Sahara Naruhiko, Maeda Sumihiro, Murayama Miyuki, Suzuki Takehiro, Dohmae Naoshi, Yen Shu-Hui, Takashima Akihiko
Laboratory for Alzheimer's Disease, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan.
Eur J Neurosci. 2007 May;25(10):3020-9. doi: 10.1111/j.1460-9568.2007.05555.x.
Abnormal accumulation of tau as filamentous structures is a neuropathological hallmark of neurodegenerative diseases referred to as tauopathies. Little is known about the role of native cysteine residues in tau assembly because their substitution with other amino acids has no effect on tau filament morphology. To understand the process involved in tau oligomerization, we analysed both heparin-induced assembly of different forms of recombinant human tau and assembly of tau from COS-7 cells transiently expressing different human tau constructs. Here, we demonstrated that tau assembly involves two distinct dimers (cysteine-dependent and cysteine-independent) that differ in resistance to reduction. During assembly, an increase of cysteine-dependent tau oligomer was observed prior to detection of increased thioflavin T fluorescence signals. The latter event was accompanied by an increase of cysteine-independent dimer. Fewer higher-order oligomers and aggregates were assembled from four-repeat tau containing two amino-terminus inserts that have either the C291A/C322A mutation (cysless-4R2N) or a hexapeptide deletion at residues 306-311 (DeltaPHF6-4R2N) compared with those assembled from wild-type tau. Assembly of distinct types of dimers was also observed in lysates from COS-7 cells expressing wild-type 4R2N and brain extracts from mice expressing P301L mutant tau. In contrast, COS-7 cells expressing cysless- or DeltaPHF6-4R2N tau contained very little cysteine-dependent dimer. Together, the results indicate that intermolecular disulfide crosslinking along with PHF6 hexapeptide facilitates tau oligomerization and that this event is accompanied by cysteine-independent intermolecular bridging of microtubule-binding domain, leading to assembly of higher-order oligomers. The levels of these dimers may be used to gauge the potential for tau assembly.
作为丝状结构的tau蛋白异常聚集是被称为tau蛋白病的神经退行性疾病的神经病理学标志。关于天然半胱氨酸残基在tau蛋白组装中的作用知之甚少，因为用其他氨基酸替代它们对tau蛋白丝形态没有影响。为了了解tau蛋白寡聚化过程，我们分析了肝素诱导的不同形式重组人tau蛋白的组装以及来自瞬时表达不同人tau蛋白构建体的COS-7细胞的tau蛋白组装。在此，我们证明tau蛋白组装涉及两种不同的二聚体（半胱氨酸依赖性和半胱氨酸非依赖性），它们在还原抗性方面有所不同。在组装过程中，在检测到硫黄素T荧光信号增加之前，观察到半胱氨酸依赖性tau蛋白寡聚体增加。后一事件伴随着半胱氨酸非依赖性二聚体的增加。与从野生型tau蛋白组装的相比，从含有两个氨基末端插入物（具有C291A/C322A突变（无半胱氨酸-4R2N）或在残基306-311处六肽缺失（DeltaPHF6-4R2N））的四重复tau蛋白组装的高阶寡聚体和聚集体更少。在表达野生型4R2N的COS-7细胞裂解物和表达P301L突变型tau蛋白的小鼠脑提取物中也观察到不同类型二聚体的组装。相反，表达无半胱氨酸-或DeltaPHF6-4R2N tau蛋白的COS-7细胞含有很少的半胱氨酸依赖性二聚体。总之，结果表明分子间二硫键交联与PHF6六肽一起促进tau蛋白寡聚化，并且该事件伴随着微管结合域的半胱氨酸非依赖性分子间桥接，导致高阶寡聚体的组装。这些二聚体的水平可用于衡量tau蛋白组装的潜力。
