Nonsaturable binding indicates clustering of Tau on the microtubule surface in a paired helical filament-like conformation
Nonsaturable binding indicates clustering of Tau on the microtubule surface in a paired helical filament-like conformation
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DOI：
10.1074/jbc.m002590200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Mandelkow, E
中科院分区：
文献类型：
--
作者：
Ackmann, M;Wiech, H;Mandelkow, E
在阿尔茨海默病中，tau蛋白调节微管动力学并形成不可溶的聚集体。由于所报道的牛磺酸与微管的亲和力之间存在差异，我们使用一种灵敏的酶联免疫吸附试验来确定广泛浓度范围内的相互作用。我们发现，相互作用是双相的，而不是像前面假设的那样是单相的。对于相同和不相互作用的结合部位，第一结合相是典型的，其解离常数约为0.1µM，化学计量比约为0.2Tau/微管蛋白二聚体。令人惊讶的是，第二相是不可饱和的，当游离Tau浓度高于2µM时，结合Tau与自由Tau几乎呈线性增长。斜率与微管浓度成正比。由此我们定义了一个超载参数，取值在50微米左右。考察了Tau异构体、磷酸化和二聚化对这两种相的影响。值得注意的是，Tau在微管上的过载导致了硫代黄素S荧光的增加，这让人想起Tau聚集成阿尔茨海默病成对的螺旋细丝的情况。由于聚阴离子刺激Tau聚集，并且微管蛋白的C-末端结构域是多阴离子的，我们认为Tau的早期构象变化导致成对螺旋微丝聚集发生在微管表面。
Tau protein modulates microtubule dynamics and forms insoluble aggregates in Alzheimer's disease. Because there is a discrepancy between reported affinities of Tau to microtubules, we determined the interaction over a wide concentration range using a sensitive enzyme -linked immunosorbent assay. We found that the interaction is biphasic and not monophasic as assumed earlier. The first binding phase is typical for identical and noninteracting binding sites, with dissociation constants around 0.1 mu M and stoichiometries around 0.2 Tau/tubulin dimer. Surprisingly, the second phase is nonsaturable and shows a nearly linear increase in bound Tau versus free Tau for free Tau concentrations higher than 2 mu M. The slope is proportional to the microtubule concentration. From this we define an overloading parameter with values around 50 mu M. The influence of Tau isoform, phosphorylation, and dimerization on both phases was investigated. Remarkably the overloading of Tau on microtubules leads to a thioflavin S fluorescence increase reminiscent of that seen with Tau aggregated into Alzheimer paired helical filaments. Because polyanions stimulate Tau aggregation and because the C-terminal domain of tubulin is polyanionic, we suggest that an early conformational change in Tau leading to paired helical filament aggregation occurs right on the microtubule surface.