Serpell, L. C. | Search bioRxiv

bioRxiv2020

Cell-derived hexameric β-amyloid: a novel insight into composition, self-assembly and nucleating properties

A key hallmark of Alzheimers disease (AD) is the extracellular deposition of amyloid plaques composed primarily of the amyloidogenic amyloid-{beta} (A{beta}) peptide. The A{beta} peptide is a product of sequential cleavage of the Amyloid Precursor Protein (APP), the first step of which gives rise to a C-terminal Fragment (C99). Cleavage of C99 by {gamma}-secretase activity releases A{beta} of several lengths and the A{beta}42 isoform in particular has been identified as being neurotoxic. The misfolding of A{beta} leads to subsequent amyloid fibril formation by nucleated polymerisation. This requires an initial and critical nucleus for self-assembly. Here, we identify and characterise the composition and self-assembly properties of cell-derived hexameric A{beta}42 and show its nucleating properties which are dependent on the A{beta} monomer availability. Identification of nucleating assemblies that contribute to self-assembly in this way may serve as therapeutic targets to prevent the formation of toxic oligomers.

biochemistry↗

bioRxiv2020

Nucleation-dependent aggregation kinetics of Yeast Sup35 fragment GNNQQNY

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY serves as an ideal model for structural understanding of amyloid assembly and kinetics. In this study, we used a reproducible solubilisation protocol that allows the generation of homogenous monomeric solution of GNNQQNY to understand the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequences follow nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the size and efficiency of nucleation was found to be inversely related to the reaction temperature. The generated nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T positivity, Congo red binding and {beta}-sheet rich structures displaying a characteristic cross-{beta} diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers exerted no significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous predictions based on theoretical and molecular dynamics simulations. These findings will provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic systems associated with many systemic and neurodegenerative diseases.

biophysics↗