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.

Brazilin Inhibits Prostatic Acidic Phosphatase Fibrillogenesis and Decreases its Cytotoxicity.

A 39-amino acid peptide fragment that is derived from prostatic acidic phosphatase (PAP), PAP248-286 , is secreted in large amounts in human semen and forms amyloid fibrils. These fibrils can capture HIV virions and increase the attachment of virions to target cells; as such, they are called a "semen-derived enhancer of virus infection" (SEVI). Therefore, the inhibition of the formation of PAP248-286 amyloid fibrils is of great significance. Herein, we demonstrate that brazilin effectively inhibits PAP248-286 aggregation. The inhibitory effect increases with increasing brazilin concentration. Thioflavin T fluorescence assays and TEM observations confirmed that a few fibrils formed when brazilin was present with PAP248-286 in an equimolar concentration. Circular dichroism spectroscopy indicated that brazilin inhibited the secondary structural transitions from α-helices and random coils into ß-sheets. Cytotoxicity assays showed that brazilin significantly decreased the cytotoxicity of the fibrils at 0.01 mmol L-1 . Isothermal titration calorimetry revealed that hydrophobic interactions were the main driving force for the binding of brazilin to the PAP248-286 monomer (dissociation constant, 4.03 µmol L-1 ), and that the binding affinity of brazilin for the fibrils was at least three orders of magnitude lower than that for the monomer. These results indicate that brazilin holds great potential as a small-molecule agent against SEVIs.

Oligomers, protofibrils and amyloid fibrils from recombinant human lysozyme (rHL): fibrillation process and cytotoxicity evaluation for ARPE-19 cell line.

Amyloid-associated diseases, such Alzheimer's, Huntington's, Parkinson's, and type II diabetes, are related to protein misfolding and aggregation. Herein, the time evolution of scattered light intensity, hydrophobic properties, and conformational changes during fibrillation processes of rHL solutions at 55 °C and pH 2.0 were used to monitor the aggregation process of recombinant human lysozyme (rHL). Dynamic light scattering (DLS), thioflavin T (ThT) fluorescence, and surface tension (ST) at the air-water interface were used to analyze the hydrophobic properties of pre-amyloid aggregates involved in the fibrillation process of rHL to find a correlation between the hydrophobic character of oligomers, protofibrils and amyloid aggregates with the gain in cross-ß-sheet structure, depending on the increase in the incubation periods. The ability of the different aggregates of rHL isolated during the fibrillation process to be adsorbed at the air-water interface can provide important information about the hydrophobic properties of the protein, which can be related to changes in the secondary structure of rHL, resulting in cytotoxic or non-cytotoxic species. Thus, we evaluated the cytotoxic effect of oligomers, protofibrils and amyloid fibrils on the cell line ARPE-19 using the MTT reduction test. The more cytotoxic protein species arose after a 600-min incubation time, suggesting that the hydrophobic character of pre-amyloid fibrils, in addition to the high prevalence of the cross-ß-sheet conformation, can become toxic for the cell line ARPE-19.

Ethanol reduces amyloid aggregation in vitro and prevents toxicity in cell lines.

BACKGROUND: Alzheimer's disease (AD) alters cognitive functions. A mixture of soluble ß-amyloid aggregates (Aß) are known to act as toxic agents. It has been suggested that moderate alcohol intake reduces the development of neurodegenerative diseases, but the molecular mechanisms leading to this type of prevention have been elusive. We show the ethanol effect in the generation of complex Aß in vitro and the impact on the viability of two cell lines. METHODS: The effect of ethanol on the kinetics of ß-amyloid aggregation in vitro was assessed by turbimetry. Soluble- and ethanol-treated ß-amyloid were added to the cell lines HEK and PC-12 to compare their effects on metabolic activity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. In addition, we used molecular modeling to assess the impact of exposure to ethanol on the structure of ß-amyloid. RESULTS: Exposure to soluble ß-amyloid was toxic to both cell lines; however, exposing the cells to ß-amyloid aggregated in 10 mmol ethanol prevented the effect. In silico modeling suggested that ethanol alters the dynamics for assembling Aß by disrupting a critical salt bridge between residues Asp 23 and Lys 28, required for amyloid dimerization. Thus, ethanol prevented the formation of complex short (∼100 nm) Aß, which are related to higher cell toxicity. CONCLUSIONS: Ethanol prevents the formation of stable Aß dimers in vitro, thus protecting the cells maintained in culture. Accordingly, in silico modelling predicts that soluble ß-amyloid molecules do not form stable multimers when exposed to ethanol.

Structure and function of amyloid in Alzheimer's disease.

This review is focused on the structure and function of Alzheimer's amyloid deposits. Amyloid formation is a process in which normal well-folded cellular proteins undergo a self-assembly process that leads to the formation of large and ordered protein structures. Amyloid deposition, oligomerization, and higher order polymerization, and the structure adopted by these assemblies, as well as their functional relationship with cell biology are underscored. Numerous efforts have been directed to elucidate these issues and their relation with senile dementia. Significant advances made in the last decade in amyloid structure, dynamics and cell biology are summarized and discussed. The mechanism of amyloid neurotoxicity is discussed with emphasis on the Wnt signaling pathway. This review is focused on Alzheimer's amyloid fibrils in general and has been divided into two parts dealing with the structure and function of amyloid.