Biochemical and cellular effects of inhibiting Nedd8 conjugation
Biochemical and cellular effects of inhibiting Nedd8 conjugation
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DOI：
10.1016/j.bbrc.2010.06.128
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发表时间：
2010-07-30
影响因子：
3.1
通讯作者：
Hagen, Thilo
中科院分区：
文献类型：
--
作者：
Leck, Yee Chin;Choo, Yin Yin;Hagen, Thilo
蛋白质与泛素样蛋白 Nedd8 的结合是一个重要的细胞过程，也是一个重要的抗癌治疗靶点。 Nedd8 已知的主要作用是附着并激活 Cullin RING E3 泛素连接酶 (CRL)。 Nedd8 与其底物的附着是通过类似于泛素转移的过程发生的，涉及 Nedd8 E1 激活酶和 Nedd8 E2 结合酶 Ubc12，后者将 Nedd8 转移到目标蛋白的赖氨酸残基上。在本研究中，我们利用显性失活 Ubc12 (dnUbc12) 和 Nedd8 E1 抑制剂 MLN4924 来抑制细胞 neddylation。我们证明 dnUbc12 通过消耗细胞 Nedd8 浓度来发挥作用。细胞neddylation的抑制导致CRL底物的快速积累以及HEK293细胞中增大且扁平的形态。抑制 Nedd8 缀合也会导致肌动蛋白细胞骨架异常。这可能至少部分是通过小 GTP 酶 RhoA（最近发现的 CRL 底物）的积累介导的。我们确实发现 siRNA 介导的 RhoA 敲低可以逆转抑制细胞 neddylation 时观察到的形态变化。总之，Nedd8通路在调节肌动蛋白细胞骨架和细胞形态方面发挥着重要作用。肌动蛋白细胞骨架的功能障碍可能有助于 Nedd8 抑制的抗癌作用。 (C) 2010 Elsevier Inc. 保留所有权利。
The conjugation of proteins with the ubiquitin-like protein Nedd8 is an essential cellular process and an important anti-cancer therapeutic target. The major known role of Nedd8 is the attachment to and activation of Cullin RING E3 ubiquitin ligases (CRL). The attachment of Nedd8 to its substrates occurs via a process analogous to ubiquitin transfer, involving a Nedd8 E1 activating enzyme and a Nedd8 E2 conjugating enzyme, Ubc12, which transfers Nedd8 onto lysine residues of target proteins. In this study, we utilize dominant-negative Ubc12 (dnUbc12) and the Nedd8 E1 inhibitor MLN4924 to inhibit cellular neddylation. We demonstrate that dnUbc12 functions by depleting cellular Nedd8 concentrations. Inhibition of cellular neddylation leads to rapid accumulation of CRL substrates and an enlarged and flattened morphology in HEK293 cells. Inhibiting Nedd8 conjugation also causes abnormalities in the actin cytoskeleton. This is likely at least partially mediated via accumulation of the small GTPase RhoA, a recently identified CRL substrate. We indeed found that siRNA mediated knockdown of RhoA can reverse the morphological changes observed upon inhibition of cellular neddylation. In conclusion, the Nedd8 pathway plays an important role in regulating the actin cytoskeleton and cellular morphology. Dysfunction of the actin cytoskeleton may contribute to the anti-cancer effect of Nedd8 inhibition. (C) 2010 Elsevier Inc. All rights reserved.