Biologische Hochschulschriften (Goethe-Universität)
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Die Physiologie des Schmerzes umfasst komplexe immunologische, sensorische und inflammatorische Prozesse im Rückenmark, im Gehirn und in der Peripherie. Wiederholte nozizeptive Stimulation induziert pathophysiologische Veränderungen bei der Schmerzweiterleitung, aus denen eine periphere oder zentrale Sensibilisierung resultiert. Diese kann bei dafür anfälligen Patienten zu der Ausbildung von chronischen Schmerzzuständen führen. Obwohl das Wissen über die genauen molekularen Vorgänge der Schmerz-Chronifizierung noch immer unvollständig ist, sind die Identifizierung von Risikofaktoren vernünftige Schritte, um die individuelle Anfälligkeit für die Entwicklung chronischer Schmerzen zu bestimmen. Das Hauptziel dieser Doktorarbeit bestand daher in der Identifikation humaner genetischer Biomarker für chronische Schmerzzustände.
Lineare sowie zyklische 3-Alkylpyridinalkaloide sind vor allem in Schwämmen der Ordnung Haplosclerida, zu der auch Haliclona viscosa zählt, weit verbreitet. Die Synthese der zuvor von C. Volk isolierten Haliclamine C und D, des Viscosamins und des Viscosalin C bildete den Ausgangspunkt dieser Arbeit.[1-4] Sie erfolgte ausgehend von den bekannten Synthesen der Cyclostellettamine und Haliclamine[5-7] und gliedert sich in drei Abschnitte: erstens Synthese eines ω-Hydroxyalkylpyridins aus einem Bromalkohol, zweitens Funktionalisierung der Monomere in Abhängigkeit der gewählten Methode zur Di- bzw. Trimerisierung und drittens Verknüpfung und gegebenenfalls Zyklisierung. Durch Anwendung und Weiterentwicklung der bekannten Synthesewege wurden so insgesamt 14 lineare Monomere, zwei zyklische Monomere, 16 Cyclostellettamine, zwei Isocyclostellettamine, sieben Haliclamine, fünf Viscosaline sowie Viscosamin[8] und ein Analogon mit Heptylkette hergestellt. Dieser synthetische Zugang ermöglichte es, sowohl den finalen Strukturbeweis für die zuvor isolierten Verbindungen zu erbringen, als auch durch die Analyse der Fragmentierungs-muster von synthetischen und natürlichen Verbindungen mehr über das Verhalten dieser Verbindungen unter MS-Bedingungen zu erfahren. Die so gewonnenen Erkenntnisse führten dazu, dass drei unbekannte Verbindungen ohne Isolierung der Reinsubstanz mit einer Kombination von MS- und HPLC-Daten identifiziert werden konnten. So konnten das erste monozyklische 3-Alkylpyridinalkaloid marinen Ursprungs und zwei neue Haliclamine identifiziert und synthetisiert werden Des Weiteren gelang es, für die von C. Volk isolierten, jedoch nicht identifizierten Verbindungen Strukturen zu ermitteln bzw. auf Grund der MS-Daten Strukturvorschläge zu machen. Die durch den synthetischen Zugang große Anzahl verfügbarer 3-Alkylpyridinalkaloide ermöglichte außerdem eine systematische Untersuchung über den Zusammenhang von biologischer Aktivität und Struktur. Die Ergebnisse der am Helmholtz Institut für Infektionsforschung durchgeführten Experimente zu den antibakteriellen sowie cytotoxischen Eigenschaften von natürlichen wie auch rein synthetischen 3-Alkylpyridinalkaloiden zeigten, dass die Aktivität sich schon beim Addieren bzw. Subtrahieren einer Methylengruppe in einer Alkylkette signifikant ändert. [1] C. A. Volk, M. Köck, Org. Lett. 2003, 5, 3567-3569. [2] C. A. Volk, M. Köck, Org. Biomol. Chem. 2004, 2, 1827-1830. [3] C. A. Volk, H. Lippert, E. Lichte, M. Köck, Eur. J. Org. Chem. 2004, 3154-3158. [4] C. A. Volk, Dissertation, Johann Wolfgang Goethe Universität (Frankfurt am Main), 2004. [5] A. Grube, C. Timm, M. Köck, Eur. J. Org. Chem. 2006, 1285-1295 und Referenzen darin. [6] J. E. Baldwin, D. R. Spring, C. E. Atkinson, V. Lee, Tetrahedron 1998, 54, 13655-13680. [7] A. Kaiser, X. Billot, A. Gateau-Olesker, C. Marazano, B. C. Das, J. Am. Chem. Soc. 1998, 120, 8026-8034. [8] C. Timm, M. Köck, Synthesis 2006, 2580-2584.
The fungal interaction with plants is a 400 million years old phenomenon, which presumably assisted in the plants’ establishment on land. In a natural ecosystem, all plant-ranging from large trees to sea-grasses-are colonized by fungal endophytes, which can be detected inter- and intracellularly within the tissues of apparently healthy plants, without causing obvious negative effects on their host. These ubiquitous and diverse microorganisms are likely playing important roles in plant fitness and development. However, the knowledge on the ecological functions of fungal root endophytes is scarce. Among possible functions of endophytes, they are implicated in mutualisms with plants, which may increase plant resistance to biotic stressors like herbivores and pathogens, and/or to abiotic factors like soil salinity and drought. Also, endophytes are fascinating microorganisms in regard to their high potential to produce a great spectrum of secondary metabolites with expected ecological functions. However, evidences suggest that the interactions between host plants and endophytes are not static and endophytes express different symbiotic lifestyles ranging from mutualism to parasitism, which makes difficult to predict the ecological roles of these cryptic microorganisms. To reveal the ecological function of fungal root endophytes, this doctoral thesis aims at assessing fungal root endophytes interactions with different plants and their effects on plant fitness, based on their phylogeny, traits, and competition potential in settings encompassing different abiotic contexts. To understand the cryptic implication of nonmycorrhizal endophytes in ecosystem processes, we isolated a diverse spectrum of fungal endophytes from roots of several plant species growing in different natural contexts and tested their effects on different model plants under axenic laboratory conditions. Additionally,we aimed at investigating the effect of abiotic and biotic variables on the outcome of interactions between fungal root endophytes and plants.
In summary, the morphological and physiological traits of 128 fungal endophyte strains within ten fungal orders were studied and artificial experimental systems were used to reproduce their interactions with three plant species under laboratory conditions. Under defined axenic conditions, most endophytes behaved as weak parasites, but their performance varied across plant species and fungal taxa. The variation in the interactions was partly explained by convergent fungal traits that separate groups of endophytes with potentially different niche preferences. According to my findings, I predict that the functional complementarity of strains is essential in structuring natural root endophytic communities. Additionally, the responses of plant-endophyte interactions to different abiotic factors, namely nutrient availability, light intensity, and substrate’s pH, indicate that the outcome of plant-fungus relationships may be robust to changes in the abiotic environment. The assessment of the responses of plant endophyte interactions to biotic context, as combinations of selected dominant root fungal endophytes with different degrees of trait similarity and shared evolutionary history, indicates that frequently coexisting root-colonizing fungi may avoid competition in inter-specific interactions by occupying specific niches, and that their interactions likely define the structure of root-associated fungal communities and influence the microbiome impacts on plant fitness.
In conclusion, my findings suggest that dominant fungal lineages display different ecological preferences and complementary sets of functional traits, with different niche preferences within root tissues to avoid competition. Also, their diverse effects on plant fitness is likely host-isolate dependent and robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont.
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
Many hominin species are best physically represented and understood by the sum of their dental morphologies. Generally, taxonomic affinities and evolutionary trends in development (ontogeny) and morphology (phylogeny) can be deduced from dental analyses. More specifically, the study of dental remains can yield a wealth of information on many facets of hominin evolution, life history, physiology and ecological adaptation; in short, the organisms paleobiomics. Functionally, teeth present information about dietary preferences, that is, the dietary niche in ecological context and, in turn, masticatory function. As the amount and types of information that can be gleaned from 2-dimensional tooth measurement exhaust themselves, 3-dimensional microscopic modeling and analysis presents a largely fertile ground for reexamination and reinterpretation of dental characteristics (Bromage et al., 2005). As such, a novel, non-destructive approach has been developed which combines the work of two established technologies (confocal microscopy and 3D modeling) adapted specifically for the purpose of mineralized tissue imaging. Through this method, 3D functional masticatory and therefore occlusal molar microwear is able to be visualized, quantified and comparatively analyzed to assess dietary preference in Javanese Homo erectus. This method differs from other microwear investigative techniques (defining 'pits'- vs- 'scratches', microtexture analysis etc.) in that it defines a molars masticatory microwear functional interactions in 3-dimensions as its baseline dataset for further interpretations and analyses. Due to poor specimen collection techniques employed during the first half of the 20th century, the very complex geologic nature of the Sangiran Dome and disagreements over its chronostratigraphy, only very few scientific works have addressed the Sangiran 7 (S7) Homo erectus molar collection (n=25) (e.g. Grine and Franzen, 1994; Kaifu, 2006). Grine and Franzen's (1994) work was a predominantly qualitative initial assessment of the specimens and identified five specimens that might better be ascribed to a fossil pongid rather than H. erectus. They also noted several molars to which tooth position (M1 or M2) was unable to be ascribed (Grine and Franzen, 1994). Kaifu (2006) comparatively examined crown sizes in several S7 molars.
The Sangiran 7 collection originates from two distinct geologic horizons: ten from the older Sangiran Formation (S7a, ~1.7 to 1.0mya) and fifteen from the younger, overlying Bapang Formation (S7b, ~1.0 to .7mya). During this million year period, Java was connected to the mainland during various glacio-eustatic low-stands in sea level. These mainland connections varied in size, extent, climatic condition and therefore in faunal and floral composition. As the S7 sample may be representative of the earliest Homo erectus migrants into Java and spans long durations of occupation, its investigation yields potential to understand the various influences climatic and ecogeographic fluctuations had on these populations. Since the sample consists only of teeth, an ecodietary approach has been deemed the most logical and appropriate investigative approach. Questions regarding the intra- and inter- S7 sample
relationships will also be addressed.
By comparing various aspects of the H. erectus dentition against that of hunter/ gatherer's (H/G) whose diet is known, functional dietary similarity can be directly correlated. Thus a comparative molar sample consisting of the below historic hunter/ gather's (n=63) has been included in order to assess H. erectus's diet in ecological context: Inuit (n=9), Pacific Northwest Tribes (n=11), Fuegians (n=11), Australian Aborigines (n=12) and Bushman (n=20). Methodologically, this approach produces a 3D facet microwear vector (fmv) signature for each molar which can then be compared for statistical similarity.
Microwear (and, as such, the fmv signatures) was defined by the regular, parallel striations found on specific cusp facets known to arise from patterned, directional masticatory movements. This differs significantly from post-mortem or taphonomic microwear which produces striations at irregular angles on multiple, non-masticatory surfaces (Peuch et al.1985, Teaford, 1988). A 'match value' is produced to determine the similarity of two molars fmv's. The 'match values' are ranked (high to low) and these rankings are used to statistically analyze and infer dietary preference: between Sangiran 7 (as an entire sample) compared against that of the historic hunter/ gatherer H. sapiens whose diet and ecogeography is known; within S7a and S7b and then among the S7 sample (eg. S7a-vs-S7b); whether the purported Pongo molars actually affiliate well with H. erectus, the hunter-gatherer's or if they demonstrate distinctly different fmv signatures altogether; whether fmv signatures are useful in distinguishing molars whose tooth position is in doubt (eg. M1 or M2).
When compared against individual H/G molars, the results show that Sangiran 7 H. erectus most closely correlates with Bushmen across all areas of fmv signature analysis. However, within broader dietary categories (yearly reliant on proteinaceous foods; seasonally reliant on proteinaceous foods; not reliant on proteinaceous foods), it was found that H. erectus most closely allied with the two hunter/ gatherer subpopulations associated with the 'Seasonally reliant on proteinaceous foods' (Australian Aboriginals and Pacific Northwest Tribes). There was also evidence for dietary change or specialization over time. As the environment changed during occupation by the earlier Sangiran to the later Bapang individuals, the dietary preference shifted from a focus on vegetative foods to a diet much more inclusive of proteinaceous resources.
These results are considered logical within the larger ecogeographic and chronostratigraphic context of the Sangiran Dome during the Pleistocene. However, a larger sample would be needed to confirm this. Although general dietary preferences can be drawn from this method, it is not possible at present to define specific foods consumed on a daily basis (eg. tubers or tortoise meat).
Out of the five specimens possibly allied with Pongo, S7-14 matched at the 'high' designation with a hunter/ gatherer, S7-62 matched 'moderately', S7-20 matched 'low' while the remaining two were not able to be matched with any other teeth for various reasons. Although designation to Pongo cannot be ruled on at this time using this method, it does demonstrate that at least two of the teeth correlate well with various hunter/ gatherer's who do not share dietary similarity with Pongo. This suggests their designation as Pongo should be more closely reevaluated. As for the four specimens whose tooth position was unsure, S7-14 matched 'highly' with 1st molars, S7-62 and S7-78 matched 'moderately' with 2nd and 1st molars respectively while S7-20 only matched at the 'low' designation. Although this approach is still exploratory, it adds another analytical tool for use in defining tooth position.
In sum, this method has demonstrated its usefulness in defining and functionally analyzing a novel 3D molar microwear dataset to interpret dietary preference. Future work would include a pan- H. erectus molar sample in order to illuminate broader populational, taxonomic and dietary correlations within and amoung all H. erectus specimens. A larger, more heterogenous historic H/G sample would also be included in order to provide a wider dietary comparative population. This method can be further extended to include and compare any and all hominins as well as any organism which produces micro wear upon it molars. Also, the data obtained and resultant fmv signature diagrams have the potential to be incorporated into 3D VR reconstructions of mandibular movement thus recreating mastication in extinct organisms and leading to more robust anatomical and physiological investigations especially when viewed in the context of larger environmental conditions or changes.
A novel role for mutant mRNA degradation in triggering transcriptional adaptation to mutations
(2020)
Robustness to mutations promotes organisms’ well-being and fitness. The increasing number of mutants in various model organisms, and humans, showing no obvious phenotype (Bouche and Bouchez, 2001; Chen et al., 2016b; Giaever et al., 2002; Kok et al., 2015) has renewed interest into how organisms adapt to gene loss. In the presence of deleterious mutations, genetic compensation by transcriptional upregulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems (El-Brolosy and Stainier, 2017; Rossi et al., 2015; Tondeleir et al., 2012); however, the molecular mechanisms underlying this response remained unclear. To investigate this phenomenon, I develop and study multiple models of transcriptional adaptation in zebrafish and mouse cell lines. I first show that transcriptional adaptation is not caused by loss of protein function, indicating that the trigger lies upstream, and find that the response involves enhanced transcription of the related gene(s). Furthermore, I observe a correlation between levels of mutant mRNA degradation and upregulation of related genes. To investigate the role of mutant mRNA degradation in triggering the response, I generate mutant alleles that do not transcribe the mutated gene and find that they fail to induce a transcriptional response and display stronger phenotypes. Transcriptome analysis of alleles displaying mutant mRNA degradation revealed upregulation of a significant proportion of genes displaying sequence similarity with the mutated gene’s mRNA, suggesting a model whereby mRNA degradation intermediates induce transcriptional adaptation via sequence similarity. Further mechanistic analyses suggested RNA-decay factors-dependent chromatin remodeling, and repression of antisense RNAs to be implicated in the response. These results identify a novel role for mutant mRNA degradation in buffering against mutations. Besides, they hold huge implications on understanding disease-causing mutations and shall help in designing mutations that lead to minimal transcriptional adaptation-induced compensation, facilitating studying gene function in model organisms.
The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
Bei endogenen Retroviren handelt es sich um feste Bestandteile des Genoms. Im Fall von PERV (porzine endogene Retroviren) existieren zusätzlich infektiöse, xenotrope Vertreter. Aufgrund dieser Tatsache ist es notwendig, diese replikationskompetenten Proviren aus dem Genom potentieller Donortiere für die Xenotransplantation zu entfernen. Mit dem Wissen um die chromosomale Lage und der damit verbundenen Möglichkeit des Nachweises per PCR wurde im Rahmen dieser Arbeit gezeigt, dass aufgrund einer polymorphen Verteilung ein Ausschluss dieser funktionellen Proviren, mittels konventioneller Züchtung, möglich ist. Allerdings stellen sowohl die deletierten und mutierten proviralen Sequenzen durch eine Rekombination oder eine Komplementation, als auch ekotrope PERV-C ein Restrisiko im Falle einer Xenotransplantation dar. Es ist eine PERV-A/C Rekombinante beschrieben ex vivo worden, welche eine höhere Infektiösität aufweist als alle bisher untersuchten PERV. Bis auf die Rezeptor-Bindedomäne stellt dieses Virus ein PERV-C dar. Deshalb sollten chromosomal PERV-C identifiziert werden, um bei polymorpher Verteilung im Schweinegenom durch entsprechende Züchtung diese aus dem Genom heraushalten zu können. Im Rahmen dieser Arbeit ist es mit Hilfe einer speziellen PCR gelungen sieben Integrationsorte von PERV-C zu identifizieren. Da das Genom des Schweins bisher noch nicht komplett sequenziert ist, war es noch nicht möglich die gefundenen chromosomalen Bereiche zu kartieren. Dies wäre wiederum die Basis für eine Durchmusterung von Schweinen auf die Anwesenheit der gefundenen Proviren. Des Weiteren ist noch nicht bekannt, ob es sich bei diesen PERV-C um vollständige Proviren handelt, da aufgrund der verwendeten PCR und der sehr hohen Homologie verschiedener PERV untereinander nur provirale 3'Enden mit entsprechenden Flanken identifiziert werden konnten. Zusätzlich wurden in den Proben transgener Schweine PERV-C env spezifische Anteile nachgewiesen. Die Verteilung dieser Sequenzen, welche ebenfalls polymorph ist, gibt zwar keinen Aufschluss über die Anwesenheit eines Volllängen Provirus, jedoch ist aufgrund dieser Verteilung gleichfalls ein Ausschluss dieses Virus durch herkömmliche Züchtung möglich. Auf der anderen Seite besteht ein weiteres Risiko nach einer Xenotransplantation, wenn ein infektiöses PERV durch Komplementation gebildet wird, welches als Erbinformation ein env-deletiertes Provirus trägt. Das komplementierte PERV könnte potentiell, nach erfolgter Xenotransplantation, menschliche Zellen infizieren. Daraufhin wäre es zwar nicht mehr in der Lage infektiöse Partikel zu bilden, jedoch besteht noch das Risiko einer Retrotransposition, welche an sich schon mutagen wirkt. Zusätzlich könnten durch diesen Vorgang Gene zerstört oder Onkogene angeschaltet werden. Um dieses Risiko abschätzen zu können, wurden im Rahmen dieser Arbeit modifizierte Proviren von PERV-B(33) und MoMLV (Positivkontrolle) hergestellt und in einem Retrotranspositions Assay getestet. Die Modifikation der Proviren beinhaltete die Deletion des für die Retrotransposition nicht notwendigen env-Leserahmens, im Austausch gegen eine inserierte Indikatorkassette für die Retrotransposition (neoint). Im Rahmen der in dieser Arbeit durchgeführten Experimente konnte im Fall des Molekularklons PERV-B(33) eine Frequenz der Retrotransposition von maximal 1,2*10-6 pro Zelle und Generation ermittelt werden. Demzufolge stellt eine Retrotransposition von env-deletierten proviralen porzinen Sequenzen nach erfolgter Xenotransplantation ein minimales Risiko dar.
Mitochondial NADH:ubiquinone oxidoreductase (complex I) the largest multiprotein enzyme of the respiratory chain, catalyses the transfer of two electrons from NADH to ubiquinone, coupled to the translocation of four protons across the membrane. In addition to the 14 strictly conserved central subunits it contains a variable number of accessory subunits. At present, the best characterized enzyme is complex I from bovine heart with a molecular mass of about 980 kDa and 32 accessory proteins. In this study, the subunit composition of mitochondrial complex I from the aerobic yeast Y. lipolytica has been analysed by a combination of proteomic and genomic approaches. The sequences of 37 complex I subunits were identified. The sum of their individual molecular masses (about 930 kDa) was consistent with the native molecular weight of approximately 900 kDa for Y. lipolytica complex I obtained by BN-PAGE. A genomic analysis with Y. lipolytica and other eukaryotic databases to search for homologues of complex I subunits revealed 31 conserved proteins among the examined species. A novel protein named “X” was found in purified Y. lipolytica complex I by MALDI-MS. This protein exhibits homology to the thiosulfate sulfurtransferase enzyme referred to as rhodanese. The finding of a rhodanese-like protein in isolated complex I of Y. lipolytica allows to assume a special regulatory mechanism of complex I activity through control of the status of its iron-sulfur clusters. The second part of this study was aimed at investigating the possible role of one of these extra subunits, 39 kDa (NUEM) subunit which is related to the SDRs-enzyme family. The members of this family function in different redox and isomerization reactions and contain a conserved NAD(P)H-binding site. It was proposed that the 39 kDa subunit may be involved in a biosynthetic pathway, but the role of this subunit in complex I is unknown. In contrast to the situation in N. crassa, deletion of the 39 kDa encoding gene in Y. lipolytica led to the absence of fully assembled complex I. This result might indicate a different pathway of complex I assembly in both organisms. Several site-directed mutations were generated in the nucleotide binding motif. These had either no effect on enzyme activity and NADPH binding, or prevented complex I assembly. Mutations of arginine-65 that is located at the end of the second b-strand and responsible for selective interaction with the 2’-phosphate group of NADPH retained complex I activity in mitochondrial membranes but the affinity for the cofactor was markedly decreased. Purification of complex I from mutants resulted in decrease or loss of ubiquinone reductase activity. It is very likely that replacement of R65 not only led to a decrease in affinity for NADPH but also caused instability of the enzyme due to steric changes in the 39 kDa subunit. These data indicate that NADPH bound to the 39 kDa subunit (NUEM) is not essential for complex I activity, but probably involved in complex I assembly in Y. lipolytica.
The enzyme acetyl-CoA carboxylase (ACC) plays a fundamental role in the fatty acid metabolism. It regulates the first and rate limiting step in the biosynthesis of fatty acids by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA and exists as two different isoforms, ACC1 and ACC2. In the last few years, ACC has been reported as an attractive drug target for treating different diseases, such as insulin resistance, hepatic steatosis, dyslipidemia, obesity, metabolic syndrome and nonalcoholic fatty liver disease. An altered fatty acid metabolism is also associated with cancer cell proliferation. In general, the inhibition of ACC provides two possibilities to regulate the fatty acid metabolism: It blocks the de novo lipogenesis in lipogenic tissues and stimulates the mitochondrial fatty acid β-oxidation. Surprisingly, the role of ACC in human vascular endothelial cells has been neglected so far. This work aimed to investigate the role of the ACC/fatty acid metabolism in regulating important endothelial cell functions like proliferation, migration and tube formation.
To investigate the function of ACC, the ACC-inhibitor soraphen A as well as an siRNA-based approach were used. This study revealed that ACC1 is the predominant isoform both in human umbilical vein endothelial cells (HUVECs) and in human dermal microvascular endothelial cells (HMECs). Inhibition of ACC via soraphen A resulted in decreased levels of malonyl-CoA and shifted the lipid composition of endothelial cell membranes. Consequently, membrane fluidity, filopodia formation and the migratory capacity were attenuated. Increasing amounts of longer acyl chains within the phospholipid subgroup phosphatidylcholine (PC) were suggested to overcompensate the shift towards shorter acyl chains within phosphatidylglycerol (PG), which resulted in a dominating effect on regulating the membrane fluidity. Most importantly, this work provided a link between changes in the phospholipid composition and altered endothelial cell migration. The antimigratory effect of soraphen A was linked to a reduced amount of PG and to an increased amount of polyunsaturated fatty acids (PUFAs) within the phospholipid cell membrane. This link was unknown in the literature so far. Interestingly, a reduced filopodia formation was observed upon ACC inhibition via soraphen A, which presumably caused the impaired migratory capacity.
This work revealed a relationship between ACC/fatty acid metabolism, membrane lipid composition and endothelial cell migration. The natural compound soraphen A emerged as a valuable chemical tool to analyze the role of ACC/fatty acid metabolism in regulating important endothelial cell functions. Furthermore, regulating endothelial cell migration via ACC inhibition promises beneficial therapeutic perspectives for the treatment of cell migration-related disorders, such as ischemia reperfusion injury, diabetic angiopathy, macular degeneration, rheumatoid arthritis, wound healing defects and cancer.