Understanding the Complexity of Alzheimer’s Disease’s Amyloid-β Fibrillization via Metal Ion Binding, Familial Mutants, and Gold Nanoparticles
Date Issued
2014
Date
2014
Author(s)
Liao, Yi-Hung
Abstract
Amyloid plagues are the major pathogenic hallmarks in many neurodegenerative diseases. Fibrillization of the primary constituents, amyloid-β (Aβ), in plagues is considered the pathogenesis of Alzheimer’s disease (AD). Conclusive evidence is still lacking to reach a comprehensive and convergent view of how Aβ is modulated in the pathogenesis of AD. Here, in three separate chapters, we examined how gold nanoparticles (AuNPs) interfere with fibrillization of Aβ, the effects of single-point N-terminal mutation, H6 and D7, on the interactions between metal ions and Aβ, and how single-point mutations, E22 and D23, influence the structures of fibril.
In Chapter 3, experimental results showed that (1) bare AuNPs inhibited Aβ fibrillization in a dose-dependent manner and redirected Aβ forming fragmented fibrils and spherical oligomers; (2) bare AuNPs bound preferentially to Aβ fibrils but not amorphous aggregates; (3) negative surface potential of AuNPs was required for inhibitive effect. In conjunction with our in vitro biophysical and biochemical data, cell studies from our lab member revealed that in our experimental settings, inhibition of Aβ fibrillization by negatively charged AuNPs also reduce Aβ-induced cytotoxicity in neuronal cells.
In Chapter 4, we aimed to reveal distinctions among structures of homopolymeric fibrils (homo-fibrils) formed from incubation of monomers of a single species, and heteropolymeric ones (hetero-fibrils) started from incubation of equal-molar-mixed monomers of wild type and an Aβ variant. We showed that E22 hetero-fibrils are distinct from E22 homo-fibrils in terms of secondary structures. Perturbed secondary structure could influence conformational packing of cross β-sheet, which were not revealed by stoichiometry between Aβ and ThT but by ThT fluorescence lifetime.
In Chapter 5, we investigated millisecond binding kinetics of four metal ions, Zn2+, Cu2+, Fe3+, and Al3+. We then focused on Cu2+ and demonstrated that mutation at residues H6 and D7 impact on Cu2+ binding affinities. We found by Bis-ANS fluorescence that diminished hydrophobic exposure was accompanied by Cu2+ binding except for D7A and H6R. Our preliminary proton NMR showed that there existed environmental variance surrounding residues H6, H13/H14, and Y10 among monomeric Aβ variants in native condition. Cu2+-binding-induced peak loss was less evident in D7 mutants. 1H-15N NMR HSQC spectroscopy provided additional information regarding the residual involvement on Cu2+ binding between D7H and wild type Aβ.
Subjects
阿茲海默症
乙型類澱粉蛋白
類澱粉蛋白纖維
家族性突變型
金屬離子
奈米金粒子
Type
thesis
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