NF-κB调控ASPP表达参与文昌鱼先天免疫应答机制研究

ASPP (apoptosis-stimulating protein of p53) plays key role in NF-κB mediated apoptosis. However, whether ASPP could participate in NF-κB mediated immune response remained unknown. In previous works, we found that NF-κB family gene REL and ASPP were involved in immune response of amphioxus. Promoter analysis and molecular modeling prediction results indicated that the amphioxus REL protein could binding to the upstream promoter of ASPP and might interact with ASPP protein. Thus, we speculated that NF-κB could regulate the ASPP expression to participate in innate immune response of amphioxus. For revealing the relationship between amphioxus REL and ASPP involved in innate immune response, we will confirm REL regulates ASPP expression through injecting REL inhibitor into body cavity of amphioxus and promoter report experiment. The immunofluorescence, GST-Pulldown and CoIP experiments will be used to testify the protein interaction between ASPP and REL. Finally, the phosphorylation level and translocation of REL protein from cytoplasm to nucleus will be investigated through injection ASPP pathway inhibitor into body cavity. This project will not only clarify the mechanism of REL regulation of ASPP expression involved in innate immune response of amphioxus, but also shows important theoretical significance for further elucidating the regulation mechanism evolutionary of immune response in chordate animals.

ASPP在NF-κB介导的细胞凋亡过程中起重要的作用，但其是否也参与了NF-κB介导的免疫响应至今仍不清楚。我们前期发现REL（NF-κB家族的一员）和ASPP参与了文昌鱼免疫应答；生物信息预测结果表明REL能结合到ASPP基因上游调控区启动子上，并且REL与ASPP蛋白能互作。因而，我们推测REL能调节ASPP表达参与文昌鱼免疫响应，但其机制尚不明确。为此，本项目拟采用体内REL抑制剂和体外启动子报告基因实验，验证REL对ASPP表达的调控；采用细胞免疫荧光技术、GST-Pulldown和CoIP等检测REL与ASPP蛋白互作关系；体腔注射ASPP抑制剂实验验证REL与ASPP蛋白互作对REL蛋白磷酸化水平及入核的影响。项目完成不仅阐明了REL调节ASPP表达及其互作在文昌鱼免疫应答中的作用机制，而且对深刻理解动物先天免疫系统调控机制的演化也具有重要的理论意义。

文昌鱼是脊索动物的基底生物，在结构和发育上都被认为与脊椎动物的祖先相关，已逐渐成为比较免疫学研究的重要模式生物。目前研究发现，尽管文昌鱼存在一些与适应性免疫相关的分子前体，但先天免疫依然是其抵抗病原物侵染的主要防线。免疫系统一旦崩溃，动物个体将会危及生命，细胞将发生凋亡。p53细胞凋亡刺激蛋白(apoptosis stimulating protein of p53, ASPP)能够参与NF-κB介导的细胞凋亡调控，抑制DNA损伤诱发的细胞凋亡。本项目主要围绕ASPP基因在文昌鱼先天免疫调控通路中的功能进行研究。通过qRT-PCR及western blot方法明确了文昌鱼ASPP参与先天免疫调控的功能；酵母双杂交实验发现ASPP能够与甘露糖受体（Mannose receptor, MR）蛋白互作，并对文昌鱼甘露糖受体基因进行了克隆和功能分析。免疫荧光共聚焦实验和Co-IP实验进一步验证文昌鱼ASPP能够与MR蛋白互作。双荧光素酶报告系统实验证实ASPP与MR蛋白互作能够激活NF-κB参与免疫应答响应。此外，还完成了文昌鱼TAK1基因的克隆、进化和在免疫应答中的功能分析。截止目前，在该项目的资助下共发表论文8篇，第一标注5篇。对文昌鱼先天性免疫应答网络和调控功能进行研究，不仅对揭示文昌鱼自身的生命活动规律具有重要意义，而且对阐明脊椎动物免疫系统的起源、进化与调控机制也具有重要的理论和实践意义。