Abstract
Elucidating the molecular mechanisms in the development of such a devastating neurodegenerative disorder as Alzheimer’s disease (AD) is currently one of the major challenges of molecular medicine. Evidence strongly suggests that the development of AD is due to the accumulation of amyloid β (Aβ) oligomers; therefore, understanding the molecular mechanisms defining the conversion of physiologically important monomers of Aβ proteins into neurotoxic oligomeric species is the key for the development of treatments and preventions of AD. However, these oligomers are unstable and unavailable for structural, physical, and chemical studies. We have recently developed a novel flexible nano array (FNA)-oligomer scaffold approach in which monomers tethered inside a flexible template can assemble spontaneously into oligomers with sizes defined by the number of tethered monomers. The FNA approach was tested on short decamer Aβ(14–23) peptides which were assembled into dimers and trimers. In this paper, we have extended our FNA technique for assembling full-length Aβ42 dimers. The FNA scaffold enabling the self-assembly of Aβ42 dimers from tethered monomeric species has been designed and the assembly of the dimers has been validated by AFM force spectroscopy experiments. Two major parameters of the force spectroscopy probing, the rupture forces and the rupture profiles, were obtained to prove the assembly of Aβ42 dimers. In addition, the FNA-Aβ42 dimers were used to probe Aβ42 trimers in the force spectroscopy experiments with the use of AFM tips functionalized with FNA-Aβ42 dimers and the surface with immobilized Aβ42 monomers. We found that the binding force for the Aβ42 trimer is higher than the dimer (75 ± 7 pN vs. 60 ± 3 pN) and the rupture pattern corresponds to a cooperative dissociation of the trimer. The rupture profiles for the dissociation of the Aβ42 dimers and trimers are proposed. Prospects for further extension of the FNA-based approach for probing of higher order oligomers of Aβ42 proteins are discussed.
Highlights
Growing evidence has revealed that the neurotoxic effects of diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) result from oligomeric forms of amyloid beta (Aβ) and α-synuclein, respectively (Sengupta et al, 2016; Lee et al, 2017; Ono, 2018)
The contour length of the flexible nano array (FNA) segment with 16 PA units corresponds to the value 32 nm (Tong et al, 2013; Krasnoslobodtsev et al, 2015), so stretching of this segment is sufficient to dissociate the FNA-Aβ42 dimer
The other end of the FNA scaffold separated from another anchoring point for Aβ42 monomer is terminated with biotin and is used in the Aβ42 dimer stretching experiments, in which the surface is functionalized with streptavidin
Summary
Growing evidence has revealed that the neurotoxic effects of diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) result from oligomeric forms of amyloid beta (Aβ) and α-synuclein, respectively (Sengupta et al, 2016; Lee et al, 2017; Ono, 2018). Considering protein aggregates are stabilized by weak interactions typically transient in nature, they are difficult to measure. Because of these challenges, only mixtures of aggregates with differing morphologies have been studied far. Teplow group has developed the photo cross-linking method to prepare discrete sizes of Aβ oligomers (Bitan and Teplow, 2004; Ono et al, 2010). These oligomers showed similar neurotoxicity as in vivo Aβ oligomers regardless of cross-linking (Ono et al, 2009). Urbanc and co-workers used a copper and hydrogen peroxide induced cross-linking method for stabilizing Aβ oligomers; oligomers remain crosslinked, so drawbacks with the mobility limitations by crosslinking remain (Williams et al, 2016)
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