Aix-Marseille Univ, CNRS, UMR 7281 BIP, Bioénergétique et Ingénierie des Protéines, Marseille, France.
Aix-Marseille Univ, CNRS, UMR 7051, INP, Institut de Neurophysiopathologie, Marseille, France.
Sci Rep. 2018 Sep 14;8(1):13846. doi: 10.1038/s41598-018-32096-9.
Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αβ-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αβ-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αβ-tubulin.
Tau 是一种微管相关蛋白，它可以诱导和稳定微管细胞骨架的形成，并在神经退行性疾病中发挥重要作用。Tau 与微管结合的区域已经确定了很长时间，但 Tau 如何以及在何处与其伴侣结合仍然存在争议。我们使用定点自旋标记结合 EPR 光谱来监测 Tau 与紫杉醇稳定的微管结合，或当 Tau 直接用作微管形成诱导剂时与 αβ-微管蛋白结合。使用接枝在 Tau 两个天然半胱氨酸上的马来酰亚胺功能化标签，我们发现这两种情况下 Tau 在这些区域都保持高度的灵活性，这证实了 Tau:MTs 复合物的模糊性。更有趣的是，使用通过二硫键连接的标签，我们首次证明了 αβ-微管蛋白或微管与 Tau 之间的巯基-二硫键交换。此外，使用含有两个天然半胱氨酸的 Tau 片段或仅含有其中一个的变体，我们确定了每个半胱氨酸的单独作用。在预形成的微管或 Tau 诱导的微管之间观察到的标记释放动力学差异，结合结构数据的比较，使我们提出了 Tau 与 αβ-微管蛋白的两个可能的结合位点。
