【作者】 卫赛赛；

【导师】 许正平；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 血管生成素(Angiogenin, ANG)具有促进血管生成和肿瘤细胞增殖的双重活性,与肿瘤的发生发展密切相关,但其作用的分子机制未明。由于蛋白质-蛋白质相互作用在生命活动中扮演着重要角色,因此研究ANG相互作用蛋白质是阐明ANG功能及分子机制的有效途径。本论文的第一部分运用免疫共沉淀与质谱技术在HeLa细胞中筛选到20个可能与ANG相互作用的蛋白质,其中包括了2个已知的ANG结合蛋白。通过对这20个蛋白质的功能注释和相互作用网络构建,发现其中4个蛋白质,即含IQ结构域的GTP酶激活蛋白(IQ motif containing GTPase activating protein 1, IQGAP1)、非肌型肌球蛋白重链(non-muscle myosin heavy chain 9, MYH9)、α-辅肌动蛋白4(alpha-actinin 4, ACTN4)及p-肌动蛋白(beta-actin, ACTB)与actin细胞骨架的调节及细胞粘附、迁移相关,并与Racl一起构成了一个与actin细胞骨架调节通路相关的ANG相互作用蛋白质子网络,提示ANG参与上述过程。随后利用免疫共沉淀技术证实,这4个蛋白质与ANG在HeLa细胞内的相互作用是真实的。由于actin骨架系统的运动与细胞粘附是细胞迁移的重要组成步骤,因此我们推测ANG很可能通过调节上述两个过程而调节了细胞的迁移。为了证实我们的猜想,第二部分首先利用免疫荧光技术检测了ANG与actin骨架蛋白在细胞中的共定位情况。结果表明,ANG与actin骨架蛋白结合于细胞-基质粘附所形成的黏着斑区域。随后,我们检测了ANG对细胞骨架及黏着斑形态、活性的影响。抑制内源ANG表达后,利用荧光标记的鬼笔环肽对F-actin进行染色以显示细胞骨架形态的变化；免疫荧光法标记黏着斑蛋白paxillin,检测黏着斑形态的变化；同时Western-blot法检测黏着斑激酶(FAK)磷酸化程度的改变。结果表明,ANG干扰后actin应力纤维显著增粗,黏着斑显著增大,但是数量减少,EGF促进的黏着斑激酶的活化受阻。最后,划线法实验结果表明,干扰内源ANG后EGF促进的细胞迁移速度减慢。综合以上结果,我们认为ANG很可能通过与actin骨架蛋白相互作用来调节actin骨架系统的运动与细胞粘附,最终影响细胞迁移能力。另一个得到验证的ANG结合蛋白为损伤DNA结合蛋白2(damage-specific DNA binding protein 2, DDB2),该蛋白在紫外诱导的DNA损伤修复中发挥作用,提示ANG也可能参与该过程。我们初步探索了紫外辐照刺激下ANG蛋白水平的变化及其对紫外诱导的细胞凋亡的影响。由于这部分结果较为初步,因此放在附呈现,进一步的研究正在进行中。综合研究结果,本论文创新地证明：1.ANG在HeLa细胞内与20个蛋白质有潜在的相互作用；2.ANG可在HeLa细胞内与IQGAP1、MYH9、ACTN4、ACTB及DDB2等相互作用；3.ANG与actin骨架蛋白在黏着斑位置相互作用,从而影响actin骨架和黏着斑形态及活性,促进EGF刺激的细胞迁移。更多还原

【Abstract】 Angiogenin (ANG) is an angiogenic factor which also promotes tumor cell proliferation directly. However, the underlying molecular mechanisms remain elusive. Protein-protein interactions play crucial roles in biological processes, therefore, we reason that identifying ANG-ineracting proteins is a feasible way to elucidate ANG’s biological functions and mechanism of action.In the first part of the thesis, we identified ANG-interacting proteins in HeLa cells using co-immunoprecipitation (Co-IP) combined with mass spectrum (MS) analysis. The results showed 20 potential ANG-binding partners, two of which had been reported. Bioinformatics analysis of the proteins, including functional annotations and constructions of protein-protein interacting (PPI) networks, revealed IQ motif containing GTPase activating protein 1 (IQGAP1), non-muscle myosin heavy chain 9 (MYH9),α-actinin 4 (ACTN4), andβ-actin (ACTB) to be involved in actin cytoskeleton regulations and cell adhesions. The four proteins also organized an actin cytoskeleton regulatory pathway-related sub-PPI network together with Rac1, indicating ANG involved in the above mentioned pathways. The interactions between the four proteins and ANG were confirmed by immunoprecipitation assay.The interaction of ANG with actin cytoskeleton proteins MYH9, ACTN4, and ACTB suggests the possibility that ANG is involved in the processes of actin cytoskeleton dynamics and cell adhesion. Since the coordination of cytoskeleton dynamics and cell-matrix adhesion is particularly important for cell migration, potential ANG functions in the two processes may explain how the protein promotes cell migration. To verify this hypothesis, in the second part of the thesis, we first identified the co-localization of ANG with actin cytoskeleton proteins and found that ANG bond to actin cytoskeleton proteins at the focal adhesion (FA) sites. Next, we stained F-actin with TRITC-conjugated phalloidin, focal adhesion with paxillin antibody, and detected phosphorylated-FAK levels using Western-blot when ANG was down-regulated. Data showed that the formation of stress fibres was enhanced, focal adhesions were enlarged, and EGF-stimulated focal adhesion kinase activation was inhibited in ANG down-regulated cells. At last, we used wound healing assay to determine the migration speed, and found that EGF-stimulated migration was retarded in ANG down-regulated cells. Altogether, our data suggested that ANG might regulate actin cytoskeleton dynamics and cell adhesion through interacting with actin cytoskeleton proteins, thus promote cell migration.Another identified ANG-associated protein was damage-specific DNA binding protein 2 (DDB2), which bind to the damaged DNA sites immediately after UV irradiation, indicating ANG involved in the same process. Thus, we primarily detected that ANG might response to UV irradiation by degradation and decrease UV-induced cell apoptosis in ANG down-regulated cells. These results are presented in the supplement. Much more studies are undergoing.According to the results above, we conclude:1. ANG interacts with 20 potential proteins in HeLa cells.2. ANG interacts with IQGAP1, MYH9, ACTN4, ACTB and DDB2 in HeLa cells; 2. ANG binds to actin cytoskeleton proteins at focal adhesions to interrupt formations and activities of stress fibres and FAs, thus to enhance EGF stimulated cell migration.更多还原