Paudel H K, Li W
Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3T 1E2, Canada.
J Biol Chem. 1999 Mar 19;274(12):8029-38. doi: 10.1074/jbc.274.12.8029.
In Alzheimer's disease, microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments. Sulfated glycosaminoglycans such as heparin and heparan sulfate were shown to accumulate in pretangle neurons, stimulate in vitro tau phosphorylation, and cause tau aggregation into paired helical filament-like filaments. The sulfated glycosaminoglycan-tau interaction was suggested to be the central event in the development of neuropathology in Alzheimer's disease brain (Goedert, M., Jakes, R., Spillantini, M. G., Hasegawa, M., Smith, M. J., and Crowther, R. A. (1996) Nature 383, 550-553). The biochemical mechanism by which sulfated glycosaminoglycans stimulate tau phosphorylation and cause tau aggregation remains unclear. In this study, disuccinimidyl suberate (DSS), a bifunctional chemical cross-linker, cross-linked tau dimers, tetramers, high molecular size aggregates, and two tau species of sizes 72 and 83 kDa in the presence of heparin. In the absence of heparin only dimeric tau was cross-linked by DSS. Fast protein liquid chromatography gel filtration revealed that 72- and 83-kDa species were formed by intramolecular cross-linking of tau by DSS. These observations indicate that heparin, in addition to causing aggregation, also induces a conformational change in tau in which reactive groups are unmasked or move closer leading to the DSS cross-linking of 72- and 83-kDa species. Heparin-induced structural changes in tau molecule depended on time of heparin exposure. Dimerization and tetramerization peaked at 48 h, whereas conformational change was completed within 30 min of heparin exposure. Heparin exposure beyond 48 h caused an abrupt aggregation of tau into high molecular size species. Heparin stimulated tau phosphorylation by neuronal cdc2-like kinase (NCLK) and cAMP-dependent protein kinase. Phosphopeptide mapping and phosphopeptide sequencing revealed that tau is phosphorylated by NCLK on Thr212 and Thr231 and by cAMP-dependent protein kinase on Ser262 only in the presence of heparin. Heparin stimulation of tau phosphorylation by NCLK showed dependence on time of heparin exposure and correlated with the heparin-induced conformational change of tau. Our data suggest that heparin-induced conformational change exposes new sites for phosphorylation within tau molecule.
在阿尔茨海默病中，微管相关蛋白tau会发生异常磷酸化，并聚集成双螺旋丝。硫酸化糖胺聚糖，如肝素和硫酸乙酰肝素，被证明会在缠结前神经元中积累，刺激体外tau磷酸化，并导致tau聚集成双螺旋丝样细丝。硫酸化糖胺聚糖与tau的相互作用被认为是阿尔茨海默病脑内神经病理学发展的核心事件（戈德特，M.，杰克斯，R.，斯皮兰蒂尼，M.G.，长谷川，M.，史密斯，M.J.，和克劳瑟，R.A.(1996)《自然》383,550 - 553）。硫酸化糖胺聚糖刺激tau磷酸化并导致tau聚集的生化机制仍不清楚。在本研究中，双功能化学交联剂辛二酸二琥珀酰亚胺酯（DSS）在肝素存在的情况下，交联了tau二聚体、四聚体、高分子量聚集体以及两种大小分别为72 kDa和83 kDa的tau蛋白。在没有肝素的情况下，只有二聚体tau被DSS交联。快速蛋白质液相色谱凝胶过滤显示，72 kDa和83 kDa的蛋白是由DSS对tau进行分子内交联形成的。这些观察结果表明，肝素除了引起聚集外，还会诱导tau的构象变化，其中反应基团被暴露或靠近，导致72 kDa和83 kDa蛋白被DSS交联。肝素诱导的tau分子结构变化取决于肝素暴露时间。二聚化和四聚化在48小时达到峰值，而构象变化在肝素暴露30分钟内完成。肝素暴露超过48小时会导致tau突然聚集成高分子量聚集体。肝素通过神经元细胞周期蛋白依赖性激酶2样激酶（NCLK）和环磷酸腺苷依赖性蛋白激酶刺激tau磷酸化。磷酸肽图谱分析和磷酸肽测序显示，只有在肝素存在的情况下，tau才会被NCLK在苏氨酸212和苏氨酸231位点磷酸化，被环磷酸腺苷依赖性蛋白激酶在丝氨酸262位点磷酸化。肝素通过NCLK刺激tau磷酸化显示出对肝素暴露时间的依赖性，并且与肝素诱导的tau构象变化相关。我们的数据表明，肝素诱导的构象变化暴露了tau分子内新的磷酸化位点。
