Background Most Alzheimer’s disease (AD) treatments focus on symptomatic relief by boosting the availability of neurotransmitters in the brain. Aside from symptomatic therapies, the amyloid‐β (Aβ) targeting approach has recently been established to reduce or postpone the production of Aβ plaques.1 Therefore, understanding the pathogenesis of AD requires regulation of Aβ fibrillation. Method We used the CamSol method and molecular dynamics simulations to find a group of residues that were significantly associated with Aβ fibrillation. To investigate the suppressed amyloid aggregation and reduced cytotoxicity of the designed mutants, we performed multiple in vitro experiments such as thioflavin T assay, circular dichroism spectroscopy measurement, transmission electron microscopy image analysis, liquid chromatography‐mass spectrometry based quantification, and cell viability test. We also characterized structural dynamics of the designed mutants using a multidisciplinary biophysical approach with small‐angle X‐ray scattering, ion mobility‐mass spectrometry, additional in silico experiments, and mass spectrometry. Result We rationally designed mutant constructs to suppress the fibrillation process using comprehensive molecular dynamics simulations and the atomic resolution structure of fibrils. Then, we used a multidisciplinary biophysical technique to investigate the physicochemical characteristics and unveil the structural basis associated with reduced self‐assembly. Lastly, cell‐based tests were performed to evaluate the alleviated cytotoxicity of designed mutant candidates. Conclusion More generally, our method for modulating the self‐assembly property of the pathologically disordered proteins offers a novel viewpoint to treating neurodegenerative diseases and other protein folding‐related issues. Reference: 1. Sevigny, J., et al. “The antibody aducanumab reduces A beta plaques in Alzheimer’s disease.” Nature 2016, 537(7618), 50‐56.
Background Intracellular tau protein tangle is a hallmark of various neurodegenerative diseases, which include Alzheimer’s disease (AD). For this reason, characterizing the physiological factors associated with tau aggregation and the aggregation mechanism has been considered crucial for understanding tau pathology. Recent evidence implies that (i) intracellular ATP level may be dysregulated and (ii) the increased ATP level may have an influence on the tau aggregation. However, the effects of ATP on tau fibrillation and its mechanistic details are not fully elucidated. Methods For characterizing K18 fibrillation mechanism catalysed by ATP, we performed multiple biophysical experiments such as thioflavin assays, circular dichroism, small‐angle X‐ray scattering, and native mass spectrometry. We further measured cellular ATP amount in presence of TNFα, and intracellular tau aggregation was examined by bimolecular fluorescence complementation experiments. Results Herein, we report the catalytic role of ATP and its mechanistic details in tau aggregation at a molecular level for the first time. Using multiple approaches including solution small angle X‐ray scattering (SAXS), native mass spectrometry (MS) and molecular dynamics (MD) simulations, we observed that nonspecific electrostatic interactions between negatively charged triphosphate moiety in ATP and basic residues in tau K18 (i.e. four‐repeat domain of tau) direct the dimerization of K18 by charge neutralization of the protein in the early stage of the aggregation. We observed that ATP was excluded in the late stage of fibrillation and acted to accelerate K18 fibrillation even at lower stoichiometric ratios for the charge neutralization, indicating that ATP acted as a catalyst for K18 fibrillation. Moreover, our bimolecular fluorescence complementation (BiFC) and immunoblotting (IB) experiments suggested a correlation between ATP dyshomeostasis and tau aggregation in cellular environment. Conclusion We believe that our observations would provide valuable insights into the mechanism of tau aggregation and the importance of ATP in tauopathies.
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