Flavonoids with Vicinal Hydroxyl Groups Inhibit Human Calcitonin Amyloid Formation.

Human calcitonin (hCT) is a 32-residue peptide hormone that can aggregate into amyloid fibrils and cause cellular toxicity. In this study, we investigated the inhibition effects of a group of polyphenolic molecules on hCT amyloid formation. Our results suggest that the gallate moiety in epigallocatechin-3-gallate (EGCG), a well-recognized amyloid inhibitor, is not critical for its inhibition function in the hCT amyloid formation. Our results demonstrate that flavonoid compounds, such as myricetin, quercetin, and baicalein, that contain vicinal hydroxyl groups on the phenyl ring effectively prevent hCT fibrillization. This structural feature may also be applied to non-flavonoid polyphenolic inhibitors. Moreover, our results indicate a plausible mechanistic role of these vicinal hydroxyl groups which might include the oxidation to form a quinone and the subsequent covalent linkage with amino acid residues such as lysine or histidine in hCT. This may further disrupt the crucial electrostatic and aromatic interactions involved in the process of hCT amyloid fibril formation. The inhibition activity of the polyphenolic compounds against hCT fibril formation may likely be attributed to a combination of factors such as covalent linkage formation, aromatic stacking, and hydrogen bonding interactions.

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation.

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3ß-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.

Vibrational Approach to the Dynamics and Structure of Protein Amyloids.

Amyloid diseases, including neurodegenerative diseases such as Alzheimer's and Parkinson's, are linked to a poorly understood progression of protein misfolding and aggregation events that culminate in tissue-selective deposition and human pathology. Elucidation of the mechanistic details of protein aggregation and the structural features of the aggregates is critical for a comprehensive understanding of the mechanisms of protein oligomerization and fibrillization. Vibrational spectroscopies, such as Fourier transform infrared (FTIR) and Raman, are powerful tools that are sensitive to the secondary structure of proteins and have been widely used to investigate protein misfolding and aggregation. We address the application of the vibrational approaches in recent studies of conformational dynamics and structural characteristics of protein oligomers and amyloid fibrils. In particular, introduction of isotope labelled carbonyl into a peptide backbone, and incorporation of the extrinsic unnatural amino acids with vibrational moieties on the side chain, have greatly expanded the ability of vibrational spectroscopy to obtain site-specific structural and dynamic information. The applications of these methods in recent studies of protein aggregation are also reviewed.

Residue-Specific Dynamics and Local Environmental Changes in Aß40 Oligomer and Fibril Formation.

Elucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue-specific dynamics and local environmental changes of Aß40 along the course of aggregation by using para-cyanophenylalanine (PheCN ) as a fluorescent and vibrational probe. Our results show that the PheCN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N-terminal region suggest that they play crucial roles in the formation of the oligomeric core. The PheCN 4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N-terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.

Gold Nanoparticles as a Probe for Amyloid-ß Oligomer and Amyloid Formation.

The process of amyloid-ß (Aß) amyloid formation is pathologically linked to Alzheimer's disease (AD). The identification of Aß amyloids and intermediates that are crucial players in the pathology of AD is critical for exploring the underlying mechanism of Aß aggregation and the diagnosis of the disease. Herein, we performed a gold nanoparticle (AuNP)-based study to detect the formation of Aß amyloid fibrils and oligomers. Our results demonstrate that the intensity of the surface plasmon resonance (SPR) absorption band of the AuNPs is sensitive to the quantity of Aß40 amyloids. This allows the SPR assay to be used for detection and semi-quantification of Aß40 amyloids, and characterization of the kinetics of Aß amyloid formation. Furthermore, our study demonstrates that the SPR band intensity of the AuNPs is sensitive to the presence of oligomers of both Aß40 and an Aß40 mutant, which forms more stable oligomers. The kinetics of the stable oligomer formation of the Aß40 mutant can also be monitored following the SPR band intensity change of AuNPs. Our results indicate that this nanoparticle based method can be used for mechanistic studies of early protein self-assembly and fibrillogenesis.

Site-specific dynamics of amyloid formation and fibrillar configuration of Aß(1-23) using an unnatural amino acid.

We identify distinct site-specific dynamics over the time course of Aß1-23 amyloid formation by using an unnatural amino acid, p-cyanophenylalanine, as a sensitive fluorescent and Raman probe. Our results also suggest the key role of an edge-to-face aromatic interaction in the conformational conversion to form and stabilize ß-sheet structure.