Distinctive contribution of two additional residues in protein aggregation of Aß42 and Aß40 isoforms.

Amyloid-ß (Aß) is one of the amyloidogenic intrinsically disordered proteins (IDPs) that self-assemble to protein aggregates, incurring cell malfunction and cytotoxicity. While Aß has been known to regulate multiple physiological functions, such as enhancing synaptic functions, aiding in the recovery of the blood-brain barrier/brain injury, and exhibiting tumor suppression/antimicrobial activities, the hydrophobicity of the primary structure promotes pathological aggregations that are closely associated with the onset of Alzheimer's disease (AD). Aß proteins consist of multiple isoforms with 37-43 amino acid residues that are produced by the cleavage of amyloid-ß precursor protein (APP). The hydrolytic products of APP are secreted to the extracellular regions of neuronal cells. Aß 1-42 (Aß42) and Aß 1-40 (Aß40) are dominant isoforms whose significance in AD pathogenesis has been highlighted in numerous studies to understand the molecular mechanism and develop AD diagnosis and therapeutic strategies. In this review, we focus on the differences between Aß42 and Aß40 in the molecular mechanism of amyloid aggregations mediated by the two additional residues (Ile41 and Ala42) of Aß42. The current comprehension of Aß42 and Aß40 in AD progression is outlined, together with the structural features of Aß42/Aß40 amyloid fibrils, and the aggregation mechanisms of Aß42/Aß40. Furthermore, the impact of the heterogeneous distribution of Aß isoforms during amyloid aggregations is discussed in the system mimicking the coexistence of Aß42 and Aß40 in human cerebrospinal fluid (CSF) and plasma. [BMB Reports 2024; 57(6): 263-272].

Genipin and pyrogallol: Two natural small molecules targeting the modulation of disordered proteins in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the aggregation of disordered proteins, such as amyloid beta (Aß) and tau, leading to neurotoxicity and disease progression. Despite numerous efforts, effective inhibitors of Aß and tau aggregates have not been developed. Thus, we aimed to screen natural small molecules from crude extracts that target various pathologies and are prescribed for patients with neurological diseases. In this study, we screened 162 natural small molecules prescribed for neurological diseases and identified genipin and pyrogallol as hit compounds capable of simultaneously regulating the aggregation of Aß and tau K18. Moreover, we confirmed the dual modulatory effects of these compounds on the reduction of amyloid-mediated neurotoxicity in vitro and the disassembly of preformed Aß42 and tau K18 fibrils. Furthermore, we observed the alleviatory effects of genipin and pyrogallol against AD-related pathologies in triple transgenic AD mice. Molecular dynamics and docking simulations revealed the molecular interaction dynamics of genipin and pyrogallol with Aß42 and tau K18, providing insights into their suppression of aggregation. Our findings suggest the therapeutic potential of genipin and pyrogallol as dual modulators for the treatment of AD by inhibiting aggregation or promoting dissociation of Aß and tau.

Self-Aggregating Tau Fragments Recapitulate Pathologic Phenotypes and Neurotoxicity of Alzheimer's Disease in Mice.

In tauopathy 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 remain unclear. In the brains of AD patients, only a minor segment of tau forms ß-helix-stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is 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) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N-terminus of tau-AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation-induced conformational changes in tau-AC. Tau-AC oligomers are 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 is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau-AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau-AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.

Decoding the Roles of Amyloid-ß (1-42)'s Key Oligomerization Domains toward Designing Epitope-Specific Aggregation Inhibitors.

Fibrillar amyloid aggregates are the pathological hallmarks of multiple neurodegenerative diseases. The amyloid-ß (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-ß (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-ß (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-ß (1-42) aggregation. Using the designed peptides, we characterized the roles of the three hydrophobic regions during amyloid-ß (1-42) fibrillar aggregation and monitored the consequent effects on its aggregation property and structural conversion. Furthermore, we used an amyloid-ß (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-ß (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-ß (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-ß (1-42) aggregation inhibitors.

Kinetic Modulation of Amyloid-ß (1-42) Aggregation and Toxicity by Structure-Based Rational Design.

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-ß 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.

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells.

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.

ATP Kinetically Modulates Pathogenic Tau Fibrillations.

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 found abundantly in the cytosol of cells, catalyzes fibrillation of tau as well as human islet amyloid polypeptide, a representative of basic intrinsically disordered proteins. Our results showed that ATP attracts multiple lysine residues of the four-repeat domain of tau (K18) via supramolecular complexation, thereby forming dimers that are converted to nuclei and accelerate fibril elongation. However, ATP was not directly incorporated into the K18 fibrils, suggesting that ATP plays the role of a catalyst, rather than a reactant, during 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 changes and fibrillation of tau.