Porat Yair, Kolusheva Sofiya, Jelinek Raz, Gazit Ehud
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Biochemistry. 2003 Sep 23;42(37):10971-7. doi: 10.1021/bi034889i.
The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.
人胰岛淀粉样多肽形成淀粉样纤维与II型糖尿病相关。虽然之前有人提出形成的纤维因膜渗透活性而对胰腺β细胞有毒性，但最近的研究表明原纤维聚集体在渗透脂质双层方面具有显著更高的效力。在此，我们以高时间分辨率专门研究了可溶性和不溶性胰岛淀粉样多肽聚集体的膜相互作用活性。使用脂质/聚二乙炔（PDA）仿生囊泡的比色分析清楚地证明了与脂质囊泡强烈相互作用的可溶性聚集体的瞬时形成。比色测定反映出，孵育约1小时后观察到可溶性部分的膜结合水平出现峰值，随后聚集体的膜相互作用水平下降。膜活性聚集体的瞬时性质通过荧光猝灭测定得到独立证实。使用透射电子显微镜的超微结构分析提供了原纤维前聚集体的形态学证据，支持了与膜相互作用的可溶性物种的瞬时存在，并便于观察溶液中无膜活性的细丝。综上所述，我们的结果为形成具有高膜活性的瞬时可溶性原纤维前聚集体提供了实验证据。结合设计的纤维化抑制剂对这些观察结果的意义进行了讨论。
