Abedini Andisheh, Raleigh Daniel P
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA.
J Mol Biol. 2006 Jan 13;355(2):274-81. doi: 10.1016/j.jmb.2005.10.052. Epub 2005 Nov 8.
Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.
胰岛淀粉样多肽（IAPP；胰淀素）是2型糖尿病中淀粉样蛋白形成的原因。并非所有生物体都会形成胰岛淀粉样蛋白，并且淀粉样蛋白的形成与一级序列的变异密切相关。对人和啮齿动物IAPP的研究表明，20 - 29位氨基酸残基区域是重要的淀粉样蛋白调节序列。大鼠的20 - 29序列含有三个脯氨酸残基，不会形成淀粉样蛋白，而人类序列不含脯氨酸且易于形成淀粉样蛋白。这导致了一种观点，即20 - 29区域构成了一个关键的淀粉样蛋白生成结构域，决定了整个序列的特性。人和大鼠IAPP的不同行为可能是由于20 - 29区域的差异，或者仅仅是因为多个脯氨酸残基会使淀粉样纤维不稳定。我们通过研究一个在该片段中与人类肽相同但在该区域外有三个脯氨酸残基的变体，来测试20 - 29区域有多关键。我们设计了一种人类IAPP淀粉样蛋白生成8 - 37区域的变体（hIAPP(8 - 37) 3xP），在第17、19和30位进行脯氨酸替换。与野生型相比，3xP变体更容易合成，并且溶解度显著更高。傅里叶变换红外光谱、透射电子显微镜、刚果红染色和硫黄素 - T结合表明，该变体形成β - 折叠结构的倾向降低，形成的沉积物结构有序程度远低于野生型。远紫外圆二色性研究表明，hIAPP(8 - 37) 3xP在长时间孵育后形成的少量β - 折叠结构在稀释到Tris缓冲液中时很容易解离为无规卷曲结构。在假定的核心结构域外进行脯氨酸替换能有效消除淀粉样蛋白形成这一观察结果表明，IAPP聚集模型必须考虑其他区域的贡献。
