Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro
Degradation of tau protein by puromycin-sensitive aminopeptidase in vitro
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DOI：
10.1021/bi061830d
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发表时间：
2006-12-19
期刊：
影响因子：
2.9
通讯作者：
Binder, Lester I.
中科院分区：
文献类型：
--
作者：
Sengupta, Soma;Horowitz, Peleg M.;Binder, Lester I.
Tau是一种微管相关蛋白，在阿尔茨海默病（AD）和其他Tau蛋白病中通过未知机制聚集成细胞内成对螺旋丝（PHF）。一个促成因素可能是神经元内游离胞质tau代谢失败。tau蛋白的积累可以通过质量作用驱动聚集过程。因此，通常降解tau的蛋白酶是非常感兴趣的。最近的遗传筛选鉴定了嘌呤霉素敏感的氨肽酶（PSA）作为tau诱导的病理学的有效修饰剂，并建议PSA作为可能的tau降解酶。在这里，我们已经扩展了这些观察使用人重组PSA纯化大肠杆菌。使用显色底物，金属离子，和几个特定的和非特异性的蛋白酶抑制剂，包括嘌呤霉素的纯化PSA的酶活性和特性进行了验证。PSA显示出在体外消化重组人全长tau，并且这种活性被嘌呤霉素阻碍。使用新的N-末端切割特异性tau抗体（Tau-C6 g，对残基13-14之间的切割特异性）和C-末端切割特异性tau抗体（Tau-C3）确认tau的氨基末端降解的机制。此外，PSA能够消化从正常人脑纯化的可溶性tau的程度大于从AD脑纯化的可溶性tau或PHF tau，表明tau的翻译后修饰和/或聚合可能影响其被PSA消化。这些结果与PSA调节体内tau水平的观察结果一致，并表明该酶可能参与人脑中的tau降解。
Tau, a microtubule associated protein, aggregates into intracellular paired helical filaments ( PHFs) by an unknown mechanism in Alzheimer's disease ( AD) and other tauopathies. A contributing factor may be a failure to metabolize free cytosolic tau within the neuron. The buildup of tau may then drive the aggregation process through mass action. Therefore, proteases that normally degrade tau are of great interest. A recent genetic screen identified puromycin-sensitive aminopeptidase ( PSA) as a potent modifier of tau-induced pathology and suggested PSA as a possible tau-degrading enzyme. Here we have extended these observations using human recombinant PSA purified from Escherichia coli. The enzymatic activity and characteristics of the purified PSA were verified using chromogenic substrates, metal ions, and several specific and nonspecific protease inhibitors, including puromycin. PSA was shown to digest recombinant human full-length tau in vitro, and this activity was hindered by puromycin. The mechanism of amino terminal degradation of tau was confirmed using a novel N-terminal cleavage-specific tau antibody ( Tau-C6g, specific for cleavage between residues 13-14) and a C-terminal cleavage-specific tau antibody ( Tau-C3). Additionally, PSA was able to digest soluble tau purified from normal human brain to a greater extent than either soluble or PHF tau purified from AD brain, indicating that post-translational modifications and/or polymerization of tau may affect its digestion by PSA. These results are consistent with observations that PSA modulates tau levels in vivo and suggest that this enzyme may be involved in tau degradation in human brain.