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The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor beta (TGF-\(\beta\)) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-beta-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 angstrom crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.
Ziel der vorliegenden tierexperimentellen Studie war es, Unterschiede im Einheilverhalten der Werkstoffe Titan und VA-Stahl (316L) anhand der Matrixproteine Kollagen Typ I (C1), Kollagen Typ III (C3) und Fibronektin im implantatumgebenden Interface zu untersuchen und darzustellen. Hierzu wurden die Einheilkapseln der Implantate nach subkutaner, intramuskulärer und intraossärer Implantation nach den Bewertungskriterien Kapselqualität, Kapseldicke und Verteilungsmuster der Matrixproteine mittels konventioneller Mikroskopie und Konfokaler Laserscanning Mikroskopie (CLSM) analysiert. Nach subkutaner Implantation zeigten beide Werkstoffe in Übereinstimmung mit den Ergebnissen von SHANNON et al. (1997) vermehrt locker angeordnete, teils parallel orientierte Kollagenfasern mit erhöhtem Zellaufkommen an Fibroblasten und Makrophagen. Nach intramuskulärer Implantation jedoch fanden sich vorwiegend parallel angeordnete, teils dicht gepackte Kollagenfasern mit nur mäßig erhöhtem Zellaufkommen. Intramuskulär eingebrachte Implantate heilten in dünneren Kapseln ein, als subkutan eingebrachte Implantate. Es ergab sich keine Korrelation zu den ermittelten Kapselqualitäten. Dies erstaunt umso mehr, da unter der fortwährenden funktionellen Beanspruchung der intramuskulären Implantate im Bereich der Bauchmuskulatur gegenüber der statischeren Platzierung im subkutanen Rückenfett eine erhöhte Zell- und Matrixreaktion erwartet worden war. Im Lokalisationsvergleich zeigte sich intramuskulär für beide Werkstoffe ein erhöhtes Aufkommen an Fibronektin. Dies könnte auf die erhöhte Stoffwechselaktivität und funktionelle Belastung im Muskelgewebe zurückgeführt werden (ROSENGREN et al. 1994). Nach intraossärer Implantation konnten dünnere Kallusformationen für VA-Stahl gegenüber Titan in allen Proteinfluoreszenzen nachgewiesen werden. Die Qualität der Kallusformation und die histologische Kallusstruktur glichen sich mit zunehmender Implantationsdauer der regulären Knochenstruktur an. Die semiquantitativ beurteilte Verteilung der Matrixproteine mittels CLSM zeigte bei deutlichen Standardabweichungen für beide Werkstoffe erhöhte Fluoreszenz-Intensitäten nur in den implantatnahen Kapselanteilen. In den mittleren und den implantatfernen Kapselabschnitten waren für beide Werkstoffe inkonstant höhere Fluoreszenzwerte gegenüber den Vergleichskollektiven messbar. Der intraossäre Materialvergleich ergab implantatnahe und implantatferne Fluoreszenzmaxima für alle Matrixproteine, die mit zunehmender Implantationsdauer abfielen. Reproduzierbare, materialspezifische Unterschiede waren in Analogie zu BERGER-GORBET et al. (1996) nicht zu finden. In den mittleren Kallusabschnitten konnten reproduzierbare Fluoreszenzunterschiede nur bei Detektion von Kollagen Typ I (C1) in allen Zeitintervallen gesehen werden. Im Vergleich zur Literatur konnte die von VIROLAINEN et al. (1997) beschriebene biphasische Proteinanhäufung, wie auch ein wechselndes Proteinaufkommen (LINDHOLM et al. 1996) nach intraossärer Implantation nicht nachvollzogen werden. Ergänzende Beobachtungen der hier vorgestellten Studie verdeutlichen, dass die lokale, intraossäre Anreicherung von Matrixproteinen, unabhängig von Implantatinsertion oder gar Werkstoffeigenschaften, nach jeglicher Traumatisierung von Knochengewebe den knöchernen Reparationsprozess begleitet. Unter dem Aspekt der Restitutio ad Integrum von Knochenwunden können diese Beobachtungen auf das implantatnahe und das implantatferne Restitutionszentrum übertragen werden. Die Aktivität dieser Restitutionszentren hält bis zum Abschluss der knöchernen Remodellierung über 12 Wochen hinaus an. Dies deckt sich mit Aussagen von STEFLIK et al. (1998), wonach der periimplantäre Knochenumbau langfristig dynamisch bestehen bleibt. Um der zunehmenden Verbreitung nicht nur dentaler Implantate gerecht zu werden, muss auch zukünftig ein besseres Verständnis der Komunikationswege zwischen Implantaten und Biosystemen gewonnen werden. Dies bedeutet für die Herstellung und Weiterentwicklung von Implantaten, dass nicht nur die Werkstoff- und Oberflächenauswahl wichtig ist, sondern auch die funktionell erforderliche Oberflächenstrukturierung auf die gewünschte Wechselwirkung mit Bestandteilen der EZM und den Zellen angepasst sein sollte (THULL 2005). Die CLSM kann hierbei aufgrund der Möglichkeit der 3-dimensionalen in-situ-Darstellung des Implantatinterface biologisch-strukturelle und molekularbiologisch-immunologische Fragestellungen beantworten.
Cytosine methylation is a conserved epigenetic feature found throughout the phylum Platyhelminthes
(2013)
Background: The phylum Platyhelminthes (flatworms) contains an important group of bilaterian organisms responsible for many debilitating and chronic infectious diseases of human and animal populations inhabiting the planet today. In addition to their biomedical and veterinary relevance, some platyhelminths are also frequently used models for understanding tissue regeneration and stem cell biology. Therefore, the molecular (genetic and epigenetic) characteristics that underlie trophic specialism, pathogenicity or developmental maturation are likely to be pivotal in our continued studies of this important metazoan group. Indeed, in contrast to earlier studies that failed to detect evidence of cytosine or adenine methylation in parasitic flatworm taxa, our laboratory has recently defined a critical role for cytosine methylation in Schistosoma mansoni oviposition, egg maturation and ovarian development. Thus, in order to identify whether this epigenetic modification features in other platyhelminth species or is a novelty of S. mansoni, we conducted a study simultaneously surveying for DNA methylation machinery components and DNA methylation marks throughout the phylum using both parasitic and non-parasitic representatives.
Results: Firstly, using both S. mansoni DNA methyltransferase 2 (SmDNMT2) and methyl-CpG binding domain protein (SmMBD) as query sequences, we illustrate that essential DNA methylation machinery components are well conserved throughout the phylum. Secondly, using both molecular (methylation specific amplification polymorphism, MSAP) and immunological (enzyme-linked immunoabsorbent assay, ELISA) methodologies, we demonstrate that representative species (Echinococcus multilocularis, Protopolystoma xenopodis, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica and Polycelis nigra) within all four platyhelminth classes (Cestoda, Monogenea, Trematoda and 'Turbellaria') contain methylated cytosines within their genome compartments.
Conclusions: Collectively, these findings provide the first direct evidence for a functionally conserved and enzymatically active DNA methylation system throughout the Platyhelminthes. Defining how this epigenetic feature shapes phenotypic diversity and development within the phylum represents an exciting new area of metazoan biology.
Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 \(\mu\)M blocked the IL-1\(\beta\)-induced nuclear translocation of NF-\(\kappa\)B/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1\(\beta\)-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1\(\beta\)-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.
In the course of a screen designed to produce antibodies (ABs) with affinity to proteins in the honey bee brain we found an interesting AB that detects a highly specific epitope predominantly in the nuclei of Kenyon cells (KCs). The observed staining pattern is unique, and its unfamiliarity indicates a novel previously unseen nuclear structure that does not colocalize with the cytoskeletal protein f-actin. A single rod-like assembly, 3.7-4.1 mu m long, is present in each nucleus of KCs in adult brains of worker bees and drones with the strongest immuno-labelling found in foraging bees. In brains of young queens, the labelling is more sporadic, and the rod-like structure appears to be shorter (similar to 2.1 mu m). No immunostaining is detectable in worker larvae. In pupal stage 5 during a peak of brain development only some occasional staining was identified. Although the cellular function of this unexpected structure has not been determined, the unusual distinctiveness of the revealed pattern suggests an unknown and potentially important protein assembly. One possibility is that this nuclear assembly is part of the KCs plasticity underlying the brain maturation in adult honey bees. Because no labelling with this AB is detectable in brains of the fly Drosophila melanogaster and the ant Camponotus floridanus, we tentatively named this antibody AmBNSab (Apis mellifera Brain Neurons Specific antibody). Here we report our results to make them accessible to a broader community and invite further research to unravel the biological role of this curious nuclear structure in the honey bee central brain.
Nowadays, computational-aided investigations become an essential part in the chemical, biochemical or pharmaceutical research. With increasing computing power, the calculation of larger biological systems becomes feasible. In this work molecular mechanical (MM) and quantum mechanical approaches (QM) and the combination of both (QM/MM) have been applied to study several questions which arose from different working groups. Thus, this work comprises eight different subjects which deals with chemical reactions or proton transfer in enzymes, conformational changes of ligands or proteins and verification of experimental data.
This work firstly deals with reaction mechanisms of aromatic inhibitors of cysteine proteases which can be found in many organisms. These enzymes are responsible for various cancer or diseases as for example Human African Trypanosomiasis (HAT) or the Chagas disease. Aromatic SNAr-type electrophiles might offer a new possibility to covalently modify these proteases. Quantum mechanical calculations have been performed to gain insights into the energetics and possible mechanisms.
The next chapter also deals with Trypanosomiasis but the focus was set on a different enzyme. The particularity of Trypanosomiasis is the thiol metabolism which can also be modified by covalent inhibitors. In this context, the wild type and point mutations of the enzyme tryparedoxin have been investigated via molecular dynamic (MD) simulations to examine the influence of specific amino acids in regard to the inhibitor. Experimental data showed that a dimerization of the enzyme occurs if the inhibitor is present. Simulations revealed that the stability of the dimer decreases in absence of the inhibitor and thus confirms these experiments.
Further investigations concerning cysteine proteases such as cruzain and rhodesain have been conducted with respect to experimental kinetic data of covalent vinylsulfone inhibitors. Several approaches such as QM or QM/MM calculations and docking, MD or MMPBSA/MMGBSA simulations have been applied to reproduce these data. The utilization of force field approaches resulted in a qualitatively accurate prediction.
The kinase AKT is involved in a range of diseases and plays an important role in the formation of cancer. Novel covalent-allosteric inhibitors have been developed and crystallized in complex with AKT. It was shown that depending on the inhibitor a different cysteine residue is modified. To investigate these differences in covalent modification computational simulations have been applied.
Enoyl-(acyl carrier) (ENR) proteins are essential in the last step of the fatty acid biosynthesis II (FAS) and represent a good target for inhibition. The diphenylether inhibitor SKTS1 which was originally designed to target the ENR’s of Staphylococcus aureus was also crystallized in InhA, the ENR of Mycobacterium tuberculosis (TB). Crystal structures indicate a change of the inhibitor's tautomeric form. This subject was investigated via MD simulations. Results of these simulations confirmed the tautomerization of the inhibitor.
This work also deals with the development of a covalent inhibitor originating from a non-covalent ligand. The target FadA5 is an essential enzyme for the degradation of steroids in TB and is responsible for chronic tuberculosis. This enzyme was crystallized in complex with a non-covalent ligand which served as starting point for this study. Computations on QM or QM/MM level and docking and MD simulations have been applied to evaluate potential candidates.
The next chapter focuses on the modification of the product spectrum of Bacillus megaterium levansucrase, a polymerase which catalyzes the biosynthesis of fructans. The covalent modification of the wild type or mutants of the enzyme lead to an accumulation of oligosaccharides but also to polymers with higher polymerization degree. To understand these changes in product spectra MD simulations have been performed.
Finally, the proton transfer in catalytic cysteine histidine dyads was investigated. The focus was set on the influence of the relaxation of the protein environment to the reaction. Calculations of the enzymes FadA5 and rhodesain revealed that the preferred protonation state of the dyade depends on the protein environment and has an impact on the reaction barrier. Furthermore, the adaptation of the environment to a fixed protonation state was analyzed via MD simulations.
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 mu m, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 mu m thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons. Pauli et al. develop targeted volumetric dSTORM in order to image large hippocampal mossy fiber boutons (MFBs) in brain slices. They can identify synaptic targets of individual MFBs and measured size and density of Bassoon clusters within individual untruncated MFBs at nanoscopic resolution.
Fanconi anemia (FA) is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs). Mutations in 17 genes (FANCA-FANCS) have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2), UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.
After a large outbreak in Brazil, novel drugs against Zika virus became extremely necessary. Evaluation of virus-based pharmacological strategies concerning essential host factors brought us to the idea that targeting the Axl receptor by blocking its dimerization function could be critical for virus entry. Starting from experimentally validated compounds, such as RU-301, RU-302, warfarin, and R428, we identified a novel compound 2′ (R428 derivative) to be the most potent for this task amongst a number of alternative compounds and leads. The improved affinity of compound 2′ was confirmed by molecular docking as well as molecular dynamics simulation techniques using implicit solvation models. The current study summarizes a new possibility for inhibition of the Axl function as a potential target for future antiviral therapies.