Dokument: Biophysical characterization of the interaction between amyloid-β (1-42) protein and ligands
| Titel: | Biophysical characterization of the interaction between amyloid-β (1-42) protein and ligands | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=50357 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20200917-085526-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Zhang, Tao [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Willbold, Dieter [Gutachter] Prof. Dr. Steger, Gerhard [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | It has been a century since the first Alzheimer’s disease (AD) patient was described and reported by the German psychiatrist Alois Alzheimer. The disease is receiving increasing attention both due to its complex pathophysiology and the great socioeconomic burden following the global ageing trend. Currently there are around 50 million people suffering from dementia worldwide. AD is the most common cause of dementia, contributing to over 60% of all cases. The aggregation of amyloid-β (Aβ) into toxic oligomers and fibrillar structures in the brain has been recognized as one of the hallmarks of the disease. The accumulation of various Aβ aggregates in the central nervous system occurs early in the progression of AD and induces functional and structural damages to the brain at multiple levels. The central role of Aβ aggregation in the etiology of AD has made it a pivotal target for disease intervention.
A lot of physiological or non-physiological factors have been documented to be able to affect the aggregation pathway of Aβ. For example, zinc ions (Zn2+), the second most abundant trace element in human body, was found to enrich around the amyloid plaque in Alzheimer’s brains. However, the detailed influence of Zn2+ on the fibril formation of Aβ remains controversial. In chapter I, we investigated the influence of Zn2+ on the aggregation of Aβ42, the major toxic isoform of Aβ products in the brain, by applying analytical ultracentrifugation (AUC) and other biophysical techniques. We found that stoichiometric Zn2+ shifts the aggregation of Aβ42 toward non-fibrillary aggregates with reduced β-sheet structures and amyloidogenic activity. The interaction between Zn2+ and Aβ42 is rapid and can be reversed by adding metal ion chelators such as EDTA. AUC was combined with atomic force microscopy (AFM) to elucidate size distributions and morphologies of Aβ42 aggregates formed with or without Zn2+. Several non-physiological factors, including two D-enantiomeric peptides (D-peptides) and three commercially available monoclonal antibodies (mAbs) against Aβ were also studied to characterize how they interact with Aβ42 and impact the aggregation process (chapters II-IV). Based on the strategy that stabilizing monomeric Aβ in unstructured conformations could prevent them from the oligomerization and even eliminate existing cytotoxic Aβ species, a series of all-D-enantiomeric peptides were developed and characterized regarding their effects on AD pathology. D3 and RD2 are two of the D-peptides investigated in the present study (Chapters II and III). We first demonstrated that D3 and RD2 bind to Aβ42 monomers with nanomolar affinities via microscale thermophoresis (MST). The high affinity binding leads to the formation of Aβ42 and D-peptides complexes at 1:1 and other stoichiometries, as revealed by fluorescence based analytical ultracentrifugation and molecular dynamics simulations. MST measurements revealed an enhancement in the interaction between D-peptides and Aβ42 when reducing the ionic strength of the buffer, indicating the involvement of electrostatic interaction between these two molecules. By employing thioflavin T (ThT) assay, circular dichroism (CD) spectroscopy and AFM, we demonstrated that D3 and RD2 significantly retard the secondary structure transition and the fibrillation of Aβ42 by interfering with the nucleation process and retaining Aβ monomers in unstructured conformations. Our research showed for the first time that D3 and RD2 are able to interact with monomeric Aβ42, which leads to the formation of highly flexible complexes at various stoichiometries. Our study substantiates the versatile effects of D-peptides on regulating Aβ aggregation and envisions the promising application of D-peptides in Aβ aggregation related pathologies. In chapter IV, the interaction between three commercially available anti-Aβ antibodies and Aβ42 was characterized using fluorescence based AUC and MST measurements. All three antibodies, 6E10, 4G8 and 12F4, are able to interact with Aβ42 monomers with low nanomolar affinities, irrespective of the difference in epitopes. The KD values are consistent with reported values from surface plasmon resonance (SPR) studies on antibodies and Aβ monomers. Besides, the affinity for 12F4 and Aβ42 monomer was quantified for the first time. The results on Aβ42 monomers and antibodies also validate that fluorescence based AUC and MST are useful and rigorous methods for the study of the interaction between aggregation-prone proteins and their binding partners. To summarize, this work characterized how several binding partners interact with Aβ42 and further affect its fibrillation process. In particular, our study figures out that the interaction and complex formation between D-peptides and Aβ monomers could be an important and early event of the whole interaction process, and that D-peptides play a diverse role in modulating the aggregation of Aβ through interfering with Aβ nucleation and eliminating toxic Aβ assemblies. These findings demonstrate that both Aβ monomers and oligomers can be involved in the interaction with D-peptides and highlight the potential of D-peptides as drug candidates for the intervention of AD. Our study may also aid to understand the interaction between small ligands and intrinsically disorder proteins. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie | |||||||
| Dokument erstellt am: | 17.09.2020 | |||||||
| Dateien geändert am: | 17.09.2020 | |||||||
| Promotionsantrag am: | 04.06.2019 | |||||||
| Datum der Promotion: | 09.07.2019 |
