Synthetic Proteins Potently and Selectively Bind the Oncoprotein Gankyrin, Modulate Its Interaction with S6 ATPase, and Suppress Gankyrin/MDM2-Dependent Ubiquitination of p53.
Synthetic Proteins Potently and Selectively Bind the Oncoprotein Gankyrin, Modulate Its Interaction with S6 ATPase, and Suppress Gankyrin/MDM2-Dependent Ubiquitination of p53.
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DOI：
10.1021/acschembio.5b00201
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发表时间：
2015-08-21
影响因子：
4
通讯作者：
McNaughton BR
中科院分区：
文献类型：
--
作者：
Chapman AM;McNaughton BR
锚蛋白重复癌蛋白gankyrin的过表达与许多癌症的发病、增殖和/或转移直接相关。Gankyrin在多种疾病相关的生化过程中的作用是深远的。除了其他细胞过程之外，gankyrin过表达通过涉及MDM 2的复合物导致p53的细胞水平降低。因此，抑制这种相互作用是一种有吸引力的策略，用于调节ganklycoprotein过表达细胞中的致癌表型。然而，缺乏明确定义的疏水性小分子结合口袋上推定的锚蛋白重复结合面对传统的小分子药物的发现提出了挑战。相比之下，由于它们的大小和相对高的折叠能量，合成的Gankyrin结合蛋白原则上可以与涉及Gankyrin的生理相关PPI竞争。以前，我们发现形状互补蛋白支架可以重新表面化，以中等亲和力（KD ~6 μM）结合gankyrin。在这里，我们使用酵母展示高通量筛选，易错PCR，DNA改组和蛋白质工程来优化这个复合物。最好的蛋白质以优异的亲和力（KD ~21 nM）结合Gankyrin，从复杂的细胞环境中选择性地与Gankyrin共纯化，调节Gankyrin和生理结合伴侣（S6 ATP酶）之间的相互作用，并抑制p53的Gankyrin/MDM 2依赖性泛素化。
Overexpression of the ankyrin repeat oncoprotein gankyrin is directly linked to the onset, proliferation and/or metastasis of many cancers. The role of gankyrin in multiple disease-relevant biochemical processes is profound. In addition to other cellular processes, gankyrin overexpression leads to decreased cellular levels of p53, through a complex that involves MDM2. Thus, inhibition of this interaction is an attractive strategy for modulating oncogenic phenotypes in gankyrin-overexpressing cells. However, the lack of well-defined hydrophobic small-molecule binding pockets on the putative ankyrin repeat binding face presents a challenge to traditional small-molecule drug discovery. In contrast, by virtue of their size and relatively high folding energies, synthetic gankyrin-binding proteins could, in principle, compete with physiologically relevant PPIs involving gankyrin. Previously, we showed that a shape-complementary protein scaffold can be resurfaced to bind gankyrin with moderate affinity (KD ~6 μM). Here, we used yeast display high-throughput screening, error-prone PCR, DNA shuffling, and protein engineering to optimize this complex. The best of proteins proteins bind gankyrin with excellent affinity (KD ~21 nM), selectively co-purify with gankyrin from a complex cellular milieu, modulate an interaction between gankyrin and a physiological binding partner (S6 ATPase), and suppress gankyrin/MDM2-dependent ubiquitination of p53.