Inhibition of amyloid fibrillation and cytotoxicity of lysozyme fibrillation products by polyphenols.

An increasing number of studies conducted under in vitro and in vivo conditions, have concluded that polyphenols, compounds frequently occurring in many herbs with antioxidant properties, prevent and reverse amyloid fibril formation. However, the mechanisms by which these natural products modulate the protein aggregation process are poorly understood. Herein, a range of techniques including thioflavin T (ThT) and ANS fluorescence assays, electron microscopy and circular dichroism have been employed to determine the efficacy of rosmarinic acid (RA) and resveratrol (Res) on the inhibition/reversion of fibrillogenesis and hindering cytotoxicity induced by protofibrils and amyloid fibrils of hen egg white lysozyme (HEWL). Results demonstrated that both polyphenols effectively inhibit fibrillogenesis and destabilize preformed fibrils of HEWL in a concentration-dependent manner. Cytotoxicity protection on PC12 cells was also observed using the MTT assay, ROS production assay, and phase-contrast microscopy. It is suggested that the mechanism underlying the inhibitory effects of RA and Res is to prevent hydrophobic interactions between HEWL amyloidogenic prefibrillar species, although additional studies is needed to elucidate the detailed mechanisms involved. A combination of antioxidative and anti-amyloidogenic properties of these molecules may provide them with the described neuroprotective capacities.

Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation.

A number of ligands with affinities for the heme binding site of apomyoglobin were tested to control amorphous and fibrillar aggregation in the protein. Several techniques, including fluorescence, dynamic light scattering, transmission electron microscopy, dot blot analysis combined with viability studies were employed for structural characterization and cytotoxicity assessment of the intermediate and final protein structures formed during the aggregation process. Of the small molecules investigated, chrysin and Nile red with high structural similarities to heme were chosen for further studies. Only fibril formation was found to be prevented by Nile red, while chrysin, with a greater structural flexibility, was able to prevent both types of aggregate formation. The two ligands were found to influence aggregation at different stages of intermediate structure formation, an ability determined by their degrees of similarities with heme. Based on structural characterization and toxicity studies, it is concluded that ligands similar in structure to heme may be effective in influencing various stages of aggregate formation and toxicity potencies of the protein structures. Since metalloproteins constitute more than thirty percent of all known proteins, it is concluded that the present strategy may be of general significance.

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: role of surface hydrophobicity.

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.