Alzheimer’s disease (AD) is associated with self-assembly of amyloid β-protein (Aβ) into soluble oligomers. Of the two predominant Aβ alloforms, Aβ40 and Aβ42, the latter is particularly strongly linked to AD. Longitudinal studies revealed a correlation between AD and type 2 diabetes (T2D), characterized by abnormal insulin levels and insulin resistance. Although administration of intranasal insulin is explored as a therapy against AD, the extent to which insulin affects Aβ dynamics and activity is unclear. We here investigate the effect of insulin on Aβ42 self-assembly and characterize the capacity of insulin, Aβ42, and Aβ42 co-incubated with insulin to disrupt the integrity of biomimetic lipid vesicles. We demonstrate that quiescently incubated insulin, which does not form amyloid fibrils, over time develops membrane-disrupting capacity, which we propose to originate in misfolded insulin monomers. These hypothetically toxic misfolded monomers might contribute to the development of insulin resistance in early stages of T2D that are associated with abnormally high insulin levels. We show that in contrast to quiescent incubation, insulin incubated under agitated conditions readily forms amyloid fibrils, which protect against membrane permeation. Insulin quiescently incubated with Aβ42 attenuates both Aβ42 fibril formation and the ability of Aβ42 to disrupt membranes in a concentration-dependent manner. Our findings offer insights into interactions between insulin and Aβ42 that are relevant to understanding the molecular basis of intranasal insulin as a therapy against Aβ-induced AD pathology, thereby elucidating a plausible mechanism underlying the observed correlations between AD and T2D.

Protein self-assembly plays an important role in cellular processes. Whereas molecular dynamics (MD) represents a powerful tool in studying assembly mechanisms, its predictions depend on the accuracy of underlying force fields, which are known to overly promote protein assembly. We here examine villin headpiece domain, HP36, which remains soluble at concentrations amenable to MD studies. The experimental characterization of soluble HP36 at concentrations of 0.05 to 1 mM reveals concentrationindependent 90% monomeric and 10% dimeric populations. Extensive allatom MD simulations at two protein concentrations, 0.9 and 8.5 mM, probe the HP36 dimer population, stability, and kinetics of dimer formation within two MD force fields, Amber ff14SB and CHARMM36m. MD results demonstrate that whereas CHARMM36m captures experimental HP36 monomer populations at the lower concentration, both force fields overly promote HP36 association at the higher concentration. Moreover, contacts stabilizing HP36 dimers are force-field-dependent. CHARMM36m produces consistently higher HP36 monomer populations, lower association rates, and weaker dependence of these quantities on the protein concentration than Amber ff14SB. Nonetheless, the highest monomer populations and dissociation constants are observed when the TIP3P water model in Amber ff14SB is replaced by TIP4P/2005, showcasing the critical role of the water model in addressing the protein solubility problem in MD.

proline hydroxyls make up 21% of the hydrogen bonds between triple helices, proline hydroxyls can also be expected to contribute to the structural integrity and the mechanical properties of fibrils. Here we investigate this hypothesis by carrying out comparative molecular dynamics studies of fibrils in the presence and absence of hydroxylation. We investigate this in both rat and human type I fibrils. The structure of the human type I fibril structure is constructed using homology modeling, and its hydroxylation sites are determined using MS/MS sequencing. Expectedly, we find that in both human and rat fibrils, the presence of hydroxylation does alter the contact interface between triple-helices. Specifically, hydroxylation affects hydrogen bonding patterns, but in different ways in the two collagens, despite the two collagens sharing 94% sequence identity. However, in both rat and human fibrils, the altered local interactions have little effect on macroscopic structural properties, including D-band lengths and gapoverlap ratios. Additionally, hydroxylation only marginally increases Young's moduli, although the effects of hydroxylation are more pronounced under large (> 5%) strains. Overall, this study suggests that although local interactions between triple helices in fibrils are sensitive to hydroxylation and that there does exist some degree of plasticity in interfacial contacts between triple helices, the hallmark macroscopic properties of fibrils are not governed by hydroxylation. 2266-Plat Superresolution Imaging of Amyloid Structures over Extended Times using Transient Binding of Single Thioflavin T Molecules

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