Molecular mapping of the recognition interface between the islet amyloid polypeptide and insulin
Molecular mapping of the recognition interface between the islet amyloid polypeptide and insulin
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DOI：
10.1002/anie.200602034
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Gazit, Ehud
中科院分区：
文献类型：
--
作者：
Gilead, Sharon;Wolfenson, Haguy;Gazit, Ehud
胰岛素由两条链 A 和 B 组成，通过两个二硫桥交联。第一步，我们评估了每条分离的链对 IAPP 淀粉样蛋白形成的影响。 IAPP 被允许单独形成淀粉样原纤维或在存在胰岛素链的情况下形成淀粉样原纤维。使用硫代黄素 T (ThT) 荧光测定法监测纤维化过程（参见支持信息）。监测人和牛胰岛素的抑制能力作为对照。牛胰岛素和人胰岛素均对高度淀粉样变的 IAPP 聚集表现出强烈的抑制作用（图 1）。还原论分析使我们能够明确指出 B 链是一种有效的抑制剂，其作用呈剂量依赖性。其抑制作用似乎与完整胰岛素分子的抑制作用相似。在 10 倍的比例下，B 链抑制似乎在整个实验过程中都是绝对的。相反，在 A 链存在的情况下，没有观察到抑制作用，反而加速了聚集。因此，我们认为胰岛素对 IAPP 的抑制主要是由 B 链介导的。为了证实我们的假设，我们通过使用远紫外圆二色性 (CD) 分析评估了胰岛素链抑制 IAPP 从随机卷曲到 β 折叠的结构转变的能力，这种转变是淀粉样蛋白形成的特征（参见支持信息）。 IAPP 单独孵育或在 A 链、B 链或胰岛素以 1:1 的比例存在的情况下孵育。 0、24和48小时后记录CD光谱（图2）。单独的IAPP的光谱显示24小时后从零时的随机卷曲构象（最小在200 nm附近）到β-折叠构象（最小在217 nm附近）的清晰转变。然而，当 IAPP 与胰岛素一起孵育时，没有观察到这种转变。这证实了在结构转变的早期阶段有效的胰岛素抑制。与 B 链一起孵育的 IAPP 的 CD 分析表明，如在存在胰岛素的情况下观察到的，随机螺旋构象没有随时间的推移而发生转变。相比之下，当 IAPP 与 A 链一起孵育时，24 小时后观察到结构转变，与单独 IAPP 的情况一样。因此，正如对 ThT 阳性原纤维形成所观察到的，只有 B 链（而不是 A 链）抑制 IAPP 的结构转变过程。
Insulin is composed of two chains, A and B, crosslinked by two disulfide bridges. As a first step, we evaluated the effect of each of the separated chains on IAPP amyloid formation. IAPP was allowed to form amyloid fibrils alone or in the presence of insulin chains. The fibrilization process was monitored by using the thioflavin T (ThT) fluorescence assay (see the Supporting Information). The inhibition abilities of human and bovine insulin were monitored as controls. Both bovine and human insulin exhibited a strong inhibitory effect on the aggregation of the highly amyloidogneic IAPP (Figure 1). The reductionist analysis allowed us to specifically pinpoint the B chain as a potent inhibitor which works in a dose-dependent manner. Its inhibition appears to be similar to that of the intact insulin molecule. At a 10-fold ratio, B-chain inhibition seems to be absolute throughout the course of the experiment. Conversely, no inhibition was observed in the presence of the A chain, but the aggregation was instead accelerated. Hence, we propose that the inhibition of IAPP by insulin is predominantly mediated by the B chain. In order to confirm our assumption, we assessed the ability of the insulin chains to inhibit the IAPP structural transition from random coils into β sheets, a transition that is characteristic of amyloid formation, by using far-UV circular dichroism (CD) analysis (see the Supporting Information). IAPP was incubated alone or in the presence of the A chain, the B chain, or insulin at a 1: 1 ratio. CD spectra were recorded after 0, 24, and 48 h (Figure 2).The spectra of IAPP alone showed a clear transition from a random-coil conformation at zero time (minimum in the vicinity of 200 nm) to a β-sheet conformation (minimum in the vicinity of 217 nm) after 24 h. However, when the IAPP was incubated with insulin, this transition was not observed. This confirms the efficient insulin inhibition at a very early stage of the structural transition. The CD analyses of IAPP incubated with the B chain indicated a random-coil conformation without a transition over time, as observed in the presence of insulin. By contrast, when IAPP was incubated with the A chain, a structural transition was observed after 24 h, as in the case of IAPP alone. Accordingly, as observed for the formation of ThT-positive fibrils, only the B chain (and not the A chain) inhibits the process of structural transition by IAPP.