Aplp1 interacts with Lag3 to facilitate transmission of pathologic α-synuclein.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid ß precursor-like protein 1 (Aplp1) interacts with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology deepens our insight about molecular mechanisms of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson's disease and related α-synucleinopathies.

Characterization and insight mechanism of an acid-adapted ß-Glucosidase from Lactobacillus paracasei and its application in bioconversion of glycosides.

Introduction: ß-glucosidase is one class of pivotal glycosylhydrolase enzyme that can cleavage glucosidic bonds and transfer glycosyl group between the oxygen nucleophiles. Lactobacillus is the most abundant bacteria in the human gut. Identification and characterization of new ß-glucosidases from Lactobacillus are meaningful for food or drug industry. Method: Herein, an acid-adapted ß-glucosidase (LpBgla) was cloned and characterized from Lactobacillus paracasei. And the insight acid-adapted mechanism of LpBgla was investigated using molecular dynamics simulations. Results and Discussion: The recombinant LpBgla exhibited maximal activity at temperature of 30°C and pH 5.5, and the enzymatic activity was inhibited by Cu2+, Mn2+, Zn2+, Fe2+, Fe3+ and EDTA. The LpBgla showed a more stable structure, wider substrate-binding pocket and channel aisle, more hydrogen bonds and stronger molecular interaction with the substrate at pH 5.5 than pH 7.5. Five residues including Asp45, Leu60, Arg120, Lys153 and Arg164 might play a critical role in the acid-adapted mechanism of LpBgla. Moreover, LpBgla showed a broad substrate specificity and potential application in the bioconversion of glycosides, especially towards the arbutin. Our study greatly benefits for the development novel ß-glucosidases from Lactobacillus, and for the biosynthesis of aglycones.

Neuroprotective effects of probiotics on anxiety- and depression-like disorders in stressed mice by modulating tryptophan metabolism and the gut microbiota.

Anxiety- and depression-like behaviors are commonly observed clinical features of depression and many other mental disorders. Recent evidence has revealed the crucial role of the microbiota-gut-brain axis in the bidirectional communication between the gastrointestinal tract and the central nervous system. Supplementation with psychobiotics may provide a novel approach for the adjunctive treatment of mental disorders by regulating the intestinal microecology. We isolated and identified a novel probiotic, Lactiplantibacillus plantarum D-9 (D-9), from traditional Chinese fermented foods in our previous work, which exhibited a high yield of gamma-aminobutyric acid (GABA). Herein, it was proved that the oral administration of D-9 could alleviate the depression- and anxiety-like behaviors of Chronic Unpredicted Mild Stress (CUMS) mice, and show non-toxicity or side-effects in the mice. Physiological and biochemical analyses demonstrated that D-9 regulated tryptophan metabolism, the HPA-axis and inflammation in CUMS mice. Moreover, D-9 modulated the structure and composition of the gut microbiota, leading to an increase in the relative abundance of Ligilactobacillus murinus and Lactobacillus johnsonii, and a decrease in the levels of Kineothrix alysoides and Helicobacter bilis compared to those in CUMS mice. Our work demonstrates that D-9 alleviated anxiety- and depression-like disorders in CUMS mice by modulating tryptophan metabolism and the gut microbiota. These findings provide an innovative strategy for the intervention and treatment of depressive disorders.

Heterologous expression and fibrillary characterization of the microtubule-binding domain of tau associated with tauopathies.

BACKGROUND: Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. METHODS AND RESULTS: Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. CONCLUSIONS: The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.

Lactobacillus kefiranofaciens ZW18 from Kefir enhances the anti-tumor effect of anti-programmed cell death 1 (PD-1) immunotherapy by modulating the gut microbiota.

Research on probiotics assisting PD-1 inhibitors in anti-tumor therapy has attracted widespread attention. Therefore, it is important to find new probiotic strains with a PD-1 inhibitor promoting effect. This study aims to find a strain with a good promoting effect on PD-1 inhibitor treatment from 5 probiotic strains with the function of modulating the gut microbiota or enhancing immunity. A preclinical study on the effect of probiotics combined with PD-1 inhibitors in murine melanoma was designed. In this study, Lactobacillus kefiranofaciens ZW18 (ZW18) was found to have the best anti-melanoma effect among the probiotic candidates in PD-1 inhibitor treatment. ZW18 inhibited the tumor growth in PD-1-treated mice with an inhibition rate of 66.16% by activating the body's immunity and promoting the tumor CD8+ T cell infiltration. Moreover, the supplement of ZW18 optimized the composition of the gut microbiota in mice treated with PD-1 inhibitors, and significantly increased the abundance of Akkermansia, the Prevotellaceae_NK3B31_group and Muribaculum. Collectively, ZW18 could be regarded as a potential candidate strain for promoting tumor immunotherapy. ZW18 combined with PD-1 inhibitors has a possibility of serving as a functional food to assist tumor immunotherapy.

Complete Genome Sequence of a Novel Lactobacillus paracasei TK1501 and Its Application in the Biosynthesis of Isoflavone Aglycones.

Lactobacillus strains are considered safe and healthy probiotics for manufacturing "natural food" products; this is due to their ability to produce bioactive compounds that reduce the incidence of various human diseases. Lactobacillus paracasei TK1501 is a novel probiotic strain isolated from naturally fermented congee; and can produce a high yield of genistein, one of the most widely studied isoflavone aglycones with plenty of physiological functions. To better understand the molecular basis of isoflavone aglycones biosynthesis, the complete 2,942,538 bp genome of L. paracasei TK1501 was sequenced and assembled; a group of genes that are involved in isoflavone aglycones production were identified. Of note, a ß-glucosidase was analyzed in the L. paracasei TK1501. Moreover, we also found that L. paracasei TK1501 could be used in soymilk fermentation; which would remarkably increase the contents of genistein, daidzein, and glycitein. This work was meaningful to the application of L. paracasei TK1501 and the molecular mechanism analysis of isoflavone aglycones biosynthesis in Lactobacillus strains.

Modulation of the Gut Microbiota and Glycometabolism by a Probiotic to Alleviate Amyloid Accumulation and Cognitive Impairments in AD Rats.

SCOPE: Regulating the gut microecology by probiotics is an efficient strategy to rational prevention and treatment of Alzheimer's disease (AD). However, there is currently a lack of well-known probiotic species in the protection against AD, and the involved mechanism has not been clearly interpreted. METHODS AND RESULTS: Herein, Lactobacillus plantarum MA2 (MA2), a functional probiotic isolated from traditional Chinese Tibetan kefir grains, is demonstrated to improve the cognitive deficits and anxiety-like behaviors in the d-galactose/AlCl3 induced AD rats, and attenuate the neuronal degeneration and Aß accumulation in the brain. Moreover, the study finds MA2 could alleviate the intestinal mucosal impairments, and impedes the activation of microglia and neuroinflammation through TLR4/MYD88/NLRP3 signaling pathway. 16S rRNA sequencing and metabolomic analysis indicate that MA2 reshapes the gut microbiota structure and composition, and remarkably modulates the glycometabolism. In that case, the exopolysaccharides (EPS) that derived from MA2 is furtherly proved with inhibitory effects on the Aß42 aggregation and amyloid-induced cytotoxicity. CONCLUSION: MA2 or MA2 EPS may be used as functional food and nutritional supplement for regulating the gut microbiota and metabolism disorders in AD. This study is of great significance to develop new intervention and therapeutic strategy on AD using probiotics and their metabolites.

Engineering of a fungal steroid 11α-hydroxylase and construction of recombinant yeast for improved production of 11α-hydroxyprogesterone.

Cytochrome P450 enzyme CYP68J5 from filamentous fungus Aspergillus ochraceus is industrially used for selective C11α-hydroxylation of canrenone and progesterone. To improve its selectivity of C11α-hydroxylation for relevant steroid substrates, a sequence-based targeted mutagenesis combined with saturation mutagenesis was conducted to search for variants with improved hydroxylation reaction specificity toward progesterone and D-ethylgonendione. Recombinant yeast expressing triple mutant V64F/E65G/N66T showed significantly increased C11α-hydroxylation selectivity (85 % VS WT 69.7 %). Saturation mutagenesis of V64, E65 and N66 resulted in the identification of single mutant V64K with greatly enhanced 11α-hydroxylation specificity toward progesterone (90.6 % VS WT 69.7 %). Furthermore, mutant N66D showed significant enhanced selectivity of C11α-hydroxylation toward D-ethylgonendione (70.8 % VS WT 58 %). Evaluation of recombinant yeast over-expressing V64K for progesterone transformation in 50 mL scale resulted in product 11α-OH progesterone concentrations of 432.5 mg/L, a 30.2 % increase compared with the CYP68J5 control. Our results also reveal that V64, E65 and N66 are key residues of CYP68J5 influencing its selectivity of C11α-hydroxylation, thus offering opportunities for further engineering of CYP68J5 for expanded industrial applications.

Identification of a novel probiotic and its protective effects on NAFLD via modulating gut microbial community.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is becoming the most common progressive liver diseases. Therapeutic strategy based on gut-liver axis and probiotics is a promising approach for the treatment of NAFLD. However, rare probiotics have been applied in NAFLD treatment, and the involved molecular mechanism is not entirely clear. RESULTS: We initially identified a novel functional probiotic, Lactobacillus kefiranofaciens ZW3, on the lipid deposition by a simple and rapid zebrafish model. Supplementation with ZW3 to the methionine and choline deficient (MCD) diet induced NAFLD rats could improve the liver impairments and reduce inflammation through TLR4-MyD88 and JNK signaling pathways. Moreover, ZW3 modulated gut microbiota by promoting relative abundance of Firmicutes and Lactobacillus, decreasing the abundance of Escherichia-Shigella and Bacteroides. Functional prediction of microbiome showed ZW3 presented potential enhancement on carbohydrate and lipid metabolism, cell process control and signal transduction processes, and reduced several human diseases. CONCLUSION: This present study identified a novel probiotic and its protective effects on NAFLD, and interpreted the interactions of ZW3 with the immune system and gut microbiota involved in gut-liver axis. © 2022 Society of Chemical Industry.

Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1.

ß-hydroxybutyric acid is the most sensitive indicator in ketoacidosis detection, and accounts for nearly 78% of the ketone bodies. Diaphorase is commonly used to detect the ß-hydroxybutyric acid in clinical diagnosis. However, the extraction of diaphorase from animal myocardium is complex and low-yield, which is not convenient for large-scale production. In this study, a diaphorase from Geobacillus sp. Y4.1MC1 was efficiently heterologous expressed and purified in E. coli with a yield of 110 mg/L culture. The optimal temperature and pH of this recombinant diaphorase (rDIA) were 55 °C and 6.5, respectively. It was proved that rDIA was a dual acid- and thermo-stable enzyme, and which showed much more accurate detection of ß-hydroxybutyric acid than the commercial enzyme. Additionally, we also investigated the molecular interaction of rDIA with the substrate, and the conformation transition in different pH values by using homology modeling and molecular dynamics simulation. The results showed that 141-161 domain of rDIA played important role in the structure changes and conformations transmission at different pH values. Moreover, it was predicted that F105W, F105R, and M186R mutants were able to improve the binding affinity of rDIA, and A2Y, P35F, Q36D, N210L, F211Y mutants were benefit for the stability of rDIA.

Dual Effect of the Acidic Polysaccharose Ulvan on the Inhibition of Amyloid-ß Protein Fibrillation and Disintegration of Mature Fibrils.

The abnormal folding and aggregation of amyloid-ß protein (Aß) is the main reason for the occurrence and development of Alzheimer's disease (AD). The discovery of novel inhibitors against Aß aggregation is still the current research focus. Herein, we report the inhibitory effect of ulvan, an acidic polysaccharide from green algae of the genus Ulva, against Aß fibrillation using thioflavin T (ThT) fluorescence and atomic force microscopy (AFM) assays. It is shown that ulvan effectively inhibits Aß fibrillogenesis in a concentration-dependent manner and actively inhibits the formation of A11-reactive Aß oligomers, the most toxic Aß species. The circular dichroism spectrum reveals that ulvan blocks the conformational transition of Aß40 from the initial random coil to a ß-sheet structure, but it only delays the conformational transition of Aß42. It is also found that ulvan greatly reduces Aß-induced cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In addition, ulvan effectively downregulates intracellular reactive oxygen species production and protects PC12 cells from the damage caused by Aß fibrillation. Moreover, ulvan disaggregates preformed mature fibrils into off-pathway oligomers and greatly decreases their associated cytotoxicity, as revealed using ThT fluorescence, AFM, MTT, and dot-blotting assays. The above results not only fully describe the inhibitory effect of ulvan on Aß fibrillation and its related cytotoxicity but also provide novel ideas for the development of functional food ingredients from seaweed to treat AD.

General Aggregation-Induced Emission Probes for Amyloid Inhibitors with Dual Inhibition Capacity against Amyloid ß-Protein and α-Synuclein.

Amyloid self-assembly is pathologically linked to many neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). While many inhibitors have been developed individually for specific amyloid proteins, there are a few effective platforms to screen on a large scale general amyloid inhibitors against different amyloid proteins. Herein, we developed a new class of amyloid inhibitor probes by site-specific conjugation of aggregation-induced emission (AIE) molecules with amyloid proteins (i.e., AIE@amyloid probes) to realize a high-throughput screening of small-molecule inhibitors. Optimization of site-specific AIE conjugation with two amyloid proteins, amyloid-ß protein (Aß) and α-synuclein (αSN), enabled us to retain their high amyloidogenic properties; i.e., AIE-amyloid probes alone exhibited strong fluorescence due to amyloid-like aggregation, but they showed no fluorescence in the presence of amyloid inhibitors to prevent amyloid aggregation. From integration of AIE@amyloid probes and computational virtual screening from a large drug database, it was found that tolcapone possessed a dual inhibition against the aggregation and cytotoxicity of both Aß and αSN. More importantly, tolcapone significantly improved the spatial cognition and recognition of Aß-treated mice. This work represents an innovative attempt to design an AIE-based anti-amyloid drug platform for identifying new small-molecule inhibitors against amyloidogenesis in both AD and PD or other amyloid diseases.

Expression and purification of amyloid ß-protein, tau, and α-synuclein in Escherichia coli: a review.

Misfolding and accumulation of amyloidogenic proteins into various forms of aggregated intermediates and insoluble amyloid fibrils is associated with more than 50 human diseases. Large amounts of high-quality amyloid proteins are required for better probing of their aggregation and neurotoxicity. Due to their intrinsic hydrophobicity, it is a challenge to obtain amyloid proteins with high yield and purity, and they have attracted the attention of researchers from all over the world. The rapid development of bioengineering technology provides technical support for obtaining large amounts of recombinant amyloidogenic proteins. This review discusses the available expression and purification methods for three amyloid proteins including amyloid ß-protein, tau, and α-synuclein in microbial expression systems, especially Escherichia coli, and discusses the advantages and disadvantages of these methods. Importantly, these protocols can also be referred to for the expression and purification of other hydrophobic proteins.

Cyanidin-3-O-glucoside inhibits Aß40 fibrillogenesis, disintegrates preformed fibrils, and reduces amyloid cytotoxicity.

Alzheimer's disease (AD) is mainly caused by the fibrillogenesis of amyloid-ß protein (Aß). Therefore, the development of effective inhibitors against Aß fibrillogenesis offers great hope for the treatment of AD. Cyanidin-3-O-glucoside (Cy-3G) is a commonly found anthocyanin that is mainly present in fruits, with established neuroprotective effects in situ. However, it remains unknown if Cy-3G can prevent Aß fibrillogenesis and alleviate the corresponding cytotoxicity. In this study, extensive biochemical, biophysical, biological and computational experiments were combined to address this issue. It was found that Cy-3G significantly inhibits Aß40 fibrillogenesis and disintegrates mature Aß fibrils, and its inhibitory capacity is dependent on the Cy-3G concentration. The circular dichroism results showed that Cy-3G and Aß40 at a molar ratio of 3 : 1 slightly prevents the structural transformation of Aß40 from its initial random coil to the ß-sheet-rich structure. Co-incubation of Aß40 with Cy-3G significantly reduced the production of intracellular reactive oxygen species induced by Aß40 fibrillogenesis and thus reduced Aß40-induced cytotoxicity. Molecular dynamics simulations revealed that Cy-3G disrupted the ß-sheet structure of the Aß40 trimer. Cy-3G was found to mainly interact with the N-terminal region, the central hydrophobic cluster and the ß-sheet region II via hydrophobic and electrostatic interactions. The ten hot spot residues D7, Y10, E11, F19, F20, E22, I31, I32, M35 and V40 were also identified. These findings not only enable a comprehensive understanding of the inhibitory effect of Cy-3G on Aß40 fibrillogenesis, but also allow the identification of a valuable dietary ingredient that possesses great potential to be developed into functional foods to alleviate AD.

Molecular Mediation of Prion-like α-Synuclein Fibrillation from Toxic PFFs to Nontoxic Species.

Braak's theory described Parkinson's disease (PD) progression as prion-like α-synuclein (αSyn) spreading, which fundamentally subverts the understanding of pathogenesis. The pathological αSyn spreading pathway includes uptake, propagation, and release. However, the previous disease models were limitedly focusing on amyloid propagation/aggregation, which significantly impedes the mechanism exploration in spreading pathways and related therapeutic development. The spreading model can be achieved using recombinant αSyn preformed fibrils (PFFs), which seeds endogenous αSyn monomer to aggregation and causes substantial pathology and neurotoxicity. Here, we determined that dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, can promote the fibrillization of prion-like PFF and induce propagation to form a distinct strain. Furthermore, administration of DHM significantly reduced prion-like PFF-induced propagation and neurotoxicity. The discovery of inducing infectious and neurotoxic PFF to a nontoxic strain resulting in neuron protection via promoting the fibrillization of PFF rather than inhibiting advances the understanding of the prion-like spreading mechanism and helps in developing treatments against PD and related α-synucleinopathies.

Brazilin Inhibits α-Synuclein Fibrillogenesis, Disrupts Mature Fibrils, and Protects against Amyloid-Induced Cytotoxicity.

The inhibitory effect of brazilin against α-synuclein (α-syn) fibrillogenesis, disruption effect against mature fibrils, and the following cytotoxicity were examined by systematical biochemical, biophysical, cellular biological, and molecular simulation experiments. It is found that brazilin inhibited α-syn fibrillogenesis and disrupted the performed fibrils with a concentration-dependent manner. Moreover, cellular experimental data showed that brazilin effectively reduced the cytotoxicity induced by α-syn aggregates. Finally, molecular dynamics simulations were performed to explore the interactions between brazilin and α-syn pentamer. It is found that brazilin directly interacts with α-syn pentamer, and the hydrophobic interactions are favorable for brazilin binding with the α-syn pentamer, while the electrostatic part provides adverse effects. Three binding regions were identified to inhibit α-syn fibrillogenesis or disrupt the preformed aggregates. Furthermore, six important residues (i.e., G51, V52, A53, E61, V66, and K80) of α-syn were also identified. We expected that brazilin is an effective agent against α-syn fibrillogenesis and associated cytotoxicity.

Inhibitory Effect of a Flavonoid Dihydromyricetin against Aß40 Amyloidogenesis and Its Associated Cytotoxicity.

Misfolding and fibrillogenesis of amyloid-ß protein (Aß) play a key role in the onset and progression of Alzheimer's disease (AD). Screening for inhibitors against Aß amyloidogenesis is helpful for rational designing and developing new anti-AD drugs and therapeutic strategies. Dihydromyricetin, a natural flavonoid extracted from a Chinese herb, Ampelopsis grossedentata, has been proven with antioxidative, anti-inflammatory, and neuroprotective effects against neurodegenerative disease. Herein, we found that dihydromyricetin could inhibit Aß40 aggregation, impede the protofibril formation, disassemble preformed Aß40 fibrils, and protect PC12 cells from the Aß40-induced cytotoxicity using a series of biochemical and biophysical assays, including thioflavin T fluorescence, atomic force microscopy, and cell toxicity assays. Circular dichroism spectroscopy data proved that dihydromyricetin delayed the Aß40 conformational conversion. In addition, the results of molecular dynamics simulations indicated that the interaction between dihydromyricetin and Aß40 trimer is mainly nonpolar interactions. Key residues (i.e., V18, A21, and D23) of the Aß40 interacting with dihydromyricetin were also identified. This study suggested that dihydromyricetin shows great potential to be developed as a novel Aß40 inhibitor.

Vitamin B12 inhibits α-synuclein fibrillogenesis and protects against amyloid-induced cytotoxicity.

Vitamin B12 (VB12) is a necessary micronutrient for growth and the development of the nervous system. Severe deficiencies of VB12 have been linked to damage of learning and memory ability and cognitive decline, along with several neurological diseases. Misfolding and aggregation of α-synuclein (αSN) into insoluble fibrils is associated with the onset and progression of Parkinson's disease (PD), which affects tens of millions of elderly patients all over the world. Developing novel inhibitors to obstruct the aggregation of αSN has become a topic of intense research. In this study, the inhibitory effect of VB12 against the fibrillogenesis and cytotoxicity of αSN was systematically analyzed using thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), circular dichroism (CD) spectroscopy and cytotoxicity assays. Both ThT and AFM results showed that VB12 could inhibit αSN fibrillogenesis in a dose-dependent manner. CD data suggested that VB12 delays the conformational conversion of αSN to ß-sheet rich structures, especially to the parallel ß-sheet conformation. As a result, VB12 greatly alleviated the cytotoxicity of αSN aggregates. Moreover, VB12 was also found to disassemble preexisting mature αSN fibrils and attenuate the consequent cytotoxicity. These findings not only provide a comprehensive understanding of the inhibitory effect of VB12 on αSN fibrillogenesis, but also identify a valuable nutrient source that possesses great potential to be developed as a new functional food ingredient to help alleviate PD.

Dihydromyricetin Inhibits α-Synuclein Aggregation, Disrupts Preformed Fibrils, and Protects Neuronal Cells in Culture against Amyloid-Induced Cytotoxicity.

Fibrillogenesis of α-synuclein (αSN) is associated with the onset and progression of Parkinson's disease (PD). Dihydromyricetin (DHM), a natural flavonoid compound extracted from Ampelopsis grossedentata, has proven antioxidative, antineuroinflammatory, and neuroprotective effects in dementia. However, it remains unclear if DHM can impede αSN fibrillogenesis and attenuate the corresponding cytotoxicity. Herein, we found that DHM could inhibit αSN fibrillogenesis and destabilize mature αSN fibrils in a dose-dependent manner. Moreover, DHM protected against αSN-induced cytotoxicity by improving the cell viability by 34.73 ± 3.68% at a 1:1 molar ratio of αSN to DHM. Molecular dynamics simulations showed that DHM interacts with the αSN trimer mainly via nonpolar mechanisms. The key residues by which αSN interacts with DHM were hydrophobic, and their side chains and main chains showed a synergistic effect via hydrophobic and hydrogen-bonding interactions. These findings suggest that DHM possesses great potential to be developed into a new aggregation inhibitor for αSN.

Hydroxylated Single-Walled Carbon Nanotubes Inhibit Aß42 Fibrillogenesis, Disaggregate Mature Fibrils, and Protect against Aß42-Induced Cytotoxicity.

The fibrillogenesis of amyloid-ß protein (Aß) is considered a crucial factor in the pathogenesis of Alzheimer's disease (AD). Hence, inhibiting Aß fibrillogenesis is regarded as the primary therapeutic strategy for the prevention and treatment of AD. However, the development of effective inhibitors against Aß fibrillogenesis has faced significant challenges. Previous studies have shown that pristine single-walled carbon nanotubes (SWNTs) can inhibit fibrillogenesis of some amyloid proteins. However, the poor dispersibility of SWNTs in an aqueous environment greatly hinders their inhibitory efficacy. Here, we examined the inhibitory activity of hydroxylated SWNTs (SWNT-OH) on the aggregation and cytotoxicity of Aß42 using thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), cellular viability assays, and molecular dynamics (MD) simulations. ThT and AFM results showed that SWNT-OH inhibits Aß42 fibrillogenesis and disaggregates preformed amyloid fibrils in a dose-dependent manner. Furthermore, the ratio of hydroxyl groups in SWNT-OH is crucial for their effect against Aß42 aggregation. SWNT-OH exerted cytoprotective effects against Aß42 fibrillation-induced cytotoxicity. The results of free-energy decomposition studies based on MD simulations revealed that nonpolar interactions, and especially van der Waals forces, contributed most of the free energy of binding in the SWNT-OH-Aß complex. Two regions of the Aß pentamer were identified to interact with SWNT-OH, spanning H13-Q15 and V36-G38. The findings presented here will contribute to a comprehensive understanding of the inhibitory effect of hydroxylated nanoparticles against Aß fibrillogenesis, which is critical for the search for more effective agents that can counteract amyloid-mediated pathologies.