Interaction Of HIAPP And Heme On Its Nitration And Aggregation

A significant pathological of Type 2 diabetes(Type 2 diabetes,T2DM)patients is the insoluble fibrils deposition in their islet-β cells.The highly aggregated polypeptide is human islet amyloid polypeptide(hIAPP).In recent years,studies have shown that it is the the oligomeric intermediates formed during the lag phase rather than mature fibers produced during plateau phase that result in islet β-cell injury or apoptosis.There is no consensus on the mechanism of the physiological toxicity of hIAPP oligomers.However,more and more experiments have shown that mitochondrial dysfunction in pancreatic tissues and its mediated oxidative stress and oxidative damage play an important role in islet β-cell injury or apoptosis.Under oxidative stress or oxidative damage conditions,the increased peroxidase activity of heme can lead to the nitration of protein tyrosine,and the structure and function of the protein after nitration will also change.Some studies have pointed that the level of protein nitration in T2 DM is significantly increased,indicating that hIAPP has a direct link with it.However,the molecular mechanism is still not clear.In addition,on the autopsy of diabetic patients,the metabolic disorder of heme in the human body is closely related to the development of T2 DM,suggesting that heme may play an important role in the islet β-cell injury induced by hIAPP.To investigate the effect of tyrosine nitration on the structure and function of hIAPP,elucidate the molecular mechanism in T2 DM,and verify the fact that the interaction of heme and hIAPP can play an important role in T2 DM,we will examine this article from the following two aspects:(1)The mechanism by which heme inhibits hIAPP aggregation and dismantles hIAPP aggregates.Three mutated hIAPP polypeptides were chosen to study the interaction of mutated polypeptides with heme.The results showed that heme strongly inhibited the aggregation of hIAPP and its mutant polypeptides and partially dismantled the already formed aggregates.His18 and Arg11 in hIAPP played an important role in inhibiting aggregation by binding to heme.The control group experiments showed that the heme iron center played a very important role in binding to hIAPP,but it was not necessary to inhibit aggregation.In addition,from the results of the control group we proposed that heme might inhibit the aggregation of hIAPP by blocking the accumulation of Phe23 aromatic-aromatic stack to form a β-sheet structure.After binding heme,hIAPP was prone to form oligomers,whereas oligomers had been thought to be important contributors to apoptosis of islet-β cells.The above experimental results revealed that the interaction of heme and hIAPP,which will help to understand the relationship between heme and T2 DM.(2)Nitration of Tyr37 in hIAPP affected its own aggregation and INS-1 cytotoxicity.The dot blot experiment verified that Tyr37 in hIAPP coule be nitrated in vitro under heme-H2O2-NO2-system.UV absorption spectra and peroxidase activity experiments showed that the nitration of Tyr37 did not affect the binding of hIAPP to heme and the peroxidase activity of the heme-hIAPP complex.A series of aggregation experiments showed that nitration of Tyr37 effectively inhibited the degree of fibrillation of hIAPP.Finally,cell experiments indicated that nitration of Tyr37 increased the toxicity of INS-1 cells.These studies revealed that nitrated hIAPP increased islet-β cell cytotoxicity by increasing the number of low weight molecular oligomers.The experimental results provided a reasonable explanation for the increase of nitration levels in the islets and T2DM.