CD44 durch die Verstärkung der Signaltransduktion die TH17-Differenzierung fördern kann. / Within the first part of this thesis, the influence of the mitochondrial Protein TCAIM (T cell activation inhibitor, mitochondrial) on T cell activation was investigated. Tcaim expression correlated negatively with the rejection of allografts and it is down-regulated during T cell activation. To study effects of TCAIM during T cell activation, we generated a T cell-specific mouse strain with a Tcaim knock-in (KI) targeted to the Rosa26 locus. Tcaim overexpression changed the mitochondrial morphology and reduced the T cell receptor (TCR)-induced mitochondrial reactive oxygen species production. In vitro activation of Tcaim KI CD4+ T cells resulted in a decreased activation, proliferation and cytokine release. Importantly, Rag-1 knock-out mice, reconstituted with Tcaim KI T cells, tolerated allogeneic skin grafts. Thus, by regulating TCR-induced mitochondrial distribution and ROS production, TCAIM controls important steps during T cell activation and memory formation. The second part dealt with the influence of CD44 surface expression level for T helper cell (Th cell) differentiation. By association with lymphocyte-specific protein kinase (LCK) it can enhance T cell signaling. Allogeneic stimulation of CD4+ T cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive TH17 cells were mainly CD44+++. This is in contrast to TH1 cells which were dominantly CD44++. Titration experiments revealed that low TCR- and co-stimulation supports TH17 rather than TH1 development. Under exactly these conditions it was reported that CD44 can act as co-stimulatory molecule and replace CD28. Indeed, CD44+++CD4+ T cells contained already more phosphorylated ZAP-70 as compared to CD44++ cells. Our results support the notion that CD44 enhances TCR signaling strength by delivering LCK, which is required to support TH17 development.

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte. / Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.

Die Krebsentstehung wurde vor rund 80 Jahren auf veränderten zellulären Energiestoffwechsel zurückgeführt. Diese Hypothese konnte bisher weder experimentell bewiesen noch widerlegt werden. Durch den Einsatz zweier Modellsysteme mit unterschiedlicher Expression des mitochondrialen Proteins Frataxin konnte in der vorliegenden Arbeit <br> gezeigt werden, dass der mitochondriale Energiestoffwechsel einen Einfluss auf die Tumorentstehung zu besitzen scheint. Eine Reduktion des mitochondrialen Energiestoffwechsels wurde durch die hepatozytenspezifische Ausschaltung des mitochondrialen Proteins Frataxin in Mäusen erreicht. Der durch das Cre-/loxP-Rekombinasesystem erreichte organspezifische Knock-out wurde auf Transkriptions- und Translationsebene nachgewiesen. Anhand verminderter Aconitaseaktivität, geringeren Sauerstoffverbrauches und reduzierten ATP-Gehaltes im Lebergewebe wurde ein signifikant verminderter Energiestoffwechsel dargestellt. Zwar entsprach die Genotypenverteilung in den Versuchsgruppen der erwarteten Mendelschen Verteilung, dennoch war die mittlere Lebenserwartung der <br> Knock-out-Tiere mit ca. 30 Wochen stark reduziert. Bereits in jungem Alter war bei diesen Tieren die Ausbildung von präneoplastischen Herden zu beobachten. Mit proteinbiochemischen Nachweistechniken konnte in Lebergewebe 4-8 Wochen alter Tiere eine verstärkte Aktivierung des Apoptosesignalweges (Cytochrom C im Zytosol, <br> verstärkte Expression von Bax) sowie eine Modulation stressassoziierter Proteine (geringere Phosphorylierungsrate p38-MAPK, vermehrte Expression HSP-25, verminderte Expression HSP-70) aufgezeigt werden. Im inversen Ansatz wurde eine Steigerung des mitochondrialen Energiestoffwechsels durch stabile transgene Frataxinüberexpression in zwei Kolonkarzinomzelllinien erreicht. Diese Steigerung zeigte sich durch erhöhte Aconitaseaktivität, erhöhten Sauerstoffverbrauch, gesteigertes mitochondriales Membranpotenzial und erhöhten ATP-Gehalt in den Zellen. Die frataxinüberexprimierenden Zellen wuchsen signifikant langsamer als Kontrollzellen und zeigten im Soft-Agar-Assay und im Nacktmausmodell ein deutlich geringeres Potenzial zur Ausbildung von Kolonien bzw. Tumoren. Mittels Immunoblot war hier eine vermehrte Phosphorylierung der p38-MAPK festzustellen. <br> Die zusammenfassende Betrachtung beider Modelle zeigt, dass ein reduzierter mitochondrialer Energiestoffwechsel durch Regulation der p38-MAPK und apoptotischer Signalwege ein erhöhtes Krebsrisiko zu verursachen vermag. / Eigthy years ago, it was suggested that impaired energy metabolism might cause cancer. Compelling experimental evidence for this hypothesis is lacking. By use of two different model systems here we show that impaired expression of the mitochondrial protein frataxin leading to impaired mitochondrial energy metabolism appears to be <br> inversely related to tumour growth. To generate mice with reduced mitochondrial energy metabolism the expression of mitochondrial protein frataxin was disrupted in a hepatocyte-specific manner by using the cre/loxP-system. Presence, efficiency and specificity of disruption were shown at transcriptional and translational levels. Decreased activity of aconitase, reduced oxygen consumption and diminished ATP level in the liver revealed diminished energy <br> metabolism. Although knock-out mice were born in the expected Mendelian frequency, they exhibited a significantly decreased life expectancy. Young mice exhibited hepatic preneoplasia. The use of proteinbiochemical techniques revealed activation of apoptotic <br> pathways (cytochrome c in the cytosol, increased expression of bax) and modulation of stress-associated cascades (decreased phosphorylation of p38-MAPK, increased expression of HSP-25 and diminished expression of HSP-70). Inversely, transgenic overexpression of frataxin in colon cancer cell lines lead to increased mitochondrial energy metabolism as demonstrated by elevated activity of aconitase, increased oxygen consumption, elevated mitochondrial membrane potential and increased ATP levels. Frataxin-overexpressing colon cancer cells exhibit a <br> concurrent decrease in replication rate. The colony forming capacity in soft-agar-assay and tumour formation in nude mice were clearly decreased. Immunoblotting revealed elevated phosphorylation of p38-MAPK. Taken together, these models suggest that reduced mitochondrial energy metabolism may promote cancer through regulation of p38-MAPK and apoptotic pathways.

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.

No description available.

No description available.

No description available.

The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.