Inhibition of Lysozyme Amyloidogenesis by Osmolytes

Abstract

Report of systemic amyloidosis by point mutants of human lysozyme paved way towards more systematic studies into general principles of amyloidosis and subsequently development of broad spectrum drugs rather than individual searches for various amyloidogenic diseases. Osmolytes are well-known protein stabilizers and known to be found in incredibly large quantities in animals living under extreme conditions. The fact that they occur in huge quantities in deep-sea animals gives hope that they alone or as cocktails can be administered to terminally ill patients (regardless of the type of amyloid disease) after preliminary safety trials. However there are not many studies in this direction and they are on different proteins thus making comparison difficult. Hen lysozyme forms amyloid under various conditions. So far we have employed alkaline and acidic conditions. We have utilized residual enzymatic activity (REA) to quantify extent of folded protein, fluorescence steady state anisotropy (rss) to quantify mean oligomer size and fluorescence based Thioflavin-T (Th-T) assay kinetics to quantify amyloid content. We have studied many osmolytes in parallel,viz, Arginine, Betaine, Trehalose, TMAO, Taurine, Ectoine, Putrescine, Spermidine & Spermine. Since majority of data is kinetic in nature, an attempt is being made to tabulate all this data in the form of a mathematical matrix so as to facilitate concise presentation and facile comparison. RESULTS (with alkaline condition): (a) Different osmolytes affect different steps with their own concentration dependence profile. (b) Ectoine and Polyamines are effective at concentrations as low as 50 mM. (c) Arginine has marked geometric concentration dependence trend in REA as well as Th-T. Polyamines have no noticeable effect on stability of lysozyme but profound effect on Th-T. (d) Trehalose reveals a different concentration profile than Arginine, thus pointing to difference in mode of action.