1.核受体EAR2在人结直肠癌中的功能和机制研究 2.TRAF6介导HIF-1α发生Lys63连接的多聚泛素化研究
文献类型：学位论文
| 作者 | 李雪冰 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-05 |
| 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 | 1. EAR2 孤儿核受体 结直肠癌 2. 肿瘤坏死因子受体相关因子6 低氧诱导因子1a 泛素化 |
| 其他题名 | 1. The functions and mechanisms of EAR2 in colorectal cancer 2. TRAF6 mediates K63-linked ubiquitination of HIF-1a |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | AR2（V-erbA-related protein 2）是鸡卵清蛋白上游启动子-转录因子（chick ovalbumin upstream promoter-transcription factor，COUP-TF）家族的成员。COUP-TF属于孤儿核受体（orphan nuclear receptor），其成员参与多种生物学过程的调节。尽管对于COUP-TF家族其他成员蛋白功能的研究已开展了很多，但EAR2在癌症中的功能仍不清楚。本课题开展的目的在于探索EAR2在人结直肠癌（colorectal cancer，CRC）中的表达情况和可能的功能。我们通过研究分析配对的人结直肠癌临床组织样本，发现了结直肠癌中EAR2的表达水平明显高于正常结直肠组织。通过基因沉默技术抑制EAR2的表达可以引起结肠癌细胞的凋亡，这表明EAR2可能对于结肠癌细胞的存活能力有重要的调节作用。同时，动物实验显示，抑制EAR2表达可以明显抑制结肠癌细胞的成瘤能力。进一步研究表明，抑制EAR2可以明显抑制X染色体连锁凋亡抑制蛋白（X-linked inhibitor of apoptosis protein，XIAP）的表达，这表明EAR2调节细胞存活的作用至少部分通过了XIAP。本课题的研究结果显示，EAR2在人结直肠癌中具有高表达特征，并且抑制EAR2表达可以降低结肠癌细胞的存活能力和成瘤性。这表明EAR2在结直肠癌的发生发展中发挥重要的作用，这些研究结果不仅有助于我们了解EAR2在结直肠癌中的作用，也帮助我们进一步认识CRC发生发展的机制。同时，我们的结果也表明EAR2很可能成为今后CRC治疗的有效靶点。 TRAF6是肿瘤坏死因子受体相关因子（tumor necrosis factor receptor associated factors，TRAFs）家族的成员之一，最初发现于CD40和IL-1R信号传导途径。TRAF6作为接头蛋白可以介导TNFR（tumor necrosis factor receptor）和IL-1R（interleukin-1 receptor）/TLR（Toll-like receptor）超家族信号传递。同时，它还可作为泛素连接酶，在泛素交联酶Ubc13和Uev1A存在时催化生成以Lys63连接的多聚泛素化。这种非降解的多聚泛素链可以促进底物激酶如TAK1和PKB（Akt）的活性。低氧诱导因子1（hypoxia inducible factor-1，HIF-1）由受氧调节的HIF-1a亚基和组成型表达的HIF-1b亚基构成。在氧气充足情况下，HIF-1a被一类需要氧气发挥催化活性的脯氨酸羟化酶（prolyl hydroxylases，PHDs）羟基化，再经pVHL（von Hipple Lindau protein）介导的VCB E3复合物泛素化降解。在低氧情况下，HIF-1a的羟基化受到抑制，因此在蛋白水平得到积累。HIF-1的下游基因在肿瘤发生发展的多个方面发挥作用，如血管生成，代谢，细胞生存，迁移和侵润等。在本课题研究中，我们发现HIF-1a是TRAF6的一个底物。研究结果显示，TRAF6可以结合在HIF-1a的氧依赖降解结构域（Oxygen Dependent Degradation domain，ODD domain）上，这一结构域中含有经典的TRAF6识别并结合的氨基酸序列。进一步研究显示，TRAF6还可以介导HIF-1a发生以Lys63连接的多聚泛素化，使其稳定性提高并促进其转录活性。这一研究结果不仅发现了HIF-1a一个全新的泛素化修饰形式，同时也揭示TRAF6有可能通过调节HIF-1a在肿瘤发生发展过程中发挥作用。 |
| 索取号 | D2010-173 |
| 英文摘要 | EAR2 (V-erbA-related protein 2) is a member of the chick ovalbumin upstream promoter-transcription factors (COUP-TFs). COUP-TFs belong to orphan nuclear receptors and regulate many biological processes. Little is known regarding EAR2 in cancer, though much progress has been made in understanding the function of other COUP-TF members. The aim of this study is to investigate the expression and possible function of EAR2 in colorectal cancer. We determined expression of EAR2 in human primary colorectal malignant tumors and their paired adjacent normal colorectal tissues. We found that expression of EAR2 was upregulated in colorectal tumors. Knockdown of EAR2 induced apoptosis of colon cancer cells, suggesting that EAR2 may function to regulate survivability of colon cancer cells. In vivo tumor study demonstrated that knockdown of EAR2 inhibited the xenograft growth of colon cancer cells. We found that knockdown of EAR2 inhibited the expression of X-linked inhibitor of apoptosis protein (XIAP), suggesting that EAR2 regulates cell survivability, at least partly, through XIAP. In this manuscript, we demonstrated that expression of EAR2 was elevated in colorectal cancer and knockdown of EAR2 reduced survivability and tumor growth of colon cancer cells. Our results suggest that EAR2 plays an important role in development of colorectal cancer. The findings also suggest that EAR2 may serve as a potential therapeutic target of colorectal cancer. TRAF6 is a member of tumor necrosis factor receptor associated factors (TRAFs) family. It was first identified in the signal transduction pathways of CD40 and IL-1R. TRAF6 functions as an adaptor protein to mediate signals from both the TNFR and the IL-1R/TLR superfamily. Also, it works as an ubiquitin ligase to mediate K63-linked ubiquitination in the presence of the ubiquitin conjugation enzyme system, Ubc13 and Uev1A. These nondegradative polyubiquitin chains have been shown to be important in the activation of protein kinases such as TAK1 and PKB(Akt), which are direct targets of TRAF6. HIF-1 is a transcription factor that consists of an O2-regulated HIF-1a subunit and a constitutively expressed HIF-1b subunit. In well-oxygenated cells, HIF-1a is hydroxylated by PHDs which require O2 for their catalytic activity. Prolyl-hydroxylated HIF-1a is bound by the von Hippel-Lindau (VHL) tumor suppressor protein, which recruits the Elongin C-Elongin B-Cullin 2-E3-ubiquitin-ligase complex, leading to proteasomal degradation of HIF-1a. Under hypoxic conditions, HIF-1a prolyl hydroxylation is inhibited, thereby stabilizing HIF-1a protein. HIF-1 activates transcription of genes encoding proteins that are involved in key aspects of cancer biology, including angiogenesis, metabolism, cell survival, invasion, and metastasis. In this manuscript, we report that HIF-1a is a target of TRAF6. We found that TRAF6 had an interaction with HIF-1a. HIF-1a bound to TRAF6 at HIF-1a’s Oxygen Dependent Degradation (ODD) domain, which contains the consensus-binding site of TRAF6. Further studies indicated that TRAF6 mediated K63-linked ubiquitination of HIF-1a. It is well known that K48-linked ubiquitination targets HIF-1a for degradation. We found K63-linked ubiquitination stabilized HIF-1a and enhanced its transcription activity. In summary, our results disclose a novel K63-linked polyubiquitination of HIF-1a that mediated by TRAF6. This study also suggests that TRAF6 may play a role in tumorigenesis, probably through HIF-1a. |
| 语种 | 中文 |
| 公开日期 | 2015-12-24 |
| 源URL | [http://202.127.25.144/handle/331004/374]  |
| 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 | 李雪冰. 1.核受体EAR2在人结直肠癌中的功能和机制研究 2.TRAF6介导HIF-1α发生Lys63连接的多聚泛素化研究[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. 2012. |
入库方式： OAI收割
来源：上海营养与健康研究所
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