Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts.
Department of Immunology, Tufts University School of Medicine, Boston, Massachusetts.
Curr Protoc. 2021 Jun;1(6):e156. doi: 10.1002/cpz1.156.
A large protein complex, containing RIPK1, RIPK3, and caspase-8 and known as Complex II, has emerged as one of the key mediators of cell death downstream from a range of innate immune triggers. This regulatory mechanism plays a prominent role in macrophages, where Complex II has been linked to apoptosis, pyroptosis, and necroptosis as well as the enhancement of inflammatory gene expression. Although core components of this complex are fairly well understood, more subtle proteomic changes that determine the direction of a response once the complex is assembled remain much less clear. In addition, Complex II components undergo a wealth of post-translational changes that modify the functions of the complex components. This necessitates development of robust and efficient methods of isolating Complex II for further interrogation of its composition and the post-translational modifications of its components. This article describes several methods that we have developed for Complex II isolation, which can be used to obtain complementary information about this signaling mechanism. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation Basic Protocol 2: Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein Alternate Protocol: Alternative methods of immunoprecipitation of RIPK1 and other Complex-II-related factors Support Protocol: Generation of stable macrophage cell lines using lentiviral expression Basic Protocol 3: Use of proximity labeling to identify necrosome components in the detergent-insoluble fraction of the cell lysates.
一种包含 RIPK1、RIPK3 和 caspase-8 的大型蛋白质复合物，被称为复合物 II，已成为一系列先天免疫触发下游细胞死亡的关键介质之一。这种调节机制在巨噬细胞中起着重要作用，复合物 II 已与细胞凋亡、细胞焦亡和坏死以及炎症基因表达的增强有关。尽管该复合物的核心成分已经相当清楚，但对于复合物组装后决定反应方向的更细微的蛋白质组变化，仍知之甚少。此外，复合物 II 成分经历了大量的翻译后修饰，这些修饰改变了复合物成分的功能。这就需要开发强大而有效的复合物 II 分离方法，以进一步研究其组成和成分的翻译后修饰。本文描述了我们开发的几种用于复合物 II 分离的方法，这些方法可用于获得有关这种信号机制的互补信息。© 2021 威立出版社有限公司。基本方案 1：使用 FADD 免疫沉淀法从坏死性和细胞焦亡性巨噬细胞中分离复合物 II 基本方案 2：分离条件表达的 3XFLAG-RIPK1 蛋白形成的复合物 可选方案：RIPK1 和其他复合物 II 相关因子免疫沉淀的替代方法 支持方案：使用慢病毒表达生成稳定的巨噬细胞细胞系 基本方案 3：使用邻近标记法鉴定细胞裂解物中去污剂不溶性部分中的坏死体成分。
