pVHL介导nCLU发生以Lys63连接的多聚泛素化研究以及ATF3在大鼠血管平滑肌细胞中的功能研究
文献类型：学位论文
| 作者 | 吕丹丹 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-05 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 方靖 |
| 关键词 | 泛素化 pVHL nCLU ATF3 凋亡 |
| 其他题名 | pVHL mediates the K63-linked ubiquitination of nCLU for nuclear translocation and the function of ATF3 in vascular smooth muscle cells. |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 本论文分为两章。 第一章pVHL介导nCLU发生以Lys63连接的多聚泛素化研究的摘要如下： 生物体内蛋白的泛素化是一个高度复杂、时空调节、精确调控的过程，作为一种重要的翻译后修饰类型参与到多种细胞活动中，包括细胞分裂、细胞分化、信号传导、囊泡转运和蛋白的质量控制等等。泛素化需要ATP供能和泛素活化酶、泛素结合酶和泛素连接酶等一系列酶的参与。pVHL是一肿瘤抑制蛋白，作为VCB泛素连接酶复合物中负责底物特异性识别的分子，通过识别和结合底物，介导底物的泛素化和蛋白酶解，它经典的底物为低氧诱导因子（HIF-a）。nCLU是clusterin基因编码的多种蛋白产物之一。它在静息的未受刺激的细胞中，以约49kDa的前体形式主要定位于胞浆中。当DNA损伤信号来临时，nCLU被诱导并以约55kDa的成熟形式转运至核内。nCLU的作用与其定位关系密切。已有的报道表明，细胞核内的nCLU在多种细胞中发挥促凋亡的作用。 我们的研究发现，pVHL与nCLU结合，且nCLU结合在pVHL的底物结合区域，暗示nCLU可能是pVHL的一个底物。有趣的是，我们发现pVHL虽然结合nCLU，但并不介导nCLU的降解。相反，pVHL的结合反而促进了nCLU的稳定性。这与以往关于pVHL作用的报道完全不同。通过进一步的研究，我们发现pVHL结合nCLU后促进其泛素化，而且是以Lys63连接的多聚泛素化，且Rbx1、Elongin B等VCB泛素连接酶复合物的其它组分亦参与到这一过程中。迄今为止，除了作为VCB泛素连接酶复合物组分的pVHL介导泛素的第48位的赖氨酸（Lys48）连接的多聚泛素化外，还未见pVHL参与以泛素的第63位的赖氨酸（Lys63）连接的多聚泛素化的报道。这一发现，使我们对pVHL的功能有了新的认识，有助于我们深入了解pVHL抑制肿瘤发生发展的作用和分子机制。我们还发现，pVHL泛素化修饰nCLU后可以促进nCLU的入核，提示pVHL可能通过泛素化修饰nCLU在抑制肿瘤过程中发挥作用。我们的工作揭示了nCLU是pVHL作为一个多功能介导蛋白的新的靶标分子，从而为pVHL参与到细胞凋亡的调控中提供了一个与nCLU相关的新思路和可能的机制。 第二章ATF3在大鼠血管平滑肌细胞中的功能研究的摘要如下： 转录激活因子3（ATF3）是ATF/CREB家族的转录因子之一。它在血管平滑肌细胞中的表达情况和功能还未见报道。我们这项工作的目的就是确定ATF3在血管平滑肌细胞中的表达情况及可能的功能。 我们发现血管平滑肌细胞能表达ATF3，并且ATF3的表达能被多种刺激诱导，包括血清、血管紧张素II和过氧化氢等。腺病毒介导的ATF3的基因沉默诱导血管平滑肌细胞的凋亡、caspase-3的剪切和细胞色素c的释放。这些结果提示我们ATF3调节血管平滑肌细胞的存活能力。除此之外，我们还发现ATF3过表达促进血管平滑肌细胞的迁移，并诱导基质金属蛋白酶1、3和13的表达。这些结果提示我们ATF3在血管平滑肌细胞的迁移中发挥作用。此外，在股动脉受损的小鼠模型中，我们发现与未受损的对照组相比，ATF3在受损的股动脉的血管平滑肌细胞中的表达显著增加。这些结果暗示ATF3很可能与相关疾病的病理学密切相关。 |
| 索取号 | D2010-005 |
| 英文摘要 | The abstract for the first part is as follows： Ubiqutination of cellular proteins is a highly complex, temporally controlled, and tightly regulated process, which has emerged as a critically important post-translational modification playing major roles in regulating a board array of basic cellular processes, such as cell division, differentiation, signal transduction, trafficking, and protein quality control. Ubiquitination is ATP dependent and involved a series of enzymes such as ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). pVHL is an important tumor suppressor protein. As a component of VCB E3 complex, pVHL is responsible for the recognition and binding of specific substrates. HIF- is one of the best-documented substrate of the pVHL ubiquitin ligase complex. nCLU is one kind of protein products encoded by the gene clusterin. In untreated cells, nCLU is localized mainly in the cytoplasm as a ~49kDa precursor form pnCLU. In response to cell damage, nCLU is induced and translocated to the nucleus as a ~55kDa mature form. The role of nCLU is highly related to its localization and it is reported that the nuclear nCLU functions as an apoptosis inducer in various cell types. In this study, we have found the specific interaction between pVHL and nCLU. nCLU binds to the subunit which is the substrate-binding region of pVHL, implying that nCLU might be one substrate of pVHL. Interestingly, we have also found that pVHL promotes the stability of nCLU instead of mediating its degradation; which is largely different from the traditional function of pVHL. Further studies show that pVHL promotes the k63-linked polyubiquitination of nCLU, and other components of VCB E3 complex such as Rbx1 and Elongin B are also involved in the pVHL-mediated k63-linked polyubiquitination of nCLU. To our knowledge, it is reported for the first time that pVHL, as a component of VCB complex, mediates k63-liked polyubiquitination besides the k48-linked polyubiquitination. Our work uncovers the new function of pVHL, which may help us to explore the tumor suppression role of pVHL in the initiantion and progression of malignancies and molecular mechanism implicated in it. It is found that the polyubiquitination of nCLU mediated by pVHL promotes the nuclear translocation of nCLU, suggesting by which pVHL may exert the role in the inhibition the progression of tumors. Our study reveals that nCLU is a new target substrate of the multiple adaptor protein pVHL, which may open doors to the studies of the function of pVHL in cell apoptosis, and its possible mechanism related to nCLU. The abstract for the second part is as follows： Activating transcription factor 3 (ATF3) is a member of the ATF/CREB (CAMP responsive element binding protein) family of transcription factors. The expression and the function of ATF3 in vascular smooth muscle cells (VSMCs) remain unknown. The aim of this work is to determine the expression and possible function of ATF3 in VSMCs. We found that VSMCs expressed ATF3, and expression of ATF3 in VSMCs was induced by a variety of stimuli including serum, angiotensin II, and H2O2. Knockdown of ATF3 induced apoptosis of VSMCs, caspase-3 cleavage, and cytochrome c release. The results suggest that ATF3 regulates survivability of VSMCs. Moreover, we found that overexpression of ATF3 promoted migration of VSMCs and induced expression of matrix metalloproteinase 1, 3, and 13. These results suggest that ATF3 plays a role in regulating migration of VSMCs. In addition, we found that the expression of ATF3 was upregulated in smooth muscle cells in the injured mouse femoral arteries compared with the uninjured control group. These results suggest that ATF3 is relevant to disease physiology. |
| 语种 | 中文 |
| 公开日期 | 2015-12-24 |
| 源URL | [http://202.127.25.144/handle/331004/373]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_营养与癌症研究组 |
| 推荐引用方式 GB/T 7714 | 吕丹丹. pVHL介导nCLU发生以Lys63连接的多聚泛素化研究以及ATF3在大鼠血管平滑肌细胞中的功能研究[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2011. |
入库方式： OAI收割
来源：上海营养与健康研究所
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