Steric crowding of the turn region alters the tertiary fold of amyloid-β18–35and makes it soluble

Aβ self-assembles into parallel cross-β fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23–29 is a defining characteristic of Aβ in its aggregated state. Major biophysical properties of Aβ, including this turn, remain unaltered in the central fragment Aβ18–35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aβ18–35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aβ18–35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe19 and Leu34 residues, observed in full-length Aβ and Aβ18–35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel β-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel β-sheet. We conclude that the self-assembly of Aβ is critically dependent on the hairpin turn and on the contact between the Phe19 and Leu34 regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions

Ordered and disordered segments of amyloid-β drive sequential steps of the toxic pathway

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-Appreciated, the mechanism of toxicity of disease-Causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aβ). Aβ oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aβ fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aβ or Aβ , is toxic by itself. The ordered part (Aβ ) is the major determinant of how Aβ attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aβ enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-Terminal. This interaction appears to commit Aβ to the toxic pathway. Our findings correlate well with Aβ sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction

A Toxicogenic Interaction between Intracellular Amyloid‑β and Apolipoprotein‑E

Alzheimer’s disease (AD) is associated with the aggregation of amyloid β (Aβ) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aβ oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aβ-ApoE interaction suppresses these changes and concomitantly reduces Aβ toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer’s patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aβ oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery