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ErbB2与其自身抑制因子的作用位点及其相互作用机制的研究

Identification of the Interaction Site of ErbB2 and Its Autoinhibitor and the Mechanism of Cell Growth Inhibition by Herstatin

【作者】 胡品良

【导师】 郭宁;

【作者基本信息】 中国人民解放军军事医学科学院 , 分子免疫学, 2005, 博士

【摘要】 ErbB2是原癌基因erbB-2编码的185kDa的细胞膜受体,为表皮生长因子受体(epidermal growth factor receptor,EGFR)家族成员之一。该家族包括ErbB-1(又称EGFR,HER1)、ErbB2(又称HER2)、ErbB-3(HER3)和ErbB-4(HER4)四个成员。ErbB2高表达肿瘤细胞中Ras-MAPK和PI3K-Akt信号传导活性较高,细胞增殖能力较强,分化成熟和凋亡机制受到抑制,细胞恶性程度高。ErbB2高表达肿瘤细胞可抵抗TNF-α、射线以及各种化疗药物引起的细胞凋亡效应。临床上ErbB2表达与患者预后密切相关,ErbB2高表达的患者易发生肿瘤转移,存活期短。由于ErbB2在正常细胞和肿瘤细胞中的表达水平具有显著的差异,因而已成为肿瘤免疫生物治疗的理想靶点,亦是目前肿瘤治疗研究领域的热点分子。Herstatin是近年来发现的唯一天然存在的ErbB2自身抑制分子,它是ErbB2mRNA加工过程中选择性剪接的产物,是一种可溶性的ErbB2受体。Herstatin能与细胞表面的ErbB2受体高亲和力结合,阻碍ErbB2同源或异源二聚体的形成,抑制受体酪氨酸磷酸化及ErbB2过表达细胞的增殖能力。但是,Herstatin与ErbB2相互作用的机制及与ErbB2受体分子的结合位点以及抑制ErbB2过表达细胞增殖的机制均不清楚。阐明ErbB2分子表达的自身调节机制,有助于理解该受体分子在生理条件下表达平衡对于细胞正常分化的重要意义,而且可能为ErbB2过表达肿瘤的治疗提供新思路。对二者相互作用位点的分析亦可为寻找到控制ErbB2的敏感靶位及有效的小分子抑制剂的设计提供依据。 本研究采用基因工程技术构建了Herstatin的真核表达载体,将构建成功的载体转染ErbB2表达水平不同的细胞,在mRNA和蛋白水平均检测到Herstatin的表达。应用MTT法证实了Herstatin对细胞的增殖具有明显的抑制作用。Hoechst和PI染色发现Herstatin转染后可诱导转染细胞凋亡,这可能是其抑制细胞增殖

【Abstract】 ErbB2, a 185 kDa receptor, is encoded by ErbB2 oncogene. It is one of the members of epidermal growth factor receptor (EGFR) family, which consists of four receptors including epidermal growth factor receptor (EGFR, also called HER-1 or ErbB-1), ErbB2 (HER-2), ErbB3 (HER-3) and ErbB4 (HER-4). ErbB2 overexpression is associated with poor progrnosis and short time to relapse. ErbB2 also enhances the metastatic potential of cancer cells. The most important intracellular signal transduction pathways activated by ErbB2 are Ras-MAPK and PI3K-Akt which play an important role in proliferation, differentiatio, even resistanse to TNF- α ,γ -radiation and chemotherapy. ErbB2 is frequently overexpressed in many human tumors. Therefore, ErbB2 is a good candidate target for immunotherapy and also a research hotspot. Recently, herstatin, an autoinhibitor of ErbB2, has been reported. It consists of subdomains L1 and S1 of ErbB2 and a novel C-terminal sequence encoded by intron 8. It has been demonstrated that Herstatin can bind to ErbB2 with high affinity. It disrupts the formation of heterodimers with EGFR or HER3 and homodimer with itself, reduces tyrosine phosphorlation of EGFR family and inhibits proliferation of cancer cells that overexpress ErbB2. However, the mechanism of the interaction between Herstatin and ErbB2 has not been fully understood and the binding sites are not clear now. The binding region of Herstatin on ErbB2 ectodomain might be a potential target region for the drug design.In the present study, the full-length sequence of Herstatin was amplified by over-lapping PCR. A plasmid containing Herstatin was constructed. The cells were transfected with the plasmid pcDNA3.1/Hst and Herstatin was expressed in the transfected cells. Herstatin mRNA and an about 60 KDa protein were detected by RT-PCR and West-blotting respectively. The results showed that Herstatin can inhibit cell proliferation and induce apoptosis by using Hoechst staining and flowcytometry.To investigate the interaction site on ErbB2 with Herstatin, the 3-D structure of Herstatin and ErbB2 complex was constructed by using computer-aided homology method. Based on the predicted secondary structure of C-terminal region of 79 aa by using GOR IV method, the 3-D model of the sequence of aa 341-419 representing C-terminal region of 79 aa was constructed by ab initio modeling method. The 3-D sequence of Herstatin from aa 1-340 were derived from the 3-D crystal structure of ErbB2 ECD. According to the 3-D crystal structure of ErbB2 ECD, the theoretical spatial structure of ErbB2 ECD was modeled with computer-aided homology method. Subsequently AUTODOCK3.0 program used to dock Herstatin- and ErbB2, the final optimized 3-D complex structure of ErbB2 ECD and Herstatin was modeled. The interaction region of ErbB2 ECD was at the C-terminal portion of S1, while the binding domain of Herstatin to ErbB2 ECD was at the 79 aa region encoded by intron 8.

【关键词】 HerstatinErbB2相互作用肿瘤治疗
【Key words】 HerstatinErbB2interactioncancer therapy
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