Identification of hNopp140 as a binding partner for doxorubicin with a phage display cloning method
Identification of hNopp140 as a binding partner for doxorubicin with a phage display cloning method
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DOI：
10.1016/s1074-5521(02)00096-0
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发表时间：
2002-02-01
影响因子：
--
通讯作者：
Yu, YG
中科院分区：
文献类型：
--
作者：
Jin, Y;Yu, J;Yu, YG
阿霉素是一种广泛使用的抗癌药物。它被认为是通过抑制DNA复制或转录发挥作用，尽管其确切的靶点和细胞毒性机制尚未得到解决。针对固定化阿霉素筛选表达人肝cDNA的T7噬菌体文库以分离阿霉素结合蛋白。所选噬菌体含有核仁磷蛋白hNopp 140的C-末端区域，这是核仁生物发生中的重要因素。将克隆序列在E.在大肠杆菌中，重组蛋白被酪蛋白激酶II磷酸化，并在镁离子和氟离子的存在下寡聚化，就像体内发生的那样。阿霉素与表达蛋白的结合解离常数为4.5 × 10（-6）M，这种相互作用被hNopp 140的磷酸化抑制。这些结果表明，阿霉素可能会破坏hNopp 140的细胞功能。
Doxorubicin is a widely used anti-cancer drug. It is assumed to act by inhibiting DNA replication or transcription, although its precise targets and mechanism of cytotoxicity remain unresolved. A T7 phage library expressing human liver cDNA was screened against immobilized doxorubicin to isolate doxorubicin binding proteins. The selected phage contained the C-terminal region of nucleolar phosphoprotein hNopp140, an important factor in the biogenesis of the nucleolus. When the cloned sequence was expressed in E. coli, the recombinant protein was phosphorylated by casein kinase II and oligomerized in the presence of magnesium and fluoride ions, as occurs in vivo. Doxorubicin bound to the expressed protein with a dissociation constant of 4.5 X 10(-6) M, and this interaction was inhibited by the phosphorylation of hNopp140. These results suggested that doxorubicin might disrupt the cellular function of hNopp140.