12 research outputs found
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
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
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