Contribution of Methionine Oxidation to Amyloid Fibril Formation by Apolipoprotein A-I

Abstract

Apolipoprotein A-I (apoA-I) is an important promoter of cardiovascular health. However, oxidation in physiological conditions can alter the structure/function of apoA-I and transform this atheroprotective protein into a potentially harmful factor. One of the possible dysfunctional fates of apoA-I is represented by protein aggregation and amyloid fibril formation; as suggested by high incidence of apoA-I amyloid deposits in atherosclerotic lesions. Recently it has been reported that upon oxidation of its three native methionines, apoA-I can produce amyloid fibrils in vitro.The aim of our study was to establish the contribution of oxidation of specific methionines to apoA-I amyloid fibril formation.Single amino acid apoA-I mutants were produced, in which each of the three native methionines (M86, M112, and M148) were replaced by leucines. After oxidation by H2O2 and removal of H2O2 by dialysis, the protein samples were incubated at 37°C and pH 6.0 to elicit protein aggregation. Amyloid fibril formation was evaluated by measuring the reactivity of the samples towards the fluorescent dye Thioflavin T (ThT).Oxidized wild-type apoA-I (WT-apoA-I) readily formed solid aggregates. ThT fluorescence developed with rapid kinetics and reached maximum intensity at 24h. Upon oxidation and incubation in similar conditions, ThT kinetics were distinct for different single M to L apoA-I mutants, suggesting that the three methionines do not equally contributes, upon oxidation, to protein destabilization and the process of amyloid fibril formation. In particular, ThT kinetics of M112L- and WT-apoA-I were not significantly different (T1/2 @ 4 and 3 h, respectively). In contrast, ThT kinetics were significantly slower for both M86L-apoA-I and M148L-apoA-I (T1/2 @ 30 h and > 48 h, respectively).These results indicate that oxidation of M86 and M148 is largely responsible for apoA-I destabilization and amyloid fibril formation.