Monoubiquitination Inhibits the Actin Bundling Activity of Fascin
Monoubiquitination Inhibits the Actin Bundling Activity of Fascin
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单泛素化抑制肌成束蛋白的肌动蛋白成束活性
DOI：
10.1074/jbc.m116.767640
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发表时间：
2016-12-30
影响因子：
4.8
通讯作者：
Yang, Shengyu
中科院分区：
文献类型：
--
作者：
Lin, Shengchen;Lu, Shuang;Yang, Shengyu
筋膜蛋白是一种肌动蛋白束蛋白，它将单个肌动蛋白细丝交叉连接成直的、致密的和僵硬的束，这对丝状足、立体纤毛和其他指状膜突起的形成至关重要。束蛋白的异常调节与癌症转移、听力损失和失明有关。在这里，我们确定单素化是一种新的机制，调节束素束的活性和动力学。单素化位点为Lys247和Lys250，这两个残基位于Fasin的肌动蛋白结合位点2的正电荷斑块中。利用化学泛素化的方法，我们合成了化学泛素化的发酵素，并测定了化学泛素化对发酵素结合活性和动力学的影响。我们的数据表明，单素化降低了束素束的EC50，延迟了束组装的启动，并加速了现有束的解体。通过分析Fasin在溶剂可及表面的静电性质，我们提出了单素化引入空间位阻干扰了肌动蛋白细丝与肌动蛋白结合位点2上带正电的斑块之间的相互作用。我们还确认SMurf1是一种E3连接酶，调节Fasin的单素化。我们的发现揭示了一种先前未知的筋膜蛋白调控机制，这将对理解生理和病理条件下肌动蛋白束的调控具有重要意义。
Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys247 and Lys250, two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC50, delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions.