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More than a century ago the phenomenon of non-Mendelian inheritance (NMI), defined as any type of inheritance pattern in which traits do not segregate in accordance with Mendel’s laws, was first reported. In the plant kingdom three genomic compartments, the nucleus, chloroplast, and mitochondrion, can participate in such a phenomenon. High-throughput sequencing (HTS) proved to be a key technology to investigate NMI phenomena by assembling and/or resequencing entire genomes. However, generation, analysis and interpretation of such datasets remain challenging by the multi-layered biological complexity. To advance our knowledge in the field of NMI, I conducted three studies involving different HTS technologies and implemented two new algorithms to analyze them.
In the first study I implemented a novel post-assembly pipeline, called Semi-Automated Graph-Based Assembly Curator (SAGBAC), which visualizes non-graph-based assemblies as graphs, identifies recombinogenic repeat pairs (RRPs), and reconstructs plant mitochondrial genomes (PMG) in a semiautomated workflow. We applied this pipeline to assemblies of three Oenothera species resulting in a spatially folded and circularized model. This model was confirmed by PCR and Southern blot analyses and was used to predict a defined set of 70 PMG isoforms. With Illumina Mate Pair and PacBio RSII data, the stoichiometry of the RRPs was determined quantitatively differing up to three-fold.
In the second study I developed a post-multiple sequence alignment algorithm, called correlation mapping (CM), which correlates segment-wise numbers of nucleotide changes to a numeric ascertainable phenotype. We applied this algorithm to 14 wild type and 18 mutagenized plastome assemblies within the Oenothera genus and identified two genes, accD and ycf2 that may cause the competitive behavior of plastid genotypes as plastids can be biparental inherited in Oenothera. Moreover, lipid composition of the plastid envelope membrane is affected by polymorphisms within these two genes.
For the third study, I programmed a pipeline to investigate a NMI phenomenon, known as paramutation, in tomato by analyzing DNA and bisulfite sequencing data as well as microarray data. We identified the responsible gene (Solyc02g0005200) and were able to fully repress its caused phenotype by heterologous complementation with a paramutation insensitive transgene of the Arabidopsis thaliana orthologue. Additionally, a suppressor mutant shows a globally altered DNA methylation pattern and carries a large deletion leading to a gene fusion involving a histone deacetylase.
In conclusion, my developed and implemented algorithms and data analysis pipelines are suitable to investigate NMI and led to novel insights about such phenomena by reconstructing PMGs (SAGBAC) as a requirement to study mitochondria-associated phenotypes, by identifying genes (CM) causing interplastidial competition as well by applying a DNA/Bisulfite-seq analysis pipeline to shed light in a transgenerational epigenetic inheritance phenomenon.
The mitochondrial chaperone complex HSP60/HSP10 facilitates mitochondrial protein homeostasis by folding more than 300 mitochondrial matrix proteins. It has been shown previously that HSP60 is downregulated in brains of type 2 diabetic (T2D) mice and patients,
causing mitochondrial dysfunction and insulin resistance. As HSP60 is also decreased in peripheral tissues in T2D animals, this thesis investigated the effect of overall reduced HSP60 in the development of obesity and associated co-morbidities.
To this end, both female and male C57Bl/6N control (i.e. without further alterations in their genome, Ctrl) and heterozygous whole-body Hsp60 knock-out (Hsp60+/-) mice, which exhibit a 50 % reduction of HSP60 in all tissues, were fed a normal chow diet (NCD) or a highfat diet (HFD, 60 % calories from fat) for 16 weeks and were subjected to extensive metabolic phenotyping including indirect calorimetry, NMR spectroscopy, insulin, glucose and pyruvate tolerance tests, vena cava insulin injections, as well as histological and molecular analysis.
Interestingly, NCD feeding did not result in any striking phenotype, only a mild increase in energy expenditure in Hsp60+/- mice. Exposing mice to a HFD however revealed an increased body weight due to higher muscle mass in female Hsp60+/- mice, with a simultaneous decrease in energy expenditure. Additionally, these mice displayed decreased fasting glycemia. Opposingly, male Hsp60+/- compared to control mice showed lower body weight gain due to decreased fat mass and an increased energy expenditure, strikingly independent of lean mass. Further, only male Hsp60+/- mice display improved HOMA-IR and Matsuda
insulin sensitivity indices.
Despite the opposite phenotype in regards to body weight development, Hsp60+/- mice of both sexes show a significantly higher cell number, as well as a reduction in adipocyte size in the subcutaneous and gonadal white adipose tissue (sc/gWAT). Curiously, this adipocyte hyperplasia – usually associated with positive aspects of WAT function – is disconnected from metabolic improvements, as the gWAT of male Hsp60+/- mice shows mitochondrial dysfunction, oxidative stress, and insulin resistance. Transcriptomic analysis of gWAT shows an up
regulation of genes involved in macroautophagy. Confirmatory, expression of microtubuleassociated protein 1A/1B light chain 3B (LC3), as a protein marker of autophagy, and direct measurement of lysosomal activity is increased in the gWAT of male Hsp60+/- mice.
In summary, this thesis revealed a novel gene-nutrient interaction. The reduction of the crucial chaperone HSP60 did not have large effects in mice fed a NCD, but impacted metabolism during DIO in a sex-specific manner, where, despite opposing body weight and
body composition phenotypes, both female and male Hsp60+/- mice show signs of protection from high fat diet-induced systemic insulin resistance.
Growth differentiation factor 15 (GDF15) is a stress-induced cytokine secreted into the circulation by a number of tissues under different pathological conditions such as cardiovascular disease, cancer or mitochondrial dysfunction, among others. While GDF15 signaling through its recently identified hindbrain-specific receptor GDNF family receptor alpha-like (GFRAL) has been proposed to be involved in the metabolic stress response, its endocrine role under chronic stress conditions is still poorly understood. Mitochondrial dysfunction is characterized by the impairment of oxidative phosphorylation (OXPHOS), leading to inefficient functioning of mitochondria and consequently, to mitochondrial stress. Importantly, mitochondrial dysfunction is among the pathologies to most robustly induce GDF15 as a cytokine in the circulation.
The overall aim of this thesis was to elucidate the role of the GDF15-GFRAL pathway under mitochondrial stress conditions. For this purpose, a mouse model of skeletal muscle-specific mitochondrial stress achieved by ectopic expression of uncoupling protein 1 (UCP1), the HSA-Ucp1-transgenic (TG) mouse, was employed. As a consequence of mitochondrial stress, TG mice display a metabolic remodeling consisting of a lean phenotype, an improved glucose metabolism, an increased metabolic flexibility and a metabolic activation of white adipose tissue.
Making use of TG mice crossed with whole body Gdf15-knockout (GdKO) and Gfral-knockout (GfKO) mouse models, this thesis demonstrates that skeletal muscle mitochondrial stress induces the integrated stress response (ISR) and GDF15 in skeletal muscle, which is released into the circulation as a myokine (muscle-induced cytokine) in a circadian manner. Further, this work identifies GDF15-GFRAL signaling to be responsible for the systemic metabolic remodeling elicited by mitochondrial stress in TG mice. Moreover, this study reveals a daytime-restricted anorexia induced by the GDF15-GFRAL axis under muscle mitochondrial stress, which is, mechanistically, mediated through the induction of hypothalamic corticotropin releasing hormone (CRH). Finally, this work elucidates a so far unknown physiological outcome of the GDF15-GFRAL pathway: the induction of anxiety-like behavior.
In conclusion, this study uncovers a muscle-brain crosstalk under skeletal muscle mitochondrial stress conditions through the induction of GDF15 as a myokine that signals through the hindbrain-specific GFRAL receptor to elicit a stress response leading to metabolic remodeling and modulation of ingestive- and anxiety-like behavior.
Die durch Phosphatidylserin Decarboxylase (PSD) katalysierte Decarboxylierung von Phosphatidylserin (PS) zu Phosphatidylethanolamin (PE) ist für Mitochondrien in Hefe und Mäusen von essentieller Bedeutung. Im Rahmen der vorliegenden Dissertation wurde erstmals die Rolle dieses PE-Syntheseweges in Pflanzen untersucht. Die drei in Arabidopsis identifizierten PSD Gene atPSD1, atPSD2, atPSD3 codieren für Enzyme, die in Membranen der Mitochondrien (atPSD1), der Tonoplasten (atPSD2) und des Endoplasmatischen Retikulums (atPSD3) lokalisiert sind. Der Beitrag der einzelnen PSDs zur PE-Synthese wurde anhand von psd Null-Mutanten untersucht. Dabei stellte sich atPSD3 als das Enzym mit der höchsten Aktivität heraus. Alternativ zum PSD-Weg wird in Arabidopsis PE auch mittels Aminoalkohol-phosphotransferase synthetisiert. Der Verlust der gesamten PSD-Aktivität, wie es in der erzeugten psd Dreifachmutante der Fall ist, wirkt sich ausschließlich auf die Lipidzusammensetzung in der Mitochondrienmembran aus. Demzufolge wird extramitochondriales PE hauptsächlich über die Aminoalkoholphosphotransferase synthetisiert. Die veränderte Lipidzusammensetzung der Mitochondrienmembran hatte jedoch keinen Einfluss auf die Anzahl, Größe und Ultrastruktur der Mitochondrien sowie auf das ADP/ATP-Verhältnis und die Respiration. Neben der Bereitstellung von Reduktionsäquivalenten beeinflusst die Funktionalität der Mitochondrien auch die Bildung von Blüten- und Staubblättern. Diese Blütenorgane waren in der psd Dreifachmutante stark verändert, und der Blütenphänotyp ähnelte der APETALA3-Mutante. Dieses homöotische Gen ist für die Ausbildung von Blüten- und Staubblättern verantwortlich. Für die Erzeugung der Mutanten psd2-1 und psd3-1 wurde ein T-DNA Vektor verwendet, der den Promotor des APETALA3 Gens enthielt, welcher in den Mutanten psd2-1, psd3-1 sowie psd2-1psd3-1 und der psd1psd2-1psd3-1 Dreifachmutante eine vergleichbare Co-Suppression des APETALA3 Gens hervorruft. Der Blütenphänotyp trat jedoch nur in der psd Dreifachmutante auf, da nur in ihr die Kombination von geringen Funktionstörungen der Mitochondrien, hervorgerufen durch veränderte Lipidzusammensetzung, mit der Co-Suppression von APETALA3 auftritt.
ATP-binding cassette (ABC) transporters are present in all kingdoms of life and enable active transport of various different molecules across biological membranes. They all share an overall architecture of two lipophilic transmembrane spanning domains (TMDs) traversing the membrane and two hydrophilic nucleotide binding domains (NBDs) usually lacking sequence identity. The multiplicity in transported molecules is accompanied by extreme diversity in TMDs. Human mitochondria harbor four ABC transporters, namely ABCB6, ABCB7, ABCB8 and ABCB10 with functional homologues in yeast and plants. Except the ones found in Rickettsiae and related bacteria mitochondrial ABC transporters are absent in bacteria. In addition to converting energy mitochondria are important platforms for biosynthesizing various cofactors as iron sulfur clusters, molybdenum cofactor (Moco) or heme. ABCB7 (Atm1 in yeast) has been shown to connect mitochondrial with cytosolic iron sulfur cluster assembly by exporting a yet unknown sulfur containing molecule. In addition, TMDs of Atm1 display a glutathione binding pocket accessible from the matrix which has been identified in all ABCB7-like transporters and also exists in a bacterial ABC transporter homologue of Atm1 in Novosphingobium aromaticivorans. In addition, ATM3, a plant mitochondrial homologous ABC transporter to human ABCB7, has been associated with biosynthesizing Moco.
In this study we used the α-proteobacterium Rhodobacter capsulatus as a model organism to characterize mitochondrial ABC transporter homologues. R. capsulatus contains two homologues to mitochondrial ABC transporters with the corresponding gene loci rcc03139 and rcc02305. They share 38 to 47 % sequence identities to human mitochondrial ABC transporters ABCB8/ABCB10 and ABCB7/ABCB6, respectively. We created interposon mutants lacking either rcc03139 or rcc02305, analyzed the physiological effects on R. capsulatus and compared the findings especially to eukaryotic deletion studies. A viable bacterial double mutant strain lacking both mitochondrial ABC transporters was constructed to investigate possible overlapping functions. Both R. capsulatus single mutants showed a severe accumulation of intracellular reactive oxygen species (ROS) in comparison to ∆nifDK which revealed to be additive in the double mutant. In the proteome of ∆rcc03139I abundancies of tetrapyrrole related proteins were significantly increased in comparison to the proteome of parental strain, which was further validated by reduced amounts of tetrapyrrole intermediates in ∆rcc03139. In contrast, in ∆rcc02305I total glutathione (GSH) was elevated when endogenous GSH biosynthesis was inhibited. In conjunction with proteomic studies we uncovered misbalanced sulfur distribution in ∆rcc02305I. Furthermore, strains lacking Rcc02305 accumulated cyclic pyranopterin monophosphate (cPMP), an intermediate of Moco biosynthesis, as it was already shown for the deletion strain of the eukaryotic counterpart ATM3 in plants. In contrast single mutant strain Δrcc03139I neither accumulated cPMP nor glutathione.
Bioinformatic analysis of the amino acid sequence of Rcc02305 revealed a pyridoxal 5´phosphate (PLP) binding site which overlaps with Walker A within the NBDs of Rcc02305 and other ABCB7-like transporters. The PLP cofactor is well studied in C-DES (L-cysteine/cystine lyase from Synechocystis) for persulfide production and in L-cysteine desulfurases such as IscS and NFS1 for its role in formation of protein-bound persulfides. Based on our findings we are able to propose a new modality for the transport of the sulfur containing molecule: first of all, the transporter produces a highly reactive persulfide which is then subsequently trapped by glutathione polysulfide, already bound within the binding pocket in TMDs. Walker A becomes accessible for ATP and after hydrolysis the mixed polysulfide is released.
Based on our studies we are convinced that both mitochondrial ABC transporter homologues fulfil distinct roles in R. capsulatus: Rcc02305 is a representative of Atm1/ABCB7-like transporters and important for proper sulfur distribution by exporting persulfides. In contrast Rcc03139 is a representative of ABCB6/ABCB10 related transporters and involved in biosynthesizing tetrapyrroles.
Identifikation des mitochondrialen Proteins Frataxin als stoffwechselmodulierenden Tumorsuppressor
(2004)
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 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 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, 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. 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.