Changing the subcellular location of the oncoprotein Bcr-Abl using rationally designed capture motifs.
Changing the subcellular location of the oncoprotein Bcr-Abl using rationally designed capture motifs.
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DOI：
10.1007/s11095-011-0654-8
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发表时间：
2012-04
影响因子：
3.7
通讯作者：
Lim CS
中科院分区：
文献类型：
--
作者：
Dixon AS;Constance JE;Tanaka T;Rabbitts TH;Lim CS
Bcr-Abl是慢性髓性白血病（CML）的病原体，定位于细胞质中，其致癌信号导致细胞增殖。如果强行进入细胞核，Bcr-Abl会导致细胞凋亡。为了实现核易位，生成了捕获Bcr-Abl的结合域，并将其附着在带有核信号的蛋白质上。这些产生的蛋白能够结合内源性Bcr-Abl并将其转运到细胞核。Bcr-Abl靶向3个不同的结构域进行捕获：通过构建高亲和力的细胞内抗体结构域（iDabs）到已知促进细胞质保留的Bcr-Abl区域，通过其卷曲结构域（CC），以及通过天然存在的蛋白质-蛋白质相互作用结构域（RIN1）。然后通过“蛋白质开关”或4个核定位信号（NLSs）的附着来测试这些结合域护送Bcr-Abl进入细胞核的能力。虽然RIN1、ABI7-iDab和CCmut3构建体都能与Bcr-Abl产生相似的共定位，但只有4NLS-CCmut3能产生Bcr-Abl的有效核易位。我们证明了一个小的结合域可以用来控制Bcr-Abl的亚细胞定位，这可能对CML治疗有影响。我们未来的终极目标是改变关键蛋白质的位置，从而改变它们的功能。
Bcr-Abl, the causative agent of chronic myelogenous leukemia (CML), localizes in the cytoplasm where its oncogenic signaling leads to proliferation of cells. If forced into the nucleus Bcr-Abl causes apoptosis. To achieve nuclear translocation, binding domains for capture of Bcr-Abl were generated and attached to proteins with signals destined for the nucleus. These resulting proteins would be capable of binding and translocating endogenous Bcr-Abl to the nucleus. Bcr-Abl was targeted at 3 distinct domains for capture: by construction of high affinity intracellular antibody domains (iDabs) to regions of Bcr-Abl known to promote cytoplasmic retention, via its coiled-coil domain (CC), and through a naturally occurring protein protein interaction domain (RIN1). These binding domains were then tested for their ability to escort Bcr-Abl into the nucleus using a “protein switch” or attachment of 4 nuclear localization signals (NLSs). Although RIN1, ABI7-iDab, and CCmut3 constructs all produced similar colocalization with Bcr-Abl, only 4NLS-CCmut3 produced efficient nuclear translocation of Bcr-Abl. We demonstrate that a small binding domain can be used to control the subcellular localization of Bcr-Abl, which may have implications for CML therapy. Our ultimate future goal is to change the location of critical proteins to alter their function.