Key stability attributes used from target protein development to process optimization

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Detektion von endokrinen Disruptoren mittels Fluoreszenzkorrelationsspektroskopie

In den letzten Jahren hat das Auftreten sogenannter endokriner Disruptoren zunehmend Besorgnis erregt. Es handelt sich hierbei um Substanzen, die das endokrine System stören und somit eine erhebliche Gefahr für Mensch und Tier darstellen. Diese Substanzen sind im menschlichen Alltag allgegenwärtig und finden sich beispielsweise in Kosmetika, Reinigungsmitteln und Kunststoffen, aber auch in Pestiziden und industriellen Chemikalien. Ihnen ist nur die biologische Aktivität, das heißt ihre hormonelle Wirkung gemein, nicht aber ihre chemische Identität. Häufig ist der Östrogenrezeptor Zielscheibe endokriner Disruption. Dieser Rezeptor gehört zur großen Familie der Steroidrezeptoren und wird normalerweise durch das Hormon Östrogen (17b-Estradiol) kontrolliert. Er ist involviert in die Morphogenese reproduktiver Organe und den Erhalt der Fortpflanzungsfähigkeit. Als nuklearer Rezeptor aktiviert er Transkriptionsfaktoren und steuert damit direkt die Genexpression und Synthese der beteiligten Proteine. Eine Störung dieser Aktivität durch endokrine Disruption kann sich in Form verschiedenster physischer Merkmale wie etwa mangelnder Geschlechtsdifferenzierung, dem Verlust der Fortpflanzungs-fähigkeit sowie Karzinomen der Fortpflanzungsorgane äußern. Die Störung hat damit nicht nur Konsequenzen für einzelne Individuen, sondern kann auch ganze Populationen betreffen, etwa durch die Herabsetzung der Fortpflanzungsrate. Schnelle und empfindliche biologische Testverfahren zur Erfassung endokriner Disruptoren sind daher unbedingt erforderlich. Um sinnvolle gesetzliche Richtlinien erstellen zu können, müssen Substanzen eindeutig als endokrine Disruptoren klassifiziert werden und ihre minimal gefährdende Konzentration bestimmt werden. Besonderer Wert muß dabei auf die tatsächliche Relevanz der Tests für östrogene Aktivität gelegt werden

In vivo investigation of estrogenic activity by fluorescence techniques

Hormone binding nuclear receptors are transcription factors that activate gene transcription by binding to specific DNA sequences in the gene promoter region and then recruiting the necessary coactivator proteins for transcriptional activation. The knowledge of the function of these receptors, e.g. how they activate or repress transcription are deduced mainly from in vitro biochemical experiments. A general model has been proposed that these receptors and the recruited coactivator proteins form relatively stable complexes with gene promoters. Only recently, in the last four years, additional in vivo data measured with fluorescence recovery after photobleaching (FRAP) have shown that many hormone receptors and coactivators are very mobile inside the nucleus and their association to chromatin is very dynamic, much faster than what has been anticipated from earlier biochemical studies. Although the mobility is reported to be high for the hormone binding receptors, there are consistent reports about reduced mobility in the presence of agonists and antagonists. However, little is known about the significance of the altered mobility. Open questions are, whether the change of mobility is due to increasing interactions with other proteins, e.g. by forming larger protein complexes or if it is due to interactions with chromatin and other relative immobile domains of the nucleus. In this work, fluorescence correlation spectroscopy (FCS) has been employed to monitor the mobility of both a hormone binding receptor, the estrogen receptor (ER), and coactivator proteins belonging to the steroid receptor coactivator (SRC)-family inside the nucleus, to investigate how their mobility is influenced by different ligands. The main advantages of FCS compared to FRAP are (i) its sensitivity which enables measurements of proteins of low abundance in live cells and (ii) its higher temporal resolution which allows us to resolve different diffusing species in the millisecond range. This work has been divided into four major parts. The first contains the construction and characterization of activity of fluorescently labeled molecules. The second part describes the optimization of the experimental conditions for measuring the mobility of yellow fluorescent protein (YFP) in living cells. The third part reports on the effects of YFP-labeled ER inside the nucleus in the presence of different ligands. Finally the fourth part investigates the effects of different ligands on the interaction between receptor and coactivator in living cells. The main results of this thesis are the following: The finding of the different mobility patterns of YFP-ER in the presence of agonists and antagonists indicate that the different ligands induce multiple discrete diffusive states; some are re-occuring for different ligands while others are characteristic for particular ligands. The potency of the different ligands to induce a lower mobility of the YFP-ER was estimated, and the relative order of the potencies to induce the lower mobility corresponds well to the relative order of their affinities for ER. ER and coactivator proteins were shown to interact in the absence of ligands and there are also clear indications of the co-existence of multiple ER-SRC complexes in the presence of agonists. The full antagonist abolishes these interactions completely in the case of the full-length coactivator and only partly when a truncated coactivator, containing only the receptor interaction domain, was used

Method for testing a substance interacting with a target molecule

The present invention relates to a method for testing a substance interacting with a target molecule in a cell by fluorescence correlation spectroscopy (FCS) by adding a test substance to the cell and determining a distribution of diffusion coefficients for the fluorescently labelled target molecule. Preferably, the target molecule is a nuclear hormone receptor, e.g. the estrogen receptor

Diffusion-Time Distribution Analysis Reveals Characteristic Ligand-Dependent Interaction Patterns of Nuclear Receptors in Living Cells

Nuclear receptors initiate transcription, interact with regulatory proteins, and are influenced by hormones, drugs, and pollutants. Herein, we discover ligand-specific mobility patterns of human estrogen receptor-a (ER) in living cells using diffusion-time distribution anal. (DDA). This novel method, based on fluorescence correlation spectroscopy (FCS), is esp. suited to unraveling multiple protein interactions in vivo at native expression levels. We found that ER forms a limited no. of distinct complexes with a varying population by dynamic interaction with other nuclear components. Dose-response curves of different ligands could be obtained for each receptor interaction. The potential to identify interacting proteins was demonstrated by comparing DDA of the ER cofactor SRC-3 attached to yellow fluorescent protein (YFP) with those of YFP-ER. Our findings open up new routes to elucidating transcription regulation and to detecting and distinguishing pharmacol. and toxicol. active compds. in vivo. Moreover, DDA provides a general approach to monitoring biochem. networks in individual living cells. [on SciFinder (R)

Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques

Novel vaccine platforms for delivery of nucleic acids based on viral and non-viral vectors, such as recombinant adeno associated viruses (rAAV) and lipid-based nanoparticles (LNPs), hold great promise. However, they pose significant manufacturing and analytical challenges due to their intrinsic structural complexity. During product development and process control, their design, characterization, and quality control require the combination of fit-for-purpose complementary analytical tools. Moreover, an in-depth methodological expertise and holistic approach to data analysis are required for robust measurements and to enable an adequate interpretation of experimental findings. Here the combination of complementary label-free biophysical techniques, including dynamic light scattering (DLS), multiangle-DLS (MADLS), Electrophoretic Light Scattering (ELS), nanoparticle tracking analysis (NTA), multiple detection SEC and differential scanning calorimetry (DSC), have been successfully used for the characterization of physical and chemical attributes of rAAV and LNPs encapsulating mRNA. Methods’ performance, applicability, dynamic range of detection and method optimization are discussed for the measurements of multiple critical physical−chemical quality attributes, including particle size distribution, aggregation propensity, polydispersity, particle concentration, particle structural properties and nucleic acid payload

Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques

Novel vaccine platforms for delivery of nucleic acids based on viral and non-viral vectors, such as recombinant adeno associated viruses (rAAV) and lipid-based nanoparticles (LNPs), hold great promise. However, they pose significant manufacturing and analytical challenges due to their intrinsic structural complexity. During product development and process control, their design, characterization, and quality control require the combination of fit-for-purpose complementary analytical tools. Moreover, an in-depth methodological expertise and holistic approach to data analysis are required for robust measurements and to enable an adequate interpretation of experimental findings. Here the combination of complementary label-free biophysical techniques, including dynamic light scattering (DLS), multiangle-DLS (MADLS), Electrophoretic Light Scattering (ELS), nanoparticle tracking analysis (NTA), multiple detection SEC and differential scanning calorimetry (DSC), have been successfully used for the characterization of physical and chemical attributes of rAAV and LNPs encapsulating mRNA. Methods’ performance, applicability, dynamic range of detection and method optimization are discussed for the measurements of multiple critical physical−chemical quality attributes, including particle size distribution, aggregation propensity, polydispersity, particle concentration, particle structural properties and nucleic acid payload.publishedVersio