Dokument: Looking for a white cat in a snowstorm - In depth thermodynamic and aggregation fingerprinting of patient-derived IgG light chains
| Titel: | Looking for a white cat in a snowstorm - In depth thermodynamic and aggregation fingerprinting of patient-derived IgG light chains | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=55874 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20210408-104942-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Sternke-Hoffmann, Rebecca [Autor] | |||||||
| Dateien: |
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| Beitragende: | Jun.-Prof. Büll, Alexander [Gutachter] Prof. Dr. Haas, Rainer [Gutachter] | |||||||
| Stichwörter: | Light chain, amyloid, aggregation | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | The deposition of immunoglobulin light chains (IgG LCs) in the form of amorphous aggregates or amyloid fibrils in different tissues is a serious problem for patients with light chain diseases, which lead to an overproduction of free light chains. A lot of scientific work has been carried out to correlate the different in vivo solubilities of LCs and their intrinsic aggregation propensity to either form highly ordered amyloid fibrils or disordered amorphous aggregates. It is generally presumed that the origin of their different solubility is to be found in the unique amino acid sequence of the respective LC.
In order to elucidate the in vivo solubility of a certain LC protein, I performed a detailed biochemical and biophysical analysis of light chains extracted and purified from the urine of a group of 20 patients with light chain disease. For all samples, the unfolding temperature of the LCs, their monomer-dimer distribution, the digestibility by trypsin and the formation of amyloid fibrils under various conditions of pH and reducing agent, were quantified. Neither of these properties correlated with the kidney damage as defined by clinical parameters. Most of the LC characteristics reported to be predictors of amyloid formation cannot be used to assess the degree of kidney damage. Nevertheless, I detected that LCs, which display very poor digestibility by trypsin, were derived from patients with the greatest impairment of kidney function. To perform a more detailed biophysical in vitro characterization, a sub-set of ten LC samples, which contained the protein at high purity, were chosen. This allows both the sequence determination, as well as a detailed biophysical study. The amino acid sequences are solved by a novel de novo sequencing workflow for patient-derived LCs, based on a combination of bottom-up and top-down proteomics. An in depth Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip) was established to characterize the behaviour of the LCs of all of which we determined the primary structure. Our results suggest that while every pathogenic LC has an unique ThAgg-fingerprint and sequence, they can all form amyloid fibrils under physiologically relevant, mildly acidic pH conditions. Therefore we suspect that extrinsic factors are the main determinants of in vivo light chain aggregation behaviour. This intrinsic amyloid propensity challenges the current paradigm of the link between sequence and amyloid fibril formation of pathogenic light chains. Since the aggregation kinetics of the LC samples are affected by external factors, I could not easily examine the influence of the anti-amyloid component EGCG on their fibril formation. For this reason I performed a detailed study on the aggregation kinetics of α-synuclein to elucidate the impact of the solution conditions on the mode of action of EGCG. The presented work makes a considerable contribution to the highly-relevant scientific field of LC amyloid pathology research. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie | |||||||
| Dokument erstellt am: | 08.04.2021 | |||||||
| Dateien geändert am: | 08.04.2021 | |||||||
| Promotionsantrag am: | 12.01.2021 | |||||||
| Datum der Promotion: | 23.03.2021 |
