Use of a Small Peptide Fragment as an Inhibitor of Insulin Fibrillation Process: A Study by High and Low Resolution Spectroscopy
Use of a Small Peptide Fragment as an Inhibitor of Insulin Fibrillation Process: A Study by High and Low Resolution Spectroscopy
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DOI：
10.1371/journal.pone.0072318
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发表时间：
2013-08-29
期刊：
影响因子：
3.7
通讯作者：
Bhunia, Anirban
中科院分区：
文献类型：
--
作者：
Banerjee, Victor;Kar, Rajiv K.;Bhunia, Anirban
NIVNVSLVK是一种无毒、九个残基的多肽，通过各种生物物理方法被证明可以干扰胰岛素纤颤。胰岛素在一定的应激条件下会发生构象变化，导致淀粉样纤维的形成。胰岛素的纤颤对其长期储存构成了一个问题，降低了其治疗II型糖尿病的效果。胰岛素低聚物向单体的解离是纤颤发生的关键步骤。用硫代黄素T荧光法测定62℃下胰岛素纤颤的时间进程显示，滞后时间从无多肽的120min增加到有多肽的236min。透射电子显微镜显示，胰岛素原纤维不存在时呈分枝状，存在时原纤维间结合较少。傅立叶变换红外光谱(FT-IR)显示，在62℃和pH 2.6的条件下，胰岛素失去了一些α-螺旋结构，但如果加入该肽，胰岛素的二级结构在3h内几乎完全保持，但在4h时部分失去。FT-IR还表明，胰岛素在2 h后形成指示纤维的交叉β结构，但在肽的存在下，α-螺旋保留到4h。尺寸排斥层析和动态光散射都表明胰岛素主要以三聚体的形式存在，其向单体的转化被肽阻止。饱和转移差核磁共振证实，多肽中的疏水残基与胰岛素疏水沟槽紧密接触。分子动力学模拟结合主成分分析揭示了该肽是如何阻断胰岛素纤颤的。体外溶血活性对HT1080细胞无明显细胞毒作用。胰岛素聚集是由于分子动力学模拟研究监测到的两个关键残基Phe(B24)和Tyr(B26)的相互作用而引起的。从这九个残基可以开发出更多的基于多肽的新的先导化合物。
A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62 degrees C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62 degrees C and pH 2.6, insulin lost some alpha-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, a-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.