Disruption of Bcr-Abl Coiled Coil Oligomerization by Design
Disruption of Bcr-Abl Coiled Coil Oligomerization by Design
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DOI：
10.1074/jbc.m111.264903
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发表时间：
2011-08-05
影响因子：
4.8
通讯作者：
Lim, Carol S.
中科院分区：
文献类型：
--
作者：
Dixon, Andrew S.;Pendley, Scott S.;Lim, Carol S.
寡聚是许多蛋白质的重要调控机制，包括癌蛋白和其他致病蛋白。癌蛋白BCR-Abl依赖于其卷曲卷曲结构域的寡聚作用，这表明设计的能够增强与BCR-Abl结合并减少自身寡聚的卷曲卷曲结构域在治疗上是有用的。BCR-Abl卷曲线圈结构域的关键突变被发现，这些突变通过分子间电荷排斥减少了同源齐聚，但通过额外的盐桥增加了与BCR-Abl卷曲线圈的相互作用，从而增强了破坏BCR-Abl低聚状态的能力。对这些突变进行了计算建模，以优化设计。体外试验证实了最佳突变的有效性和功能性，发现其同源齐聚减少，与bcr-Abl卷曲螺旋结构域的结合增加。将突变的螺旋线圈导入K562细胞后，bcr-Abl的磷酸化水平降低，细胞增殖受阻，caspase-3/7活性和DNA片段增加。重要的是，在所有实验中，突变的螺旋线圈结构域比野生型更有效。这种修饰的卷曲结构域通过寡聚破坏改善了对bcr-Abl的抑制，为治疗干预提供了一种可行的小分子抑制剂替代方案。
Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.