Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.
Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.
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DOI：
10.1158/0008-5472.can-14-3812
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发表时间：
2016-02-01
期刊：
影响因子：
11.2
通讯作者：
Panchenko AR
中科院分区：
文献类型：
--
作者：
Li M;Kales SC;Ma K;Shoemaker BA;Crespo-Barreto J;Cangelosi AL;Lipkowitz S;Panchenko AR
在许多肿瘤中都发现了单体 Casitas B 系淋巴瘤 (Cbl) 基因的致癌突变，但其意义仍然很大程度上未知。最近已经解决了几种人类 c-Cbl (CBL) 结构，描述了该蛋白质在其激活周期的不同阶段，从而提供了癌症相关蛋白质的稳定性与活性权衡如何影响疾病发作和进展的机制见解。在这项研究中，我们通过计算模拟了错义癌症突变对代表 CBL 激活周期四个阶段的结构的影响，以确定影响 CBL 稳定性、结合和活性的驱动突变。我们发现，复发性、纯合性和白血病特异性突变比随机非癌症突变对 CBL 状态具有更大的不稳定影响。我们通过在细胞中进行盲目的 CBL 介导的 EGFR 泛素化测定，进一步测试了这些计算模型评估 CBL 稳定性变化及其与泛素缀合酶 E2 结合的能力。实验性 CBL 泛素连接酶活性与 CBL 稳定性的预测变化一致，并且在较小程度上与 CBL-E2 结合亲和力一致。所有实验测试的突变中，三分之二通过破坏 CBL 稳定性或破坏 CBL-E2 结合来影响泛素连接酶活性，而约三分之一的测试突变被发现是中性的。总的来说，我们的研究结果表明，结合多种蛋白质构象以及稳定性和结合亲和力评估的计算方法可以成功预测癌症突变对蛋白质活性的功能影响，并为 CBL 突变提供概念证明。
Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.