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Analyse der Funktion der dualen Lokalisation der 3-Mercaptopyruvat Sulfurtransferase im Menschen
(2017)
Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA).
A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products.
The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering.
Mathematical models of bacterial growth have been successfully applied to study the relationship between antibiotic drug exposure and the antibacterial effect. Since these models typically lack a representation of cellular processes and cell physiology, the mechanistic integration of drug action is not possible on the cellular level. The cellular mechanisms of drug action, however, are particularly relevant for the prediction, analysis and understanding of interactions between antibiotics. Interactions are also studied experimentally, however, a lacking consent on the experimental protocol hinders direct comparison of results. As a consequence, contradictory classifications as additive, synergistic or antagonistic are reported in literature.
In the present thesis we developed a novel mathematical model for bacterial growth that integrates cell-level processes into the population growth level. The scope of the model is to predict bacterial growth under antimicrobial perturbation by multiple antibiotics in vitro.
To this end, we combined cell-level data from literature with population growth data for Bacillus subtilis, Escherichia coli and Staphylococcus aureus. The cell-level data described growth-determining characteristics of a reference cell, including the ribosomal concentration and efficiency. The population growth data comprised extensive time-kill curves for clinically relevant antibiotics (tetracycline, chloramphenicol, vancomycin, meropenem, linezolid, including dual combinations).
The new cell-level approach allowed for the first time to simultaneously describe single and combined effects of the aforementioned antibiotics for different experimental protocols, in particular different growth phases (lag and exponential phase). Consideration of ribosomal dynamics and persisting sub-populations explained the decreased potency of linezolid on cultures in the lag phase compared to exponential phase cultures. The model captured growth rate dependent killing and auto-inhibition of meropenem and - also for vancomycin exposure - regrowth of the bacterial cultures due to adaptive resistance development. Stochastic interaction surface analysis demonstrated the pronounced antagonism between meropenem and linezolid to be robust against variation in the growth phase and pharmacodynamic endpoint definition, but sensitive to a change in the experimental duration.
Furthermore, the developed approach included a detailed representation of the bacterial cell-cycle. We used this representation to describe septation dynamics during the transition of a bacterial culture from the exponential to stationary growth phase. Resulting from a new mechanistic understanding of transition processes, we explained the lag time between the increase in cell number and bacterial biomass during the transition from the lag to exponential growth phase. Furthermore, our model reproduces the increased intracellular RNA mass fraction during long term exposure of bacteria to chloramphenicol.
In summary, we contribute a new approach to disentangle the impact of drug effects, assay readout and experimental protocol on antibiotic interactions. In the absence of a consensus on the corresponding experimental protocols, this disentanglement is key to translate information between heterogeneous experiments and also ultimately to the clinical setting.
Chloroplast membranes have a unique composition characterized by very high contents of the galactolipids, MGDG and DGDG. Many studies on constitutive, galactolipid-deficient mutants revealed conflicting results about potential functions of galactolipids in photosynthetic membranes. Likely, this was caused by pleiotropic effects such as starvation artefacts because of impaired photosynthesis from early developmental stages of the plants onward. Therefore, an ethanol inducible RNAi-approach has been taken to suppress two key enzymes of galactolipid biosynthesis in the chloroplast, MGD1 and DGD1. Plants were allowed to develop fully functional source leaves prior to induction, which then could support plant growth. Then, after the ethanol induction, both young and mature leaves were investigated over time.
Our studies revealed similar changes in both MGDG- and DGDG-deficient lines, however young and mature leaves of transgenic lines showed a different response to galactolipid deficiency. While no changes of photosynthetic parameters and minor changes in lipid content were observed in mature leaves of transgenic lines, strong reductions in total chlorophyll content and in the accumulation of all photosynthetic complexes and significant changes in contents of various lipid groups occurred in young leaves. Microscopy studies revealed an appearance of lipid droplets in the cytosol of young leaves in all transgenic lines which correlates with significantly higher levels of TAGs. Since in young leaves the production of membrane lipids is lowered, the excess of fatty acids is used for storage lipids production, resulting in the accumulation of TAGs.
Our data indicate that both investigated galactolipids serve as structural lipids since changes in photosynthetic parameters were mainly the result of reduced amounts of all photosynthetic constituents. In response to restricted galactolipid synthesis, thylakoid biogenesis is precisely readjusted to keep the proper stoichiometry and functionality of the photosynthetic apparatus. Ultimately, the data revealed that downregulation of one galactolipid triggers changes not only in chloroplasts but also in the nucleus as shown by downregulation of nuclear encoded subunits of the photosynthetic complexes.
In this work the human AOX1 was characterized and detailed aspects regarding the expression, the enzyme kinetics and the production of reactive oxygen species (ROS) were investigated. The hAOX1 is a cytosolic enzyme belonging to the molybdenum hydroxylase family. Its catalytically active form is a homodimer with a molecular weight of 300 kDa. Each monomer (150 kDa) consists of three domains: a N-terminal domain (20 kDa) containing two [2Fe-2S] clusters, a 40 kDa intermediate domain containing a flavin adenine dinucleotide (FAD), and a C-terminal domain (85 kDa) containing the substrate binding pocket and the molybdenum cofactor (Moco). The hAOX1 has an emerging role in the metabolism and pharmacokinetics of many drugs, especially aldehydes and N- heterocyclic compounds.
In this study, the hAOX1 was hetereogously expressed in E. coli TP1000 cells, using a new codon optimized gene sequence which improved the expressed protein yield of around 10-fold compared to the previous expression systems for this enzyme. To increase the catalytic activity of hAOX1, an in vitro chemical sulfuration was performed to favor the insertion of the equatorial sulfido ligand at the Moco with consequent increased enzymatic activity of around 10-fold. Steady-state kinetics and inhibition studies were performed using several substrates, electron acceptors and inhibitors. The recombinant hAOX1 showed higher catalytic activity when molecular oxygen was used as electron acceptor. The highest turn over values were obtained with phenanthridine as substrate. Inhibition studies using thioridazine (phenothiazine family), in combination with structural studies performed in the group of Prof. M.J. Romão, Nova Universidade de Lisboa, showed a new inhibition site located in proximity of the dimerization site of hAOX1. The inhibition mode of thioridazine resulted in a noncompetitive inhibition type. Further inhibition studies with loxapine, a thioridazine-related molecule, showed the same type of inhibition. Additional inhibition studies using DCPIP and raloxifene were carried out.
Extensive studies on the FAD active site of the hAOX1 were performed. Twenty new hAOX1 variants were produced and characterized. The hAOX1 variants generated in this work were divided in three groups: I) hAOX1 single nucleotide polymorphisms (SNP) variants; II) XOR- FAD loop hAOX1 variants; III) additional single point hAOX1 variants. The hAOX1 SNP variants G46E, G50D, G346R, R433P, A439E, K1231N showed clear alterations in their catalytic activity, indicating a crucial role of these residues into the FAD active site and in relation to the overall reactivity of hAOX1.
Furthermore, residues of the bovine XOR FAD flexible loop (Q423ASRREDDIAK433) were introduced in the hAOX1. FAD loop hAOX1 variants were produced and characterized for their stability and catalytic activity. Especially the variants hAOX1 N436D/A437D/L438I, N436D/A437D/L438I/I440K and Q434R/N436D/A437D/L438I/I440K showed decreased catalytic activity and stability. hAOX1 wild type and variants were tested for reactivity toward NADH but no reaction was observed.
Additionally, the hAOX1 wild type and variants were tested for the generation of reactive oxygen species (ROS). Interestingly, one of the SNP variants, hAOX1 L438V, showed a high ratio of superoxide prodction. This result showed a critical role for the residue Leu438 in the mechanism of oxygen radicals formation by hAOX1. Subsequently, further hAOX1 variants having the mutated Leu438 residue were produced. The variants hAOX1 L438A, L438F and L438K showed superoxide overproduction of around 85%, 65% and 35% of the total reducing equivalent obtained from the substrate oxidation.
The results of this work show for the first time a characterization of the FAD active site of the hAOX1, revealing the importance of specific residues involved in the generation of ROS and effecting the overall enzymatic activity of hAOX1. The hAOX1 SNP variants presented here indicate that those allelic variations in humans might cause alterations ROS balancing and clearance of drugs in humans.
Im Sinne des Refinements von Tierversuchen sollen alle Bedingungen während der Zucht, der Haltung und des Transports von zu Versuchszwecken gehaltenen Tieren und alle Methoden während des Versuchs so verbessert werden, dass die verwendeten Tiere ein minimales Maß an potentiellem Distress, Schmerzen oder Leiden erfahren. Zudem soll ihr Wohlbefinden durch die Möglichkeit des Auslebens speziesspezifischer Verhaltensweisen und die Anwendung tierschonender Verfahren maximal gefördert werden. Zur Etablierung von Grundsätzen des Refinements sind grundlegende Kenntnisse über die physiologischen Bedürfnisse und Verhaltensansprüche der jeweiligen Spezies unabdingbar. Die Experimentatoren sollten das Normalverhalten der Tiere kennen, um potentielle Verhaltensabweichungen, wie Stereotypien, zu verstehen und interpretieren zu können. Standardisierte Haltungsbedingungen von zu Versuchszwecken gehaltenen Mäusen weichen in diversen Aspekten von der natürlichen Umgebung ab und erfordern eine gewisse Adaptation. Ist ein Tier über einen längeren Zeitraum unfähig, sich an die gegebenen Umstände anzupassen, können abnormale Verhaltensweisen, wie Stereotypien auftreten. Stereotypien werden definiert als Abweichungen vom Normalverhalten, die repetitiv und ohne Abweichungen im Ablauf ausgeführt werden, scheinbar keiner Funktion dienen und der konkreten Umweltsituation nicht immer entsprechen.
Bisher war unklar, in welchem Ausmaß stereotypes Verhalten den metabolischen Phänotyp eines Individuums beeinflusst. Ziel dieser Arbeit war es daher, das stereotype Verhalten der FVB/NJ-Maus erstmals detailliert zu charakterisieren, systematisch zusammenzutragen, welche metabolischen Konsequenzen dieses Verhalten bedingt und wie sich diese auf das Wohlbefinden der Tiere und die Verwendung stereotyper Tiere in Studien mit tierexperimentellem Schwerpunkt auswirken.
Der Versuch begann mit der Charakterisierung der mütterlichen Fürsorge in der Parentalgeneration. Insgesamt wurden 35 Jungtiere der F1-Generation vom Absatz an, über einen Zeitraum von 11 Wochen einzeln gehalten, kontinuierlich beobachtet, bis zum Versuchsende wöchentlich Kotproben gesammelt und das Körpergewicht bestimmt. Zusätzlich erfolgten begleitende Untersuchungen wie Verhaltenstests und die Erfassung der physischen Aktivität und metabolischer Parameter. Anschließend wurden u.a. die zerebralen Serotonin- und Dopamingehalte, fäkale Glucocorticoidlevels, hepatisches Glykogen und muskuläre Glykogen- und Triglyceridlevels bestimmt.
Nahezu unabhängig von der mütterlichen Herkunft entwickelte sich bei mehr als der Hälfte der 35 Jungtiere in der F1-Generation stereotypes Verhalten. Diese Daten deuten darauf hin, dass es keine Anzeichen für das Erlernen oder eine direkte genetische Transmission stereotypen Verhaltens bei der FVB/NJ-Maus gibt. Über den gesamten Beobachtungszeitraum zeichneten sich die stereotypen FVB/NJ-Mäuse durch ein eingeschränktes Verhaltensrepertoire aus. Zu Gunsten der erhöhten Aktivität und des Ausübens stereotypen Verhaltens lebten sie insgesamt weniger andere Verhaltensweisen (Klettern, Graben, Nagen) aus. Darüber hinaus waren Stereotypien sowohl im 24-Stunden Open Field Test als auch in der Messeinrichtung der indirekten Tierkalorimetrie mit einer erhöhten Aktivität und Motilität assoziiert, während die circadiane Rhythmik nicht divergierte. Diese erhöhte körperliche Betätigung spiegelte sich in den niedrigeren Körpergewichtsentwicklungen der stereotypen Tiere wieder. Außerdem unterschieden sich die Körperfett- und Körpermuskelanteile.
Zusammenfassend lässt sich sagen, dass das Ausüben stereotypen Verhaltens zu Differenzen im metabolischen Phänotyp nicht-stereotyper und stereotyper FVB/NJ-Mäuse führt. Im Sinne der „Guten Wissenschaftlichen Praxis“ sollte das zentrale Ziel jedes Wissenschaftlers sein, aussagekräftige und reproduzierbare Daten hervorzubringen. Jedoch können keine validen Resultate von Tieren erzeugt werden, die in Aspekten variieren, die für den vorgesehenen Zweck der Studie nicht berücksichtigt wurden. Deshalb sollten nicht-stereotype und stereotype Individuen nicht innerhalb einer Versuchsgruppe randomisiert werden. Stereotype Tiere demzufolge von geplanten Studien auszuschließen, würde allerdings dem Gebot des zweiten R’s – der Reduction – widersprechen. Um Refinement zu garantieren, sollte der Fokus auf der maximal erreichbaren Prävention stereotypen Verhaltens liegen. Diverse Studien haben bereits gezeigt, dass die Anreicherung der Haltungsumwelt (environmental enrichment) zu einer Senkung der Prävalenz von Stereotypien bei Mäusen führt, dennoch kommen sie weiterhin vor. Daher sollte environmental enrichment zukünftig weniger ein „Kann“, sondern ein „Muss“ sein – oder vielmehr: der Goldstandard. Zudem würde eine profunde phänotypische Charakterisierung dazu beitragen, Mausstämme zu erkennen, die zu Stereotypien neigen und den für den spezifischen Zweck am besten geeigneten Mausstamm zu identifizieren, bevor ein Experiment geplant wird.