【作者】 张力民；

【导师】 杨晓明；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 本论文包括两部分内容,一是羟苯丙酮酸二加氧酶(hydroxyphenylpyruvate dioxygenase, HPD)正调控NF-κB通路促进肺癌细胞存活和迁移。二是NLK通过Smad4(?)印制TGFβ信号通路活性。中国有句成语：物以类聚,人以群分。在生物学研究中也适用这样的准则。功能相关的蛋白质通常以相互联系的方式调节复杂的生物学过程。因此,蛋白质相互作用是发现未知蛋白功能或已知蛋白质新功能的重要手段。我们实验室前期利用酵母双杂交技术平台构建了大规模的蛋白质相互作用网络,发现了大量新的蛋白质间相互作用[1]。这个蛋白质相互作用网络为我们提供了很多线索和提示。本课题选取该网络中发现的可能具有重要生物学意义的两对蛋白质间相互作用—HPD与NEMO和NLK与Smad4进行了深入研究。HPD是酪氨酸分解代谢途径的关键酶[2]。NEMO是NF-κB信号通路的核心分子[3]。采用外源和内源免疫共沉淀实验我们验证了HPD与NEMO的相互作用,确定HPD通过N端与NEMO的N端发生相互作用,并证明HPD可能与IKK组成蛋白质复合体。过表达HPD加速IKBα的降解,促进NF-κB报告基因活性,增加TNFa诱导的NF-κB靶基因BCL-xL、cIAP2、ICAM-1、VCAM-1的表达；利用RNAi敲低HPD表达则抑制NF-κB报告基因的活性；HPD酶活性突变不影响HPD调节NF-κB通路的作用,HPD的催化底物-羟苯丙酮酸(HPP)和催化产物-尿黑酸(HGA)对NF-κB通路也无影响。这些结果说明HPD以不依赖酶活性的方式正调控NF-κB通路。机制上,我们发现过表达HPD可以增强TNFα刺激引起的NEMO泛素化修饰。利用RT-PCR技术检测HPD在小鼠各器官组织的表达,结果显示HPD仅在肝脏和肾脏表达,这与文献报道一致[4]。有意义的是,检测多种细胞系中HPD的表达,发现除肝实质细胞来源细胞系外,多株肺癌细胞中也表达HPD。为进一步验证HPD在肺癌组织中的表达及与NF-κB活性的关系,我们利用免疫组织化学技术检测了HPD及磷酸化p65(S276)在肺癌病人癌组织和癌旁组织中的表达情况,发现HPD在肺癌组织中的表达强度和阳性细胞率都显著高于癌旁肺组织,并与磷酸化p65(S276)的表达呈正相关。进一步,利用慢病毒介导的RNAi敲低肺癌细胞A549和H1299的HPD表达,结果发现HPD下调对肺癌细胞的细胞周期和增殖没有明显影响,但克隆形成实验和Transwell实验结果显示,敲低HPD显著抑制了肺癌细胞的克隆形成能力和迁移能力。综上,我们的实验结果发现HPD在肺癌组织中异常表达,下调其表达可减低肺癌细胞的恶性表型,机制上可能与HPD通过与NEMO的相互作用,以不依赖酶活性的方式激活NF--κB通路有关,提示HPD可能在肺癌的发生发展中起重要作用。NLK是一种保守的丝／苏氨酸蛋白激酶,识别的保守序列为S/TP基序,通过磷酸化底物的方式参与多种生物学过程[5-9]。采用免疫共沉淀和GST pull down我们证明NLK能与Smad4发生相互作用。进一步,我们初步实验的结果发现NLK以不依赖激酶的方式负调控TGFβ信号通路,机制上可能与促进Smad4的泛素化修饰,降低Smad4(?)勺稳定性有关,但其生物学意义尚有待深入研究。更多还原

【Abstract】 This essay includes two parts, the first is hydroxyphenylpyruvate dioxygenase (HPD) up-regulates NF-κB signaling pathway and promotes the survival and migra-tion of lung cancer cell, the second is NLK inhibits TGF-β signaling pathway by interacting with Smad4.It is said that birds of a feather flock together. The principle is also applicable in biology study. Therefore, Finding out the proteins that interact with your interested protein is an important approach to discover its function. Using yeast two-hybrid system, our lab built large scale protein-protein interaction network which included many newly found protein-protein interactions. The interaction network provides a lot of useful information to us. Two pairs of protein-protein interactions discovered by the network that we believed having important function was further studied. The two interactions are that between HPD and NEMO and that between NLK and Smad4.HPD is the key enzyme of tyrosine catabolism pathway. NEMO is the core protein that involves in NF-κB signaling pathway. Using in vitro and in vivo coimmunoprecipitation assay, we confirmed the interaction between HPD and NEMO through their N terminals, and HPD may form a protein complex with IKK complex. Ectopic expression of HPD accelerated the degradation of IκBα, enhanced the activity of NF-κB reporter and increased the expression of NF-κB target gene induced by TNFα, including BCL-xL, cIAP2, ICAM-1, VCAM-1; knock down of HPD inhibited the activity of NF-κB reporter; lacking of the enzyme activity of HPD didn’t influence its function in up-regulating NF-κB pathway, hydroxyphenylpyruvic acid(HPP), the substrate of HPD, and homogentisic acid(HGA), the product of HPD, had little influence on NF-κB pathway. These results indicate HPD up-regulates NF-κB pathway independent of its enzyme activity. We found over-expression of HPD augments the ubiquitination of NEMO stimulated by TNFα. RT-PCR was used to test the expression of HPD in various organs of mice, the result showed that HPD express in liver and kidney, which is consistent with previous report. Intriguingly, besides the cell lines originated from liver, HPD was detected in many lung cancer cell lines. In order to understand the relationship between HPD and NF-κB signaling pathway in lung cancer, we compared the expression of HPD and phosphorylated p65(S276) in the lung tumor tissue and relative normal lung tissue of lung cancer patients, and found lung tumor tissue expressed higher level of HPD than normal lung tissue, the expression of HPD correlate with that of phosphorylated p65(S276). Subsequently, using lentivirus system, knock-down of HPD inhibited the colonic formation ability and migration of H1299and A549and had no influence on the cell cycle and proliferation of H1299and A549. To sum up, our data show lung cancer tissue express HPD, knock-down of HPD alleviate the malignant phenotype of lung cancer, these effects maybe relevant to the interaction between HPD and NEMO and thereby activation of NF-κB pathway. These results indicated HPD may play important role in the initiation and development of lung cancer.NLK is a conserved S/T protein kinase, recognizes S/TP motif of the target proteins, involves in many biology process through phosphorylating its target proteins. The result from CoIP and GST-pull down showed that NLK interact with Smad4. NLK down-regulate TGFβ pathway independent of its kinase activity through enhancing the ubiquitination of Smad4and decrease the stability of Smad4. The biological significance of the interaction between NLK and Smad4requires further study.更多还原