A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications.
A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications.
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DOI：
10.1074/jbc.ra120.015882
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发表时间：
2020-12-25
期刊：
影响因子：
--
通讯作者：
Mandelkow E
中科院分区：
文献类型：
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作者：
Drepper F;Biernat J;Kaniyappan S;Meyer HE;Mandelkow EM;Warscheid B;Mandelkow E
神经元 Tau 蛋白的异常变化，例如磷酸化和聚集，被认为是阿尔茨海默病认知缺陷的标志。异常磷酸化被认为先于聚集，因此促进聚集，但磷酸化的性质和程度仍然不明确。 Tau 包含~85 个潜在的磷酸化位点，这些位点可以被各种激酶磷酸化，因为 Tau 的未折叠结构使它们易于接近。然而，方法学的局限性（例如磷酸肽或磷酸表位抗体的 MS）导致关于细胞中 Tau 磷酸化程度的相互矛盾的结果。在这里，我们展示了基于真核细胞中表达的完整 Tau (Sf9) 的天然 MS 的新方法的结果。磷酸化的程度是异质的，每个分子最多~20 个磷酸盐分布在 51 个位点上。中等磷酸化部分 Pm 显示总体占据量为 ∼8 Pi (± 5)，呈钟形分布；高度磷酸化的部分 Ph 有 14 Pi (± 6)。位点的分布高度不对称（所有 P 位点的 71% 位于 Tau 的 C 端一半）。所有位点都位于 Ser 或 Thr 残基上，但没有一个位点位于 Tyr 上。其他已知的翻译后修饰接近或低于我们的检测限（例如乙酰化、泛素化）。这些发现表明，正常细胞 Tau 蛋白显示出非常高程度的磷酸化，而其他修饰几乎不存在。这意味着某些位点的异常磷酸化可能不会显着影响磷酸化的程度，并且并不代表过度磷酸化。言外之意，Tau 蛋白的病理性聚集不太可能是高磷酸化的结果。
Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.