Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated By Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Yuzhong District, Chongqing, 400016, China.
Cell Commun Signal. 2021 Jul 3;19(1):71. doi: 10.1186/s12964-021-00752-9.
The fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear.
Herein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis.
HSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells.
Our study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies. Video abstract.
融合癌蛋白 Bcr-Abl 主要位于细胞质中，导致慢性髓性白血病（CML）。进入细胞核后，融合蛋白可诱导 CML 细胞凋亡。Bcr-Abl 蛋白的卷曲螺旋结构域（CC 结构域）在亚细胞定位中起核心作用。然而，CC 结构域如何影响 Bcr-Abl 的亚细胞定位尚不清楚。
通过免疫沉淀结合质谱筛选与 Bcr-Abl CC 结构域相互作用的关键蛋白。通过 DeepViewer 预测 Bcr-Abl CC 结构域与靶蛋白结合的特定部位。免疫沉淀实验用于证实蛋白结合的特定部位。IF 和 Western blot 用于观察靶蛋白的亚细胞定位。Western blot 用于检测蛋白变化。CCK-8、克隆形成试验和 FCM 周期检测用于观察抑制剂对 CML 细胞增殖能力的影响。FCM 凋亡检测用于观察细胞凋亡水平。
HSP90AB1 通过 N 端结构域（NTD）与 Bcr-Abl CC 结构域相互作用，阻止 Bcr-Abl 蛋白向细胞核转运，维持 Bcr-Abl 酪氨酸激酶的激活。核内截留的 Bcr-Abl 通过激活 p73 并抑制 Bcr-Abl 细胞质致癌信号通路的表达，显著抑制 CML 细胞的增殖并诱导其凋亡。此外，17AAG（坦那西普）与 Leptomycin B（LMB）联合使用可显著降低 CML 细胞的增殖。
本研究为将 Bcr-Abl 转入细胞核作为 CML 治疗的替代策略提供了证据，靶向 HSP90AB1 的 NTD 以抑制与 Bcr-Abl 的相互作用，为 HSP90 抑制剂在 CML 及其他 Bcr-Abl 依赖性恶性肿瘤的开发和应用提供了更准确的策略。
