Aminopeptidases do not directly degrade tau protein
Aminopeptidases do not directly degrade tau protein
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DOI：
10.1186/1750-1326-5-48
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发表时间：
2010-11-05
影响因子：
15.1
通讯作者：
Hersh, Louis B.
中科院分区：
文献类型：
--
作者：
Chow, K. Martin;Guan, Hanjun;Hersh, Louis B.
背景：Tau 过度磷酸化和聚集形成细胞内神经原纤维缠结在许多 tau 病中普遍存在。因此，目前人们对 Tau 清除所涉及的机制很感兴趣。最近有报道称，Tau 可以被一种称为嘌呤霉素敏感氨肽酶 (PSA) 的氨肽酶降解。到目前为止，据报道 PSA 只能切割肽，报道的最大底物具有 30-50 个氨基酸。我们使用多种不同的 PSA 制剂研究了这种独特的 PSA 裂解反应。结果：从 Sf9 昆虫细胞中表达并纯化了 N 末端 His 标记的 PSA。尽管这种 PSA 制剂裂解了 Tau，但使用 N 和 C 末端 Tau 抗体结合质谱进行的产物分析表明，氨肽酶存在非典型的内切蛋白水解裂解。此外，该反应不被一般氨肽酶抑制剂贝他汀或特异性PSA抑制剂嘌呤霉素阻断。为了测试 Tau 水解是否可能由蛋白酶污染物引起，该酶在大肠杆菌中表达为谷胱甘肽 S-转移酶和麦芽糖结合蛋白融合蛋白，或在 Sf9 细胞中表达为 C 末端 His 标记蛋白。纯化至接近均质后，这些其他重组形式的 PSA 均未裂解 Tau。此外，源自Sf9细胞表达系统的Tau切割活性和氨肽酶活性可通过分子筛色谱法分离。当在细胞环境中进行测试时，我们再次未能看到 Tau 的 PSA 依赖性裂解。相关氨肽酶氨肽酶N的商业制剂也表现出Tau裂解活性，但该活性也可以与氨肽酶活性分开。结论：得出结论：PSA不直接裂解Tau。
Background: Tau hyperphosphorylation and aggregation to form intracellular neurofibrillar tangles is prevalent in a number of tauopathies. Thus there is current interest in the mechanisms involved in Tau clearance. It was recently reported that Tau can be degraded by an aminopeptidase known as the puromycin sensitive aminopeptidase (PSA). Until now PSA has been reported to only cleave peptides, with the largest reported substrates having 30-50 amino acids. We have studied this unique PSA cleavage reaction using a number of different PSA preparations.Results: An N-terminally His tagged-PSA was expressed and purified from Sf9 insect cells. Although this PSA preparation cleaved Tau, product analysis with N and C terminal Tau antibodies coupled with mass spectrometry showed an endoproteolytic cleavage atypical for an aminopeptidase. Furthermore, the reaction was not blocked by the general aminopeptidase inhibitor bestatin or the specific PSA inhibitor puromycin. In order to test whether Tau hydrolysis might be caused by a protease contaminant the enzyme was expressed in E. coli as glutathione S-transferase and maltose binding protein fusion proteins or in Sf9 cells as a C-terminally His-tagged protein. After purification to near homogeneity none of these other recombinant forms of PSA cleaved Tau. Further, Tau-cleaving activity and aminopeptidase activities derived from the Sf9 cell expression system were separable by molecular sieve chromatography. When tested in a cellular context we again failed to see a PSA dependent cleavage of Tau. A commercial preparation of a related aminopeptidase, aminopeptidase N, also exhibited Tau cleaving activity, but this activity could also be separated from aminopeptidase activity.Conclusion: It is concluded that PSA does not directly cleave Tau.