CHIP 介导的 tau 蛋白超泛素化促进其自组装成不溶性 tau 丝,Chemical Science

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CHIP 介导的 tau 蛋白超泛素化促进其自组装成不溶性 tau 丝
Chemical Science ( IF 7.6 ) Pub Date : 2021-3-17 , DOI: 10.1039/d1sc00586c
Ji Hyeon Kim ₁ , Jeeyoung Lee ₁ , Won Hoon Choi ₁ , Seoyoung Park ₂ , Seo Hyeong Park ₁ , Jung Hoon Lee ₂ , Sang Min Lim ₃ , Ji Young Mun ₄ , Hyun-Soo Cho ₅ , Dohyun Han ₆ , Young Ho Suh _{1,

2} , Min Jae Lee _{1,

2}

Affiliation

tau 蛋白是一种高度可溶且天然未折叠的蛋白质。在病理条件下，tau 蛋白经历多次翻译后修饰 (PTM) 和构象变化，形成不溶性丝，这是 tau 蛋白病的蛋白质特征。为了剖析聚集过程中 tau PTM 之间的串扰，我们分别使用 GSK3β 和 CHIP在体外磷酸化和泛素化重组 tau。由此产生的磷酸化 ub-tau 包含具有赖氨酸 48 连接的常规多泛素链，足以用于蛋白酶体降解，而未磷酸化的 ub-tau 物种仅保留一到三个泛素部分。对体外重建的磷酸-ub-tau的质谱分析揭示了七个额外的泛素化位点，其中一些已知可以稳定患有 tau 病的人脑中 tau 原丝堆积。当泛素化反应延长时，磷酸-ub-tau 转化为不溶性超泛素化 tau 物种，具有纤维状形态和体外接种活性。我们通过生物物理筛选，开发出CHIP小分子抑制剂；这在体外有效抑制了 tau 泛素化，并延迟了其在培养细胞（包括原代培养神经元）中的聚集。我们的生化发现指向“多重打击模型”，其中 tau 磷酸化和超泛素化的连续事件是纤维化过程的关键驱动因素，因此表明靶向 tau 泛素化可能是缓解 tau 病进程的有效策略。

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CHIP-mediated hyperubiquitylation of tau promotes its self-assembly into the insoluble tau filaments

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.

更新日期：2021-03-17

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