From the State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China and.
the Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 2010 Feb 5;285(6):3592-3599. doi: 10.1074/jbc.M109.035691. Epub 2009 Dec 3.
Neurofibrillary tangles, principally composed of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer disease. Polyanionic cofactors such as heparin can induce Tau filament formation in vitro. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau(244-372) and heparin (average molecular mass = 7 kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays, and transmission electron microscopy. Our data clearly show that at physiological pH, heparin 7K, and human Tau(244-372) form a tight 1:1 complex with an equilibrium association constant exceeding 10(6) m(-1) under reducing conditions, triggering Tau fibrillization. In the absence of dithiothreitol, heparin shows a moderate binding affinity (10(5) m(-1)) to Tau(244-372), similarly triggering Tau fibrillization. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of 2:1 and then increases at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau and then decreases to some extent with ratios of >1:1. The retarding effect of heparin in excess is attributed to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of the 1:1 complex of Tau monomer and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases.
神经原纤维缠结主要由微管相关蛋白 Tau 组成的纤维束构成，是一组神经退行性疾病的特征，如阿尔茨海默病。多阴离子辅助因子如肝素可以在体外诱导 Tau 纤维的形成。在这里，我们使用静态光散射、等温滴定量热法、浊度测定法和透射电子显微镜定量地描述了重组人 Tau 片段 Tau(244-372)与肝素(平均分子量=7 kDa)之间的相互作用以及肝素诱导的纤维形成。我们的数据清楚地表明，在生理 pH 值下，肝素 7K 和人 Tau(244-372)在还原条件下形成一个紧密的 1:1 复合物，平衡结合常数超过 10(6) m(-1)，触发 Tau 纤维形成。在没有二硫苏糖醇的情况下，肝素对 Tau(244-372)显示出中等的结合亲和力(10(5) m(-1))，同样触发 Tau 纤维形成。进一步的纤维形成动力学分析表明，在摩尔比为 2:1 之前，滞后时间似乎大致不变，然后在更大的肝素/Tau 摩尔比下增加。代表纤维生长表观速率常数的最大斜率随着亚化学计量比的肝素/Tau 急剧增加，然后在 >1:1 的比例下有所下降。过量肝素的滞后效应归因于介质中自由肝素引起的离子强度的大幅增加。总之，这些结果表明 Tau 单体和肝素的 1:1 复合物的形成在诱导剂介导的 Tau 纤维形成中起着重要作用，为理解神经退行性疾病的发病机制提供了线索。
