Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors.
Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors.
复制标题
测量 MAVS 的单体到细丝的转变作为 RIG-I 样受体的体外活性测定。
DOI：
10.1007/978-1-4939-3335-8_9
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Hur,Sun
中科院分区：
文献类型：
--
作者：
Wu,Bin;Huoh,Yu-San;Hur,Sun
在病毒感染过程中，先天免疫装备样受体(RLR)识别病毒双链RNA(DsRNA)并触发连接蛋白线粒体抗病毒信号蛋白(MAV)的微丝组装。MAVS丝随后激活抗病毒信号事件，包括上调I型干扰素的表达。近年来，对RLRs如何识别dsRNA有了很多深入的研究，但RLRs激活如何刺激MAV细丝形成的确切机制仍不清楚。在本章中，我们描述了我们先前开发的用于研究RLR催化的MAV微丝组装的体外重建实验。我们为纯化MAVSCARD(MAVSCARD)的caspase激活招募结构域(CARD)提供了技术指导，作为一种功能单体和预制的丝状种子。我们还描述了在刺激时监测MAVSCARD单体到细丝转变的方法。这一方案为研究RLR信号事件提供了一种最低限度的方法，并有可能被用于阐明其他先天免疫受体的信号机制，如Toll样受体和炎症体，这些受体涉及更高阶的卡片或相关结构域的组装，以实现其下游信号激活。
During viral infection, the innate immune RIG-I like receptors (RLRs) recognize viral double stranded RNA (dsRNA) and trigger filament assembly of the adaptor protein Mitochondrial Anti-viral Signaling protein (MAVS). The MAVS filament then activates anti-viral signaling events including the up-regulation of type I interferon expression. In recent years, much insight has been gained into how RLRs recognize dsRNA, but the precise mechanism of how activated RLRs stimulate MAVS filament formation remains less understood. In this chapter, we describe an in vitro reconstitution assay that we have previously developed to study the RLR-catalyzed filament assembly of MAVS. We provide technical guidance for purifying the caspase activation recruitment domain (CARD) of MAVS (MAVSCARD) as a functional monomer and also preformed filament seed. We also describe the methods to monitor the monomer-to-filament transition of MAVSCARDupon stimulation. This protocol provides a minimalist approach to studying RLR signaling events and can potentially be applied to elucidate signaling mechanisms of other innate immune receptors, such as Toll-like receptors and inflammasomes, that involve higher order assemblies of CARDs or related domains for their downstream signal activation.
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