作者姓名：陈丹英   
  论文题目：转录因子NF-κB激活的调节机理   
  作者简介：陈丹英，女， 1972年5月出生，1999年9月师从于北京大学翟中和教授，于2003年6月获博士学位。   
     
  中  文  摘  要   
    

NF-κB（nuclear factor-κB）是广泛存在于各种类型细胞中的一种转录因子。 它调节大量与细胞应急反应，如免疫应答、炎症反应和细胞抗凋亡作用相关的基因的转录。NF-κB功能的失调与许多人类病症如类风湿性关节炎、癌症等直接相关，因此自1986年发现以来，NF-κB一直是细胞生物学及免疫学领域的研究热点。NF-κB能够被多种刺激因子如肿瘤坏死因子（TNF，tumor necrosis factor）、白介素-1（IL-1，interlukin-1）、细菌脂多糖（LPS，lipopolysaccharide）等激活。研究资料表明，在NF-κB活化的信号转导途径中，两种丝氨酸/苏氨酸蛋白激酶：RIP（receptor interacting protein）和NIK（NF-κB inducing kinase）发挥了关键作用。

RIP是一种具有死亡结构域（death domain）的蛋白激酶，在TNF的作用下被招募到TNF受体1（TNF-R1，TNF receptor 1）信号复合物中，是TNF-R1诱导NF-κB激活的信号通路中不可或缺的分子；另一个激酶分子NIK（NF-κB inducing kinase）是丝裂原相关蛋白激酶MAPKKK（mitogen association protein kinase kinase kinase）家族成员之一，是淋巴毒素β（LT-β，lymphotoxin-β）诱导NF-κB激活所必需的因子。由于NIK的激酶失活突变体能够抑制多种刺激因素诱发的NF-κB的活化，研究者们推测NIK可能参与了多条NF-κB活化的信号途径。RIP和NIK在NF-κB活化途径中的作用机制至今仍不清楚，我们希望通过筛选与RIP和NIK相互作用的蛋白，寻找NF-κB活化途径中新的信号传递分子和调控因子，同时进一步分析RIP和NIK在NF-κB活化途径中的功能。

在酵母双杂交筛选与RIP相互作用蛋白的过程中，我们克隆了两个新的蛋白分子：ZIN（zinc finger protein inhibits NF-κB）和B2。其中ZIN是锌指蛋白家族成员，分子中部具有4个类似Ring锌指的结构域（RLD，ring finger like domain），C端具有富含脯氨酸的结构域（proline rich domain）。免疫共沉淀实验表明，ZIN能够与RIP特异性结合。当在293细胞中过量表达时，ZIN对RIP、IKKβ（IκB kinase β）、TNF和IL-1诱导的NF-κB活化均具有剂量依赖性的抑制作用。结构域分析实验证明，ZIN的RLDs是它与RIP结合及抑制RIP介导的NF-κB活化所必需的。同时，过量表达ZIN能够促进RIP和TNF诱导的细胞凋亡。我们进一步用免疫荧光实验证明，ZIN主要分布在细胞质，其定位与RIP重叠。以上结果表明，ZIN是TNF和IL-1诱导NF-κB激活途径中的一种新的抑制因子。

同时获得的另一个新基因B2与已知基因没有明显的序列同源性。尽管在酵母细胞中B2能够与RIP结合，但免疫共沉淀实验表明，在哺乳动物细胞中B2不能与RIP特异性地结合。尽管如此，在293细胞中过量表达B2，能够剂量依赖性地活化NF-κB并促进RIP诱导的NF-κB的激活。反义RNA实验进一步证明，B2表达量的下降能够抑制NF-κB的活化，并使TNF 和RIP诱导NF-κB激活的能力明显降低。我们用B2抗体进行免疫荧光实验的结果证明，B2定位于细胞核内。以上结果表明，B2是一个NF-κB活化的新的调节因子。B2的作用机制尚需进一步研究。

为了分析NIK可能参与的信号途径，我们用NIK蛋白作为“钓饵”进行了抗体阵列筛选，以期在已知的信号分子中得到与NIK有相互作用的分子。从筛选中我们获得了ErbB4 （epidermal growth factor receptor family protein 4）和Grb7（growth receptor bound 7）。ErbB4是表皮生长因子（EGF，epidermal growth factor）/heregulin受体家族的成员之一，而Grb7是与该受体家族结合的一种接头蛋白（adaptor protein）。免疫共沉淀实验证实，NIK能够与Grb7家族成员Grb7，Grb10和Grb14结合，而不与另一种接头蛋白Grb2结合；Grb7分子中部的GM结构域负责其与NIK的结合。免疫共沉淀实验实验结果还表明，Grb7和NIK能够持续地被招募到EGF/heregulin受体家族成员，包括EGFR, ErbB2, ErbB3 和 ErbB4形成的信号复合物中。荧光报告基因检测结果表明，NIK能够促进Grb7, ErbB2/ErbB4和EGF诱导的 NF-κB的活化，并且NIK的激酶失活突变体能够抑制ErbB2/ErbB4 和EGF诱导的NF-κB的活化。我们进一步证实，在NIK-/-小鼠胚胎成纤维细胞中，EGF/heregulin受体不能够诱导NF-κB的激活。以上研究结果表明，NIK是EGF/heregulin受体信号复合物的成员之一，并且是EGF/heregulin受体激活NF-κB的必需分子。

通过上述研究，我们克隆了调节NF-κB活化的两个新基因，其中ZIN能够与RIP结合并抑制TNF和IL-1诱导的NF-κB的活化；B2能够促进NF-κB的活化。同时我们证明，NIK是EGF/heregulin受体信号复合物的成份之一，并在EGF/heregulin受体激活NF-κB的信号途径中发挥关键作用。我们的研究结果对于了解NF-κB活化调控及相关疾病的分子机理具有重要意义。

关键词：  TNF，TNF-R1，NF-κB，RIP，NIK，ZIN，B2，Grb7，ErbB4，EGF，酵母双杂交筛选，抗体阵列筛选，细胞凋亡，信号转导
  
  The regulatory Mechanism mechanism of NF-κB activation

Danying chen

    ABSTRACT   
NF-κB is a ubiquitously expressed transcription factor that regulates the expression of an exceptionally large number of genes, including those involved in immune regulation, inflammatory responses and anti-apoptosis effects.  De-regulation of NF-κB is involved in a wide range of human diseases, such as rheumatoid arthritis and cancer.  NF-κB is activated by divergent stimuli, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and LPS.  Previous studies suggest that two serine/threonine kinases, receptor interacting protein (RIP) and NF-κB inducing kinase (NIK), are critically involved in signaling pathways leading to NF-κB activation.

RIP is a death domain-containing serine threonine kinase.  RIP is recruited to TNF receptor 1 (TNF-R1) signaling complex in a TNF dependent process and is required for TNF-R1-induced NF-κB activation.  NIK is a member of the MAP kinase kinase kinase family and is required for lymphotoxin-β receptor-induced NF-κB activation.  A NIK kinase inactive mutant is a potent inhibitor of NF-κB activation triggered by various stimuli, suggesting that NIK is involved in broad ranges of NF-κB activation pathways.  The signaling mechanisms of RIP and NIK are not well understood, and which are the focus of this dissertation research.

In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified two novel genes designated as ZIN and B2.  ZIN (zinc finger protein inhibiting NF-κB) contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus.  Co-immunoprecipitation indicated that ZIN specifically interacts with RIP.  Overexpression of ZIN inhibited RIP-, IKKβ-, TNF-, and IL1-induced NF-κB activation in a dose-dependent manner in 293 cells.  Domain mapping experiments indicated that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-κB activation.  Overexpression of ZIN also potentiated RIP- and TNF-induced apoptosis.  Moreover, immunofluorescent staining indicated that ZIN is an inhibitor of TNF- and IL1-induced NF-κB activation pathways.

B2 has no obvious sequence homology with any known proteins.  Although B2 interacts with RIP in yeast, B2 does not interact with RIP in mammalian cells in transient transfection and co-immunoprecipitation experiments.  When overexpressed in 293 cells, B2 activated NF-κB and potentiated RIP-induced NF-κB activation in a dose dependent manner. Moreover, down-regulation of B2 by antisence RNA inhibited RIP- and TNF-induced NF-κB activation.  Immunofluorecent staining indicated that B2 is localized in nucleus.  Our findings suggest that B2 is a novel regulator of NF-κB activation.

To unambiguously identify signaling pathways that NIK participates, we screened antibody arrays for proteins that are associated with NIK.  This effort identified ErbB4, one of the EGF/heregulin receptors, and Grb7, an adapter protein associated with ErbB4. Co-immunoprecipitation experiments demonstrated that NIK interacts with Grb7, as well as Grb10 and Grb14, but not Grb2. Domain mapping experiments indicated that the central GM domain of Grb7 is sufficient for its interaction with NIK. Co-immunoprecipitation experiments also indicated that Grb7 and NIK could be simultaneously recruited into signaling complexes of all known EGF/heregulin receptors, including EGFR, ErbB2, ErbB3 and ErbB4. In reporter gene assays, NIK could potentiate Grb7, ErbB2/ErbB4, and EGF-induced NF-κB activation.  A NIK kinase inactive mutant could block ErbB2/ErbB4 and EGF-induced NF-κB activation.  Moreover, EGF/heregulin receptors activated NF-κB in wild-type, but not NIK-/- embryonic fibroblasts.  Our findings suggest that NIK is a component of the EGF/heregulin receptor signaling complexes and required for NF-κB activation triggered by these receptors.

In conclusion, we cloned and characterized two novel regulators of NF-κB activation: ZIN interacts with RIP and inhibits TNF- and IL-1-induced NF-κB activation; B2 potentiates the activation of NF-κB.  We also provided evidence that NIK is a component of the EGF/heregulin receptor signaling complexes and is required for EGF/heregulin receptor-mediated NF-κB activation.  Our findings help to understand the molecular mechanisms of regulation of NF-κB activation and its related diseases.

 Key word:   TNF, TNF-R1, NF-κB, RIP, NIK, ZIN, B2, Grb7, ErbB4, EGF, yeast two-hybrid screening, antibody array screening, apoptosis, signal transduction