鱼类炎症复合体的分子结构组成与功能调节机制研究

In?ammasomes are molecular platforms activated upon cellular infection or stress that trigger the activation of inflammatory caspases-1 or caspase-5 and the maturation of proin?ammatory cytokines, such as interleukin-1β (IL-1?), IL-18 and IL-33 to engage innate immune defenses as well as adaptive immunity. In view of considerable complexity of NLRs family, little information is available on inflammasome from lower vertebrates, although mammalian inflammasome is relatively well characterized. To this end, this project contributes to the first investigation of the molecular composition and functional mechanisms of a NLRP1-like (NLRP1-L) inflammasome in teleost fish. To understand the activation passways and regulation mechanisms of inflammatory caspase (caspase-1 and caspase-5) in teleost, the molecular compositions and assembly patterns, and the positive and negative regulatory mechanisms underlying fish inflammasomes are waited to be further investigated. It is suggested that there might be two functional NLRP1-like genes in teleost fish, the NLRP1-L-casA and NLRP1-L-casB, which can trigger the activation of caspase-A and caspase-B, respectively. According to a serises of bioinformatics analysis, and the similarity in structure and function between mammalian NLRP1 inflammasome and teleost inflammasome, it is proposed that NLRP1-L-casA and NLRP1-L-casB might be two ancestral NLRP1 moleculars, which were assembled into one NLRP1 molecular by a series of domain reorganization among NACHT, CARD, PYD, LRR and FIIND during the vertebrate evolution. Thus, the mammalian NLRP1 inflammasome was supposed to be evolutionarily originated from the NLRP1-L-casA and NLRP1-L-casB ancestors. In addition, it is also anticipated to reveal the co-evolutionary relationships among NLRP1 inflammasom, caspase-1/5 and IL-1β moleculars during the innate immunity evolutionary history as a whole. For this popurse, the mechanisms underlying IL-1β maturation will be further identified, which would be benefit to the better understaning of IL-1β synthesis and processing. We hope that the results from this project could enrich the current knowledge of fish immunology, and provide potential application for developing new immune adjuvants in the preventation of fish diseases.

本项目拟以大黄鱼等经济鱼类为研究对象，结合斑马鱼等模式生物，在鱼类中系统开展炎症复合体的分子鉴定与功能研究。重点探索鱼类NLRP1样炎症复合体（NLRP1-L）的分子结构、组装方式、作用途径及其信号传递和功能调节机制，利用生物信息学和比较基因组学等技术，探讨NLRP1炎症复合体的起源与进化，揭示鱼类NLRP1-L炎症复合体以NLRP1-L-CasA和NLRP1-L-CasB亚单位相互组合或相对独立方式活化caspase-A和-B的独特效应机制，阐明NLRP1-L-CasA和NLRP1-L-CasB作为炎症复合体进化的原始分子，通过基因重组演化为NLRP1复合体的进化规律。通过项目实施，填补鱼类炎症复合体研究的不足和空白，丰富鱼类免疫学内容，加深对免疫系统起源进化的认识。研究成果不仅具有原创性理论意义，而且为鱼类疫苗和分子佐剂的设计和开发应用提供新的思路和靶点，具有重要潜在应用价值。

炎性小体是近年免疫学研究的热点和重要科学命题。炎性小体在启动炎症发生、调节先天免疫以及诸多疾病的形成中发挥重要的作用。其中NLRP1是最为受到关注的经典炎性小体之一，目前的研究主要见诸人类和部分哺乳动物，低等脊椎动物中尚鲜有研究报道。鱼类作为早期脊椎动物的代表性物种，利用鱼类开展炎性小体研究，对揭示炎性小体分子与功能演化机制、丰富先天免疫理论、开辟并建立新的实验研究体系等，都具有重要的意义。本项目首次在鱼类中分离鉴定出NLRP1炎性小体，阐明了鱼类NLRP1复合体的分子组成、结构特征、活化信号、装配途径、工作原理及其功能调节机制。通过揭示鱼类NLRP1炎性小体对两种关键的炎性半胱氨酸天冬氨酸蛋白酶（Caspase-A/-B）以及IL-1β成熟加工的协同机制，提出了一种全新的顺序性活化工作原理和作用模式，并深入阐释了相关的分子机制。通过体内基因敲减等研究，揭示了鱼类NLRP1炎性小体在抵抗细菌感染中发挥重要的免疫防御作用。本项目揭示了NLRP1炎性小体在硬骨鱼类中的起源及其在早期脊椎动物中的功能机制，不仅丰富了鱼类免疫学内容，为炎性小体的功能与机制等研究提供了新的实验动物模型，而且为深入阐明以炎性小体为核心的炎症发生与调节机制的演化规律，提供了重要的科学依据。同时，项目在学科前沿水平开展鱼类免疫学理论研究的同时，还兼顾到了这一成果对于我国渔业生产中的病害防治具有重要潜在应用价值，可为鱼类病害防治中针对炎症发生与调控分子设计提供新思路和开辟新途径。