血红素与hIAPP相互作用在hIAPP诱导胰岛β细胞损伤中的作用研究

Human islet amyloid polypeptide (hIAPP) has been found to induce mitochondrion dysfunction and oxidative damage to islet ββ-cell. However, the mechanism is unclear. It was found that hIAPP can bind to heme, leading to the increase of heme’s catalytic activity on oxidation. Considering the close relationship between heme metabolism and mitochondrion function, and the roles of catalyzing protein oxidation and nitration, in combination with that hIAPP misfolding, lose of heme homeostasis and oxidative damage are important pathology features of type 2 diabetes (T2D), we hypothesized that hIAPP might affect heme metabolism through binding to heme and subsequently induce mitochondrion dysfunction and oxidative damage. To confirm our hypothesis, we’ll: (1) design a series of truncated and mutated hIAPP and study the binding properties between hIAPP and heme; (2) study the aggregation property of hIAPP upon binding and the peroxidative activities of the forming heme-hIAPP complex, as well as it catalyzed tyrosine nitration; (3) study the roles of the interaction between hIAPP and heme in heme metabolism, mitochondrion function and protein oxidation and nitration on rat insulinoma INS-1 cell. These studies are not only important for the better understanding of the pathogenic mechanism of hIAPP in T2D, but also important in providing scientific basis and new therapeutic targets for preventing and curing of T2D.

人胰岛淀粉样多肽(hIAPP)与2型糖尿病（T2D）发生发展密切相关。近年来发现，hIAPP可与血红素结合，促进血红素催化氧化活性。基于血红素代谢与线粒体功能的密切关系以及在催化蛋白质发生氧化、硝化损伤中的作用，结合T2D存在hIAPP错误折叠、血红素代谢失调及氧化硝化损伤的特点，我们推测hIAPP可能通过与血红素结合，影响血红素代谢进而诱导胰岛细胞线粒体功能障碍及氧化损伤，血红素与hIAPP相互作用可能是联系hIAPP与T2D发生发展的纽带。为证实这个假说，本项目拟设计系列突变hIAPP，研究其与血红素结合的特性、hIAPP聚集、复合物过氧化物酶活性及其对蛋白质酪氨酸硝化程度及位点选择性影响等规律，然后在胰岛细胞INS-1上研究hIAPP与血红素相互作用对血红素代谢、线粒体功能及蛋白质氧化和硝化损伤的影响。本研究有助于深刻认识hIAPP在T2D中的致病机理，为防治T2D提供科学依据。

血红素升高与2型糖尿病（T2D）发生发展密切相关，而人胰岛淀粉样多肽(hIAPP)的聚集也是引起T2D的因素之一。本项目系统研究了血红素与hIAPP的相互作用及其对胰岛β细胞损伤的协同作用。发现hIAPP 的Arg11和His18在与血红素结合起重要作用。血红素能够有效的抑制hIAPP及其突变多肽的聚集并能对已经形成聚集体进行部分解聚。hIAPP中His18和Arg11在与血红素结合后抑制聚集中起到了重要的作用，血红素可能通过阻碍Phe23芳香环-芳香环堆积形成的β-折叠结构的作用从而抑制hIAPP的聚集。血红素和hIAPP结合后，可增强血红素的过氧化物酶活性，提升血红素催化蛋白质酪氨酸硝化的能力，改变其催化催化H2O2-NO2-体系硝化MnSOD的位点分布，使得MnSOD中与活性密切相关的Tyr34硝化水平增高。hIAPP经heme-H2O2-NO2-处理可以使Tyr37硝化。Tyr37硝化的hIAPP聚集性显著降低，其原因是硝化后的hIAPP增加了hIAPP的Zeta电位，增加了肽间的静电斥力，使其寡聚体肽链不易紧密堆积而形成松散结构，从而阻碍其进一步聚集成纤维。此外，酪氨酸硝化可以通过阻断分子间氢键从而抑制hIAPP聚集。血红素与hIAPP结合后，可以通过两种途径增强其细胞毒性：一是血红素与hIAPP结合后，增强其过氧化物酶活性，导致氧化损伤进一步增强，二是促进hIAPP硝化，硝化后的hIAPP通过增加低分子寡聚物的数量进而增加胰岛β细胞毒性。本研究为体内血红素升高易于发生T2D提供了合理的解释。此外，本项目还有两个发现：一是血红素的水溶性衍生物同样具有抑制hIAPP聚集的作用，但却可以抑制hIAPP的细胞毒性，可望成为潜在的减缓T2D发生发展药物；二是酪氨酸硝化降低聚集性多肽的聚集可以用于人降钙素，酪氨酸硝化的人降钙素可以不聚集而保持其活性，可望用于临床应用。