Nitration of Tyr37 alters the aggregation pathway of hIAPP and enhances its cytotoxicity

The amyloid aggregation of hIAPP and the increased level of oxidative stress are closely related to the occurrence and development of type 2 diabetes (T2D). Protein tyrosine nitration is a common post-translational modification under oxidative stress conditions. We previously found that tyrosine nitrated hIAPP (3-NT-hIAPP) has higher cytotoxicity than wild type hIAPP. In order to further elucidate the mechanism by which tyrosine nitration enhances the toxicity of hIAPP, we systematically studied the effect of tyrosine nitration on hIAPP aggregation and its impact on INS-1 cells. Collective experimental data from ThT, RLS, DLS, zeta potentials, Bis-ANS, ¹H NMR, TEM, dye leakage and hemolysis confirmed that tyrosine nitration accelerates hIAPP aggregation, consistent with tyrosine nitration reducing hIAPP zeta potential, but 3-NT-hIAPP mainly undergoes an off-pathway aggregation to form amorphous aggregates, even in the presence of POPC/POPG LUVs. Further, our results confirmed that the most toxic species are the small amorphous aggregates formed by 3-NT-hIAPP, which is more stable and toxic than hIAPP oligomers. Collectively, these data suggest that tyrosine nitration can increase cytotoxicity of hIAPP by modulating its amyloidogenicity. This study provides new support for the fact that oxidative stress promotes the development of T2D from the view of nitrative stress.