In Vitro Induction of Amyloidosis and its Counter Measure

Incorrect folding or mis-folding of proteins results in the formation of protein aggregates. Protein aggregates in general can be categorized into two types: amorphous aggregates, the ones with no long-range order, and amyloid fibrils, the ones with highly ordered structure. Aggregation of proteins leading to malfunction of organs leads to amyloidosis. About 17 different proteins have been found to form amyloid in vivo. Amyloid fibrils which are formed from those proteins share some common morphological features, but these proteins do not possess a conserved sequence or native structural motif. Recent studies show that amyloid formation is not only possible with disease-associated proteins, but also with proteins that are not associated with any known amyloid diseases under certain conditions. Non diseaserelated proteins can be induced in vitro to polymerize into amyloid fibrils under certain favorable conditions such as heating, agitation, low pH, pressure, and the presence of cosolvent. In the present study, an egg white lysozyme was used as protein for inducing amyloidosis through formation of amyloid fibrils. The amyloidosis was induced by applying extreme acidic environment (pH 2.0) by hydrochloric acid. Briefly, the lysozyme was isolated from the egg white by ethanol precipitation method. Results revealed that 40% ethanol concentration precipitation gave highest content of protein lysozyme. Lysozyme was treated hydrochloric acid (pH=2) along with salt. Amyloid fibril formation was verified by CD spectroscopy at far UV (200-260 nm). It revealed the presence of secondary beta sheet structure. Further, nicotinamide was used at serial concentrations of concentration of 22.5 mg and 45 mg to inhibit the amyloid formation. The outcome was also verified by CD spectroscopy. Nicotinamide successfully reduced the amyloid fibril formed in the egg white lysozyme. It was concluded that the inhibition of lysozyme amyloid formation by Nicotinamide is dose-dependent. Thus the current study shed light on a rational design of effective therapeutics for amyloidogenic diseases