Department of Pathology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Centre of Biotherapy, Chengdu, China.
Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, Division of Neurology, West China Second University Hospital, Sichuan University, Chengdu, China.
Protein Sci. 2020 Jun;29(6):1366-1372. doi: 10.1002/pro.3724. Epub 2019 Sep 14.
Chromosome region maintenance 1 (CRM1) exports nuclear export signal (NES) containing cargos from nucleus to cytoplasm and plays critical roles in cancer and viral infections. Biochemical and biophysical studies on this protein were often obstructed by its low purification yield and stability. With the help of PROSS server and NES protection strategy, we successfully designed three small fragments of CRM1, each made of four HEAT repeats and capable of binding to NESs in the absence of RanGTP. One of the fragments, C7, showed dramatically improved purification yield, thermostability, mechanostability, and resistance to protease digestion. We showed by isothermal titration that the protein kinase inhibitor NES binds to C7 at 1.18 μM affinity. Direct binding to C7 by several reported CRM1 inhibitors derived from plants were verified using pull-down assays. These fragments might be useful for the development of CRM1 inhibitors towards treatment of related diseases. The strategy applied here might help to tackle similar problems encountered in different fields.
染色质区域维持蛋白 1（CRM1）将含有核输出信号（NES）的货物从细胞核输出到细胞质，在癌症和病毒感染中发挥着关键作用。对这种蛋白质的生化和生物物理研究常常受到其低纯化产量和稳定性的阻碍。在 PROSS 服务器和 NES 保护策略的帮助下，我们成功设计了 CRM1 的三个小片段，每个片段由四个 HEAT 重复组成，在没有 RanGTP 的情况下能够结合 NES。其中一个片段 C7 显示出显著提高的纯化产量、热稳定性、机械稳定性和对蛋白酶消化的抵抗力。我们通过等温滴定量热法表明，蛋白激酶抑制剂 NES 以 1.18 μM 的亲和力与 C7 结合。使用下拉测定法验证了几种来自植物的报道的 CRM1 抑制剂与 C7 的直接结合。这些片段可能有助于开发针对相关疾病的 CRM1 抑制剂。这里应用的策略可能有助于解决不同领域遇到的类似问题。
