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Auf der Suche nach neuen Vitamin D-responsiven Genen wurde die Methodik des Differential Display verwendet. Die eingesetzten Oligonukleotid-Primer waren homolog zur 24-Hydroxylase, einem für den Vitamin D-Stoffwechsel wichtigen Enzym, das von Vitamin D selbst reguliert wird. Mit diesem Ansatz sollten neue Mitglieder aus der Familie der P450-Enzyme gefunden werden. Mehrere differentiell exprimierte DNA-Fragmente wurden in der Folge isoliert und aufgearbeitet, die Sequenzierung eines 550 bp großen Fragments ergab beim Datenbankabgleich keine signifikanten Homologien und ließ daher auf ein noch unbekanntes Gen schließen. Die Klonierung und weitere Charakterisierung dieses Gens wies letztlich in eine nicht erwartete Richtung. Mit der Puromycin-sensitiven Aminopeptidase war ein Gen aus der Familie der Metallopeptidasen gefunden worden, eine Exopeptidase mit einem Zink-Ion im katalytischen Zentrum. Proteinchemisch schon vor einigen Jahren isoliert, war über die PSA (Puromycin-sensitive Aminopeptidase) bekannt, daß sie N-terminale Aminosäuren hydrolysiert mit einer Spezifität für basisch / neutrales und hydrophobes Substrat und überwiegend im Cytoplasma lokalisiert ist. Namengebend ist die starke Hemmbarkeit der Enzymaktivität durch Puromycin. Vorstehendes summiert die wichtigsten biochemischen Eigenschaften des Enzyms, aus molekularbiologischer Sicht war zum Zeitpunkt der Aufnahme der Arbeiten jedoch noch wenig über die PSA bekannt. Dies hat sich in letzter Zeit geändert, während der Sequenzierarbeiten im Rahmen dieser Arbeit konnte eine schweizer Arbeitsgruppe die Klonierung und Sequenz-Charakterisierung der murinen und später humanen cDNA des Gens veröffentlichen. Mit den Ergebnissen dieser Arbeitsgruppe wie auch den im Zuge der vorliegenden Dissertation erarbeiteten Resultaten konnte das molekularbiologische Wissen um die PSA erheblich erweitert werden. So liegt mittlerweile die komplette Nukleotid- und Aminosäurensequenz des Gens vor, das Auffinden des Zink-Bindungsmotives HEXXH(X)18E erlaubte die Einordnung des Enzyms in die Familie M1 der Metallopeptidasen, weiterhin konnte das Gen auf dem Chromosom 17q21 lokalisiert werden. Weitere Daten zur Gewebe- und Zellinienexpression sind nun ebenfalls vorhanden, interessant hierbei neben der erwartet starken Expression im Gehirn das deutliche Vorhandensein in Hoden und Nebennierenrinde. Auch in Bezug auf die Regulation der PSA wurden neue Ergebnisse erzielt, wichtigstes Resultat im Rahmen dieser Arbeit ist hier, daß sich die früher festgestellte Vitamin D –Responsivität nicht bestätigen ließ. Für die Zukunft von außerordentlichem Interesse dürfte das kürzlich gelungene Erstellen PSA-defizienter Mäuse sein, das nun erste Beobachtungen von Auswirkungen der Gen-Deletion am lebenden Organismus erlaubt. Trotz alledem sind in Bezug auf die physiologische Rolle der Puromycin-sensitiven Aminopeptidase weiterhin viele Fragen ungeklärt. Wie hoffentlich in dieser Dissertation herausgearbeitet, ergeben sich dabei interessante Perspektiven für die mögliche Funktion des Enzyms im Organismus.
Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.
Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).
Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.
In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.
The embryonic vertebrate heart tube develops an atrioventricular canal that divides the atrial and ventricular chambers, forms atrioventricular conduction tissue and organizes valve development. Here we assess the transcriptional mechanism underlying this localized differentiation process. We show that atrioventricular canal-specific enhancers are GATA-binding site-dependent and act as switches that repress gene activity in the chambers. We find that atrioventricular canal-specific gene loci are enriched in H3K27ac, a marker of active enhancers, in atrioventricular canal tissue and depleted in H3K27ac in chamber tissue. In the atrioventricular canal, Gata4 activates the enhancers in synergy with Bmp2/Smad signalling, leading to H3K27 acetylation. In contrast, in chambers, Gata4 cooperates with pan-cardiac Hdac1 and Hdac2 and chamber-specific Hey1 and Hey2, leading to H3K27 deacetylation and repression. We conclude that atrioventricular canal-specific enhancers are platforms integrating cardiac transcription factors, broadly active histone modification enzymes and localized co-factors to drive atrioventricular canal-specific gene activity.
In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage.
The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.
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.
Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet‐specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti‐GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators.
The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include 200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.
Highlights
• Loss of DNAJC19's DnaJ domain disrupts cardiac mitochondrial structure, leading to abnormal cristae formation in iPSC-CMs.
• Impaired mitochondrial structures lead to an increased mitochondrial respiration, ROS and an elevated membrane potential.
• Mutant iPSC-CMs show sarcomere dysfunction and a trend to more arrhythmias, resembling DCMA-associated cardiomyopathy.
Background
Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy.
Methods
We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca\(^{2+}\) kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tv\(_{HeLa}\)).
Results
Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca\(^{2+}\) concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation.
Conclusions
Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca\(^{2+}\) kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.