GNNQQNY: Methodology for biophysical and structural understanding of aggregation

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size ~7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

A Chemical Chaperone That Prevents Insulin Fibrillation

Insulin, a peptide hormone, is susceptible to amyloid formation upon exposure to aberrant physiological conditions, result-ing in a loss of its bioactivity. For mitigating insulin aggregation, we report a molecule called PAD-S, which completely inhibit-ed insulin fibril formation, and preserved insulin in its soluble form. Circular Dichroism spectroscopy showed that PAD-S was able to maintain the native structure of insulin, thus acting as a chemical chaperone. Seeded aggregation kinetics suggest that PAD-S inhibited primary nucleation events during aggregation. This is consistent with molecular docking results which suggest that PAD-S binds strongly to native insulin monomers/dimers. Through a competitive binding experiment with ‘LVEALYL’ peptide, we conclude that PAD-S likely binds to the amyloid prone B11-B17 residues of insulin thereby prevent-ing its aggregation. PAD-S was also effective in disaggregating preformed insulin fibrils to non-toxic species. PAD-S treated insulin was functional as indicated by its ability to phosphorylate Akt. PAD-S was also highly effective in preventing the ag-gregation of insulin biosimilars. The low cellular cytotoxicity of PAD-S, and amelioration of aggregation-induced toxicity by PAD-S treated insulin further highlights its potential as an effective chemical chaperone.

In vitro Aggregation Ability of Five Commercially Available Aβ42 Peptides

Background: As the most basic material, synthetic human Amyloid-β (1-42) (Aβ42) pep- tides from different manufacturers have been widely used. Their aggregation ability is vital to the reliability, repeatability and comparability of studies on Aβ42 physiology and pathology. However, it has not been evaluated and compared. Objectives: To analyze the consistency of the aggregation ability of 5 commercially available Aβ42 peptides. Methods: 5 Aβ42 peptides represented as A, B, C, D and E were pretreated by HFIP. The pretreated Aβ42 peptides were dissolved in Thioflavin T (ThT) solution, and their aggregation kinetics was monitored for 30 h with the aggregation kinetics test. Meanwhile, the pretreated peptides were ag- gregated in phosphate buffered saline. After aggregated for 12 h, they were detected by methods of ThT fluorescence, far-UV circular dichroism (CD), SDS-PAGE, western blot, and transmission electron microscopy (TEM), respectively. After aggregation for 8 h and 12 h, their cytotoxicity to SH-SY5Y cells was further evaluated using Cell Counting Kit-8. Results: For aggregation kinetics, peptides A, C and E remained low level curves, while peptides B and D presented typical sigmoidal kinetics curves. In CD measurement, the aggregates of pep- tides B and D showed relatively high negative CD peaks with the height of -8.09 mdeg and -14.37 mdeg, while the height of peptides A, C and E was -1.04, -3.55, and -3.88. In ThT assay, relative fluorescence intensity of the aggregates of peptides B and D were 7.79 and 8.82, higher than 1.19, 1.71, and 2.70 of peptide A, C and E, respectively. In SDS-PAGE, all aggregates contained monomers and eleven polymers. Moreover, peptide B-E presented a trapezoidal distribution from dimers to trimers, and peptide A aggregated to dimers. By western blot, the bands of monomers re- mained in all aggregates. Furthermore, peptides B and D aggregated to dimers and trimers, pep- tides A and C only aggregated to dimers, and peptide E showed a strong band of trimers. By TEM, protofibrils were observed only in peptide B, while substantial spherical aggregates were formed in other peptides. Additionally, peptides B, D and E exhibited higher cytotoxicity after being aggregat- ed for 8 h, whereas peptides A, B and D presented relatively high cytotoxicity after 12-hour aggre- gation. Conclusion: Commercially available Aβ42 peptides showed obvious differences in aggregation abil- ity, which should arouse enough attention in the field of basic study related to Aβ42. The aggrega- tion ability evaluation with the various assay methods has some discrepancies, and it is highly ur- gent to establish a reasonable and uniform measurement strategy.

Collusion of α-Synuclein and Aβ aggravating co-morbidities in a novel prion-type mouse model

Background The misfolding of host-encoded proteins into pathological prion conformations is a defining characteristic of many neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, and Lewy body dementia. A current area of intense study is the way in which the pathological deposition of these proteins might influence each other, as various combinations of co-pathology between prion-capable proteins are associated with exacerbation of disease. A spectrum of pathological, genetic and biochemical evidence provides credence to the notion that amyloid β (Aβ) accumulation can induce and promote α-synuclein pathology, driving neurodegeneration. Methods To assess the interplay between α-synuclein and Aβ on protein aggregation kinetics, we crossed mice expressing human α-synuclein (M20) with APPswe/PS1dE9 transgenic mice (L85) to generate M20/L85 mice. We then injected α-synuclein preformed fibrils (PFFs) unilaterally into the hippocampus of 6-month-old mice, harvesting 2 or 4 months later. Results Immunohistochemical analysis of M20/L85 mice revealed that pre-existing Aβ plaques exacerbate the spread and deposition of induced α-synuclein pathology. This process was associated with increased neuroinflammation. Unexpectedly, the injection of α-synuclein PFFs in L85 mice enhanced the deposition of Aβ; whereas the level of Aβ deposition in M20/L85 bigenic mice, injected with α-synuclein PFFs, did not differ from that of mice injected with PBS. Conclusions These studies reveal novel and unexpected interplays between α-synuclein pathology, Aβ and neuroinflammation in mice that recapitulate the pathology of Alzheimer’s disease and Lewy body dementia.

The N terminus of α-synuclein dictates fibril formation

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson’s disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41–140). This behavior can be explained by the unique fibril structure that is adopted by 41–140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent β-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended β-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

Polysorbate 80 Controls Morphology, Structure and Stability of Human Insulin Amyloid-Like Spherulites

Amyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

S100A9 Alters the Pathway of Alpha-Synuclein Amyloid Aggregation

The formation of amyloid fibril plaques in the brain creates inflammation and neuron death. This process is observed in neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases. Alpha-synuclein is the main protein found in neuronal inclusions of patients who have suffered from Parkinson’s disease. S100A9 is a calcium-binding, pro-inflammation protein, which is also found in such amyloid plaques. To understand the influence of S100A9 on the aggregation of α-synuclein, we analyzed their co-aggregation kinetics and the resulting amyloid fibril structure by Fourier-transform infrared spectroscopy and atomic force microscopy. We found that the presence of S100A9 alters the aggregation kinetics of α-synuclein and stabilizes the formation of a particular amyloid fibril structure. We also show that the solution’s ionic strength influences the interplay between S100A9 and α-synuclein, stabilizing a different structure of α-synuclein fibrils.