FADD形成淀粉样蛋白纤维及其在果蝇免疫信号传递中的作用

Formation of FADD amyloid fiber and its role in immune signaling in Drosophila melanogaster

摘要:

探究果蝇FADD (Fas-associated death domain-containing protein) 淀粉样蛋白纤维的形成及对IMD (Immune deficiency) 信号通路中信号传递的影响，有助于更清楚地了解昆虫先天免疫信号通路中的调节机制，为其他物种的免疫调控提供参考。通过原核表达纯化dFADD蛋白、硫黄素T结合和透射电子显微镜观察等鉴定dFADD在体外纤维的形成；通过构建dFADD真核表达载体，SDD-AGE检测、共聚焦显微镜观察等探究dFADD在果蝇S2细胞内纤维聚合物的形成；构建dFADD结构域突变体，检测纤维形成的关键结构域及对IMD信号传递的影响。结果表明，dFADD在体外和细胞水平上都能聚合形成淀粉样蛋白纤维聚合物；纤维的形成是由dFADD的DED (Death-effector domain) 结构域决定的，当DED结构域缺失时，dFADD以单体形式存在；双荧光素酶报告系统的检测结果显示，dFADD只有形成纤维时，才能诱导下游抗菌肽的表达，表明纤维形成是IMD信号传递的关键。本研究揭示了dFADD通过形成淀粉样蛋白纤维参与IMD信号通路中介导IMD与Dredd级联传导的作用，进一步加深了对淀粉样蛋白纤维不仅在哺乳动物，也在昆虫的免疫信号通路中传递信号这一保守功能的认识。

Abstract:

In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.