Abstract
BackgroundThe 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.MethodsHerein, 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.ResultsHSP90AB1 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.ConclusionOur 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.F7yTRqYkfkXw_k-2eiVFmgVideo abstract
Highlights
The fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML)
The target strip was cut, and the specific proteins were identified by the protein facility, which led to the identification of HSP90AB1 as a differential protein related to protein subcellular localization and associated with the CC domain of Bcr-Abl (Fig. 1b, c; Additional file 2: Table S1)
Association between the Bcr/Abl CC‐domain and N‐terminal domain of HSP90AB1 To confirm the intracellular binding of HSP90AB1 and CC domain, we enforced the expression of HA-Bcr/Abl and DYK-HSP90AB1 in 293 T cells for reciprocal immunoprecipitation and confirmed the associations between HSP90AB1 and Bcr-Abl proteins (Fig. 2a)
Summary
The fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). Chronic myeloid leukemia (CML) is a myeloid leukemia subtype represented by the formation of Bcr-Abl fusion gene [1, 2] This fusion gene plays a critical role in CML pathology, encoding Bcr-Abl oncoprotein which accommodates tyrosine kinase activity that can activate multiple downstream signal targets involved in the regulation. Based on our previous work, we have designed a drug transduction system to direct the oncogenic Bcr-Abl into the nucleus and induce the apoptosis of CML cells by tyrosine kinase activity [15]. The localization of Bcr-Abl plays a crucial role in the development of CML disease, and the coiled-coil domain (CC domain) at the N-terminal of Bcr-Abl is a major determinant for the location in the cytoplasm [19, 20]. How CC domain affects subcellular localization of Bcr-Abl remains unclear
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