Immunoprecipitation Strategies to Isolate RIPK1/RIPK3 Complexes in Mouse Macrophages
Ioannis Siokas1, Dingqiang Zhang1, Alexander Poltorak2
1Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, Massachusetts.
Abstract:
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.
Insights
This study details methods for isolating Complex II, a protein complex crucial for cell death and inflammation in macrophages. These techniques enable deeper investigation into its composition and post-translational modifications, advancing our understanding of innate immunity signaling.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Complex II, comprising RIPK1, RIPK3, and caspase-8, is a key mediator of cell death and inflammation in macrophages.
- While core components are known, subtle proteomic changes and post-translational modifications influencing Complex II function remain unclear.
- Robust methods for isolating Complex II are needed to study its composition and modifications.
Purpose of the Study:
- To develop and describe robust and efficient methods for isolating Complex II from macrophages.
- To enable further interrogation of Complex II composition and post-translational modifications.
- To provide complementary information about this critical innate immune signaling mechanism.
Main Methods:
- Isolation of Complex II in necroptotic and pyroptotic macrophages via FADD immunoprecipitation.
- Isolation of complexes formed by conditionally expressed 3XFLAG-RIPK1 protein.
- Proximity labeling to identify necrosome components in detergent-insoluble fractions.
Main Results:
- Several methods for Complex II isolation have been developed and are described.
- These methods allow for the study of Complex II in different cellular contexts, including necroptosis and pyroptosis.
- The described protocols facilitate the investigation of protein complexes involved in innate immune signaling.
Conclusions:
- The developed isolation methods provide valuable tools for studying Complex II.
- These techniques will aid in elucidating the subtle proteomic and post-translational changes that regulate cell death and inflammation.
- Further research into Complex II composition and function is facilitated by these robust isolation protocols.
