Digitalisierung und Demokratie

Die Digitalisierung spielt bei den Prozessen und Entwicklungen in einer Demokratie eine immer größere Rolle. Denn Digitalisierung erweitert die Möglichkeiten der Information, Kommunikation und Partizipation. Gleichzeitig können digitale Technologien zu einer schnellen Verbreitung von Falschinformationen beitragen und bergen ein Potenzial für Meinungsmanipulation, zum Beispiel vor Wahlen. Dieses Spannungsfeld ist Thema der Stellungnahme "Digitalisierung und Demokratie". Darin analysieren die Autorinnen und Autoren Aspekte des Zusammenspiels von Digitalisierung und Demokratie. Darauf aufbauend formulieren sie Handlungsempfehlungen zur Gestaltung künftiger Entwicklungen durch Politik, Recht und Zivilgesellschaft

GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGF beta signalling pathway and regulates the activity of the TGF beta receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGF beta signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.Activation of thermogenic adipocytes is a strategy to combat metabolic diseases. Here the authors report that GPR180 is a component of TGF beta signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1, and contributes to the regulation of glucose and energy metabolism

In vitro selection of streptavidin binding peptides

#### Titelblätter #### Inhaltsverzeichnis #### 1\. Einleitung 1 #### 2\. Zielsetzung 17 #### 3\. Methoden 18 #### 4\. Ergebnisse 48 #### 5\. Diskussion 97 #### 6\. Zusammenfassung 110 #### 7\. Literaturverzeichnis 112 #### 8\. Anhang 118Das überaus große Interesse an der Identifikation von Protein-Protein Wechselwirkungen gipfelte bereits Mitte der 80er Jahre mit dem 'Phage Display' in einem ersten Selektionssystem. Die Anfang der 90er Jahre aufkommende Methode zur in vitro Selektion von Nukleinsäuren, welche die schnelle Generierung von Aptameren und Ribozymen ermöglicht, verdeutlichte die enormen Vorteile und Möglichkeiten eines in vitro Systems, die von den zur Verfügung stehenden Bibliotheken mit bis zu 1016 verschiedenen Molekülen herrühren. Da Proteine im Gegensatz zu Nukleinsäuren eine größere Palette an strukturellen und katalytischen Eigenschaften in der Biologie bereitstellen und wesentlich intensiver in der Diagnostik, Therapie und bei industriellen Applikationen verwendet werden, bestand großes Interesse in der Entwicklung von Methoden zur in vitro Selektion von Proteinen. Ein Ende der 90er Jahre entwickelter Ansatz ermöglicht die in vitro Selektion und Evolution von Proteinen durch Kopplung des Genotyps, in Form der mRNA, mit dem Phänotyp, in Form des Proteins und erhielt den Namen 'Ribosome Display'. Diese Arbeit beschreibt die Selektion streptavidinbindender Peptide, sowie die Entwicklung der dafür benötigten Technologien. Streptavidin wurde als Zielmolekül ausgewählt, da eine Vielzahl von kommerziell erhältlichen Streptavidinkonjugaten eine breite Anwendung in den Biowissenschaften finden. Basierend auf einem zellfreien, prokaryontischen Translationssystem erfolgte die Etablierung des 'Ribosome Display' anhand eines Modellsystems, dessen Grundlage erstmals kein Antikörper, sondern ein His-tag, also ein einfaches Affinitätspeptid war. Es wurden zwei Nukleinsäure kodierte Peptidbibliotheken generiert, wobei der randomisierte Anteil in einem Fall aus 16 NNS-Codons besteht und die 'Loopstruktur' des Chymotrypsin- Inhibitors 2 aus dem Gerstenkorn kodiert. Die zweite Bibliothek umfaßt 15 NNS- Codons und kodiert den N-Terminus des fettsäurebindenden Proteins (FABP) aus Rinderherz. Aus der kombinatorischen FABP-Bibliothek konnten mit Hilfe des 'Ribosome Display' nach sieben in vitro Selektionsrunden streptavidinbindende Peptide isoliert werden. Klonierung und Sequenzierung ergaben eine Reihe unterschiedlicher Peptide, die sich im wesentlichen in zwei Gruppen unterteilen ließen. Bei der größeren Gruppe handelt es sich um Peptide mit einem HPQ-Motiv, das bereits aus anderen Arbeiten bekannt ist. Bei der anderen Gruppe handelt es sich um völlig neuartige Peptide, die am N-terminus ein DVEAW-Motiv aufweisen. Aus dieser Gruppe geht der beste Binder mit der Sequenz DVEAWLDERVP-LVET und einem Kd-Wert von etwa 84 nM hervor, der sich zudem hervorragend als Affinitätspeptid zur Aufreinigung rekombinanter Proteine eignet. [if !supportEmptyParas] [endif]The great interest in the identification of protein-protein interactions culminated in the first selection system which was phage display in the middle of the eighties. Beginning of the nineties a method for the in vitro selection of nucleic acids was introduced, which enables the rapid generation of aptamers and ribozymes and highlighted the enormous advantages and possibilities of an in vitro system stemming from the large initial libraries with up to 1016 different members. Because proteins in contrast to nucleic acids carry out a wider range of structural and catalytic roles in biology and are much more extensively used in diagnostic, therapeutic, and industrial applications, great interest has been generated in the development of methods for the in vitro selection of proteins. At the end of the nineties a method for the in vitro selection and evolution of proteins was developed and designated ribosome display. This work describes the selection of streptavidin-binding peptides and the development of the required technologies. Streptavidin was chosen as target molecule, because a large number of commercial available streptavidin-conjugates is widely used in the field of life science. A cell-free, prokaryotic translation system was the basis for establishing the ribosome display technique and took place with help of a model system. For the first time such a model system was based on a simple affinity tag, namely His-tag and not an antibody. Two nucleic acid encoded peptide libraries had been generated. The random portion of the one library consisted of 16 NNS-codons and encodes the loopstructure of the chymotrypsin inhibitor 2 from barley seeds. The other library contained 15 NNS-codons and encodes the N-terminus of the fatty acid binding protein (FABP) from bovine heart. It was possible to isolate streptavidin-binding peptides from the combinatorial FABP-library after seven rounds of in vitro selection. Cloning and sequencing revealed a number of different peptides which could be divided in two groups. The larger group involves peptides with a HPQ-motif which is known from other selections. The other group involves totally new peptides with a DVEAW-motif at their N-terminus. Out of this group the best binder evolves with the sequence DVEAWLDERV-PLVET and a Kd value of about 84 nM. This peptide is outstandingly suitable as an affinity peptide for the purification of recombinant proteins. [if !supportEmptyParas] [endif

The cell-free protein biosynthesis - applications and analysis of the system.

The in vitro protein biosynthesis has the potentials to become a powerful technology for biochemical research. Beside the determination of structure and function the in vitro evolution of proteins is also of great interest. The system described was used to produce bovine heart fatty acid binding protein (FABP) and bacterial chloramphenicol acetyltransferase (CAT) with and without fusion of the Strep-tag II affinity peptide. The proteins were purified after and during protein biosynthesis by using a StrepTactin Sepharose matrix. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the protein was observed. The in vitro evolution of proteins is feasible by means of ribosome display. The selection of a specific mRNA coding for a shortened FABP with a N-terminal His-tag via the accompanying protein property was shown. Goal of the selection was to bind the FABP via the His-tag on Ni(II)-IDA-agarose. After nine cycles of transcription, translation, affinity selection and RT-PCR the protein with the His-tag could be enriched 108-fold. In order to correlate a possible relationship between changes in protein population and biological function studies were initiated in which 2-dimensional protein patterns of the total in vitro system were compared after 0 and 2 h reaction time. The very interesting findings are that a number of proteins disappear, while others are newly formed during protein synthesis

Riboswitch-mediated Attenuation of Transgene Cytotoxicity Increases Adeno-associated Virus Vector Yields in HEK-293 Cells

Cytotoxicity of transgenes carried by adeno-associated virus (AAV) vectors might be desired, for instance, in oncolytic virotherapy or occur unexpectedly in exploratory research when studying sparsely characterized genes. To date, most AAV-based studies use constitutively active promoters (e.g., the CMV promoter) to drive transgene expression, which often hampers efficient AAV production due to cytotoxic, antiproliferative, or unknown transgene effects interfering with producer cell performance. Therefore, we explored artificial riboswitches as novel tools to control transgene expression during AAV production in mammalian cells. Our results demonstrate that the guanine-responsive GuaM8HDV aptazyme efficiently attenuates transgene expression and associated detrimental effects, thereby boosting AAV vector yields up to 23-fold after a single addition of guanine. Importantly, riboswitch-harboring vectors preserved their ability to express functional transgene at high levels in the absence of ligand, as demonstrated in a mouse model of AAV-TGFβ1-induced pulmonary fibrosis. Thus, our study provides the first application-ready biotechnological system-based on aptazymes, which should enable high viral vector yields largely independent of the transgene used. Moreover, the RNA-intrinsic, small-molecule regulatable mode of action of riboswitches provides key advantages over conventional transcription factor-based regulatory systems. Therefore, such riboswitch vectors might be ultimately applied to temporally control therapeutic transgene expression in vivo.publishe

AAV-mediated expression of a new conformational anti-aggregated α-synuclein antibody prolongs survival in a genetic model of α-synucleinopathies

International audiencePrion-like transmission of pathology in α-synucleinopathies like Parkinson’s disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier

FAM3D: A gut secreted protein and its potential in the regulation of glucose metabolism

The number of diabetic patients is rising globally and concomitantly so do the diabetes associated complications. The gut secretes a variety of proteins to control blood glucose levels and/or food intake. As the drug class of GLP-1 agonists is based on a gut secreted peptide and the positive metabolic effects of bariatric surgery are at least partially mediated by gut peptides, we were interested in other gut secreted proteins which have yet to be explored. In this respect we identified the gut secreted protein FAM3D by analyzing sequencing data from L- and epithelial cells of VSG and sham operated as well as chow and HFD fed mice. FAM3D was overexpressed in diet induced obese mice via an adeno-associated virus (AAV), which resulted in a significant improvement of fasting blood glucose levels, glucose tolerance and insulin sensitivity. The liver lipid deposition was reduced, and the steatosis morphology was improved. Hyperinsulinemic clamps indicated that FAM3D is a global insulin sensitizer and increases glucose uptake into various tissues. In conclusion, the current study demonstrated that FAM3D controls blood glucose levels by acting as an insulin sensitizing protein and improves hepatic lipid deposition.ISSN:1873-5169ISSN:0196-978

GPR180 is a component of TGFβ signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGFβ signalling pathway and regulates the activity of the TGFβ receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGFβ signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism

GPR180 is a component of TGF beta signalling that promotes thermogenic adipocyte function and mediates the metabolic effects of the adipocyte-secreted factor CTHRC1

Activation of thermogenic brown and beige adipocytes is considered as a strategy to improve metabolic control. Here, we identify GPR180 as a receptor regulating brown and beige adipocyte function and whole-body glucose homeostasis, whose expression in humans is associated with improved metabolic control. We demonstrate that GPR180 is not a GPCR but a component of the TGFβ signalling pathway and regulates the activity of the TGFβ receptor complex through SMAD3 phosphorylation. In addition, using genetic and pharmacological tools, we provide evidence that GPR180 is required to manifest Collagen triple helix repeat containing 1 (CTHRC1) action to regulate brown and beige adipocyte activity and glucose homeostasis. In this work, we show that CTHRC1/GPR180 signalling integrates into the TGFβ signalling as an alternative axis to fine-tune and achieve low-grade activation of the pathway to prevent pathophysiological response while contributing to control of glucose and energy metabolism.ISSN:2041-172