Several point mutations can modulate protein structure and dynamics, leading to different natures. Especially in the case of amyloidogenic proteins closely related to neurodegenerative diseases, structural changes originating from point mutations can affect fibrillation kinetics. Herein, we rationally designed mutant candidates to inhibit the fibrillation process of amyloid-β with its point mutants through multistep in silico analyses. Our results showed that the designed mutants induced kinetic self-assembly suppression and reduced the toxicity of the aggregate. A multidisciplinary biophysical approach with small-angle X-ray scattering, ion mobility-mass spectrometry, mass spectrometry, and additional in silico experiments was performed to reveal the structural basis associated with the inhibition of fibril formation. The structure-based design of the mutants with suppressed self-assembly performed in this study could provide a different perspective for modulating amyloid aggregation based on the structural understanding of the intrinsically disordered proteins.

Advanced understanding of Alzheimer's disease (AD) and several tauopathies over the past decades indicates the pathological importance of tau aggregation in these diseases. Herein, we demonstrated that adenosine triphosphate (ATP), a highly charged anionic molecule abundant in the cytosol of cells, catalyses tau fibrillation via supramolecular complexation with basic residues of tau. Our results showed that ATP attracts multiple lysine residues of four-repeat domain of tau (K18), thereby immediately forming dimers which convert to nuclei to accelerate fibril elongation. However, ATP was not directly incorporated in the K18 fibrils suggesting a catalytic role of ATP in K18 fibrillation. We also characterized the correlation between ATP dyshomeostasis and tau aggregation in the cellular environment. Our multiple biophysical approaches, including native mass spectrometry (MS), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation, provided insights into the molecular-level influence of ATP on the structural change and fibrillation of tau. File list (2) download file view on ChemRxiv 200314_TauATP_Preprint.pdf (1.62 MiB) download file view on ChemRxiv 200314_SI_TauATP_Preprint.pdf (1.47 MiB)

Fibrillar amyloid aggregates are the pathological hallmarks of multiple neurodegenerative diseases. The amyloid-β (1–42) protein, in particular, is a major component of senile plaques in the brains of patients with Alzheimer’s disease and a primary target for disease treatment. Determining the essential domains of amyloid-β (1–42) that facilitate its oligomerization is critical for the development of aggregation inhibitors as potential therapeutic agents. In this study, we identified three key hydrophobic sites (17LVF19, 32IGL34, and 41IA42) on amyloid-β (1–42) and investigated their involvement in the self-assembly process of the protein. Based on these findings, we designed candidate inhibitor peptides of amyloid-β (1–42) aggregation. Using the designed peptides, we characterized the roles of the three hydrophobic regions during amyloid-β (1–42) fibrillar aggregation and monitored the consequent effects on its aggregation property and structural conversion. Furthermore, we used an amyloid-β (1–42) double point mutant (I41N/A42N) to examine the interactions between the two C-terminal end residues with the two hydrophobic regions and their roles in amyloid self-assembly. Our results indicate that interchain interactions in the central hydrophobic region (17LVF19) of amyloid-β (1–42) are important for fibrillar aggregation, and its interaction with other domains is associated with the accessibility of the central hydrophobic region for initiating the oligomerization process. Our study provides mechanistic insights into the self-assembly of amyloid-β (1–42) and highlights key structural domains that facilitate this process. Our results can be further applied toward improving the rational design of candidate amyloid-β (1–42) aggregation inhibitors.

Summary Neuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

Advanced understanding of Alzheimer’s disease (AD) and several tauopathies over the past decades indicates the pathological importance of tau aggregation in these diseases. Herein, we demonstrated that adenosine triphosphate (ATP), a highly charged anionic molecule abundant in the cytosol of cells, catalyses tau fibrillation via supramolecular complexation with basic residues of tau. Our results showed that ATP attracts multiple lysine residues of four-repeat domain of tau (K18), thereby immediately forming dimers which convert to nuclei to accelerate fibril elongation. However, ATP was not directly incorporated in the K18 fibrils suggesting a catalytic role of ATP in K18 fibrillation. We also characterized the correlation between ATP dyshomeostasis and tau aggregation in the cellular environment. Our multiple biophysical approaches, including native mass spectrometry (MS), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation, provided insights into the molecular-level influence of ATP on the structural change and fibrillation of tau.

Advanced understanding of Alzheimer’s disease (AD) and several tauopathies over the past decades indicates the pathological importance of tau aggregation in these diseases. Herein, we demonstrated that adenosine triphosphate (ATP), a highly charged anionic molecule abundant in the cytosol of cells, catalyses tau fibrillation via supramolecular complexation with basic residues of tau. Our results showed that ATP attracts multiple lysine residues of four-repeat domain of tau (K18), thereby immediately forming dimers which convert to nuclei to accelerate fibril elongation. However, ATP was not directly incorporated in the K18 fibrils suggesting a catalytic role of ATP in K18 fibrillation. We also characterized the correlation between ATP dyshomeostasis and tau aggregation in the cellular environment. Our multiple biophysical approaches, including native mass spectrometry (MS), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation, provided insights into the molecular-level influence of ATP on the structural change and fibrillation of tau.

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.

In tauopathic conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process are not clear. In AD brains, only a small segment of tau forms -helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, we demonstrated that the tau AD nucleation core (tau-AC) sufficiently induced self-aggregation and recruited full-length tau to filaments. Unexpectedly, phospho-mimetic forms of tau-AC (at Ser324 or Ser356) showed markedly reduced aggregation and seeding propensities. Biophysical analysis revealed that the N-terminus of tau-AC facilitated the fibrillization kinetics, while its phosphorylation induced conformation changes, sterically shielding the nucleation motif. Tau-AC oligomers were efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau-AC impaired axon initial segment plasticity upon chronic depolarization and was mislocalized in the somatodendritic compartments. Furthermore, we observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponded to the neuropathological staining and neuronal loss in the brain. These findings identified tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.