HER2同源二聚体的分子结构基础研究

发布时间：2018-03-04 05:19

  本文选题：癌基因　切入点：II型表皮因子受体　出处：《第二军医大学》2015年博士论文　论文类型：学位论文

【摘要】：人表皮生长因子受体(Human epidermal growth factor receptor,EGFR)超家族是一大类在生物体生命活动中发挥重要作用的生长因子受体超家族。该家族有四个成员,第一个成员即广为人知的EGFR受体。其中,II型表皮生长因子受体(Human epidermal growth factor receptor II,HER2)是近年来肿瘤学及肿瘤治疗学中的研究热点。在多种肿瘤中,如乳腺,胃肠道,以及生殖腺体肿瘤中均有HER2受体过表达、过度激活以及异常突变激活等方式的报道。在乳腺癌中,HER2作为独立的预后生物标志物已经引起广泛的关注。在乳腺癌和胃癌的靶向治疗领域,针对HER2的抗体药物及小分子治疗手段已经成为目前主流的放化疗联合手段。HER2为分子量大约为180KDa的单次跨膜I型酪氨酸激酶受体,其胞内段的酪氨酸激酶活性相较其他三个家族成员为最强。和其他EGFR受体家族成员相比较,HER2具有比较明显的生理及病理特征:一是目前尚未鉴定出HER2的特异性可溶性配体。二是在该家族内的受体进行异元二聚化时,HER2为最常见的二聚化对象。三是早期的晶体结构发现HER2的单独蛋白质结构并没有在其他家族成员中报道中那样,具有闸门封闭态到激活态的结构基础,而是直接处于二聚化臂暴露的激活态。但目前对于HER2的激活方式,二聚化的模型仍然存在很多模糊的争议。争议的核心在于目前尚无EGFR家族成员异源二聚体的分子模型以及HER2同源二聚体的分子模型。而仅利用目前报道的EGFR二聚化模型来推测其他成员的二聚化机制具有无法突破的理论限制性。在本课题中,我们首先报道了三种不影响HER2同源二聚体的抗体Fab片段与HER2的共结晶复合物。对三种HER2/Fab复合物的数据解析最终的结果为3.3?,3.5?和3.1?。针对相位问题,本文利用分子置换的方法,成功的解析三种晶体复合物的三维晶体结构。对三种复合物的分析确认了一种HER2同源相互作用。基于对三种复合物中的HER2结构提示,通过体外点突变实验,细胞膜表面化学交联实验,免疫共沉淀实验以及磷酸化检测试验,我们揭示并证实了HER2同源二聚体的分子结构模型。并通过对多种治疗性抗体对HER2二聚体的影响,从阐述了不同表位治疗性抗体对HER2同源二聚体影响的分子机制。最后,在基于对EGFR同源二聚体及HER2同源二聚体深入的分析比较,课题最终建立了一种新的EGFR家族受体的二聚方式模型,即新的“头靠背”的分子模型,此模型补充了EGFR家族受体二聚化的机制,并对EGFR在细胞膜上的相对位置以及相互作用力方式提出了新的分子模型和理论,并对传统观点提出挑战。本论文的工作对于理解HER2基本的生理过程二聚化具有重要的意义。
[Abstract]:The human epidermal growth factor receptor superfamily is a large class of growth factor receptor superfamilies that play an important role in the life of organisms. There are four members of the superfamily. The first member, known as the EGFR receptor, Human epidermal growth factor receptor II HER2, has been a hot topic in oncology and oncology in recent years. In many kinds of tumors, such as mammary gland and gastrointestinal tract, And the overexpression of HER2 receptor in gonadal tumors. Overexpression and abnormal mutation activation are reported. As an independent biomarker for prognosis, HER2 has attracted wide attention in breast cancer and gastric cancer. Antibody drugs and small molecule therapy for HER2 have become the mainstream combination of radiotherapy and chemotherapy. Her2 is a single transmembrane type I tyrosine kinase receptor with molecular weight of about 180KDa. The activity of tyrosine kinase in the intracellular segment is stronger than that of the other three family members. Compared with other members of the EGFR receptor family, the activity of cytosolic tyrosine kinase has obvious physiological and pathological characteristics. One is that the specificity of HER2 has not been identified. The second is that HER2 is the most common dimerization object when the receptor in the family is dimerized. Third, the early crystal structure found that the individual protein structure of HER2 has not been reported in other family members. It has the structural basis from the gate closed state to the active state, but is directly exposed to the active state of the dimer arm. However, for the current activation mode of HER2, The core of the controversy lies in the absence of a molecular model of heterodimer for members of the EGFR family and a molecular model for the homologous dimer of HER2. Only the currently reported EGFR dimer is used. The model is used to speculate that the dimerization mechanism of other members has theoretical limitations that cannot be broken through. We first report three cocrystalline complexes of Fab fragments with HER2 which do not affect the HER2 homologous dimer. The results of data analysis for the three HER2/Fab complexes are 3.3? ,3.5? And 3.1? In this paper, we use the method of molecular permutation to solve the phase problem. The three dimensional crystal structures of the three crystal complexes were successfully analyzed. An HER2 homologous interaction was confirmed by the analysis of the three kinds of complexes. Based on the HER2 structure cues of the three kinds of complexes, the point mutation experiment in vitro was carried out. The molecular structure model of HER2 homologous dimer was revealed and confirmed by chemical cross-linking of cell membrane, immunoprecipitation and phosphorylation assay. The effects of various therapeutic antibodies on HER2 dimer were also studied. The molecular mechanism of the effect of different epitope therapeutic antibodies on HER2 homologous dimer was discussed. Finally, based on the analysis and comparison of EGFR homologous dimer and HER2 homologous dimer, Finally, a new model of dimerization of EGFR family receptors is established, that is, a new molecular model of "head back", which complements the mechanism of dimerization of EGFR family receptors. A new molecular model and theory about the relative position of EGFR in the cell membrane and the mode of interaction force are proposed, and the traditional view is challenged. The work of this paper is of great significance for understanding the dimerization of the basic physiological process of HER2.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R73-3
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本文编号：1564295