Abstract
The apoptosis-associated speck-like protein with a caspase-recruitment domain (ASC) adaptor protein bridges inflammasome sensors and caspase-1. Upon inflammasome activation, ASC nucleates in a prion-like manner into a large and single platform responsible for the recruitment and the activation of caspase-1. Active caspase-1 will in turn promote the proteolytic maturation of the pro-inflammatory cytokine IL-1β. ASC oligomerization is direct evidence for inflammasome activation and its detection allows a read-out independent of caspase-1 and IL-1β. This protocol describes how to detect the oligomerization of ASC by Western blot.
Keywords: Inflammasome, Caspase-1, Pyroptosis, Biochemistry, Auto-inflammation
Background
Inflammasomes are large multiprotein platforms that sense a variety of microbial, endogenous and environmental stressors leading to the maturation of the pro-inflammatory IL-1 family of cytokines (Martinon et al., 2002; Sharma and Kanneganti, 2016). Upon activation, inflammasome sensors recruit the adaptor protein ASC through pyrin domain (PYD)-PYD homotypic interactions. ASC will in turn bind to caspase-1 via caspase activation and recruitment domain (CARD)-CARD interactions and favor auto-proteolytic cleavage of caspase-1, leading to maturation of IL-1β and IL-18 (Hoss et al., 2016). Inflammasome activation triggers supramolecular oligomerization of ASC dimers into large interweaving fibrils also termed ‘ASC-specks’ or ‘pyroptosome’ (Fernandes-Alnemri et al., 2007). ASC-speck/pyroptosomes is a hallmark of inflammasome activation that correlates with caspase-1 cleavage and release of mature IL-1β (Dick et al., 2016). Recently we showed that Nelfinavir, an HIV-protease inhibitor, promotes the release of self-DNA into the cytosol, activates the DNA sensing inflammasome AIM2 and subsequent ASC oligomerization (Di Micco et al., 2016). This protocol aims at detecting endogenous ASC oligomerization in immortalized bone marrow-derived macrophages (iBMDMs) by Western blot analysis. It is adapted from the publication of Fernandes-Alnemri et al. (2007) that used this technique to detect ASC oligomerization in THP-1 cells.
Materials and Reagents
Equipment
Procedure
Data analysis
Immunoreactive bands for ASC oligomers appear at molecular weights corresponding to ASC monomers, dimers, and higher orders of oligomers. A positive control for ASC expression in the total cell lysates of each condition needs to be done. Other positive controls for equal protein loading such as tubulin can be included. Three independent experiments should be done. An example of data analysis can be found in Figure 1 and in Figure 3A of (Di Micco et al., 2016). In this study immortalized bone marrow-derived macrophages (iBMDMs) left unchallenged (lane 1) or challenged with NLRP3 inflammasome activator Nigericin (lane 2) or AIM2 inflammasome activators poly(dA:dT) (lane 3) and Nelfinavir (lane 4). In the first lane, resting iBMDMs did not exhibit ASC oligomerization in the pelleted fraction. In lanes 2, 3 and 4, iBMDMs challenged with inflammasome activators exhibited strong ASC oligomerization in their cross-linked pellets. Figure 1. BMDMs were primed with LPS and treated for 6 h with vehicle, DMSO (Lane1) Nelfinavir, (Lane 2), Lipofectamine (Lipo) (lane 3) Lipofectamine plus poly(dA:dT) (Lane 4), as indicated. Cross-linked pellets (Pell) or soluble lysates (Lys) were immunoblotted for ASC or caspase-1. More details on the experiment can be found in Di Micco et al., 2016.
Notes
Recipes
Acknowledgments
This work was supported by European Research Council Starting Grant 281996 and is adapted from (Di Micco et al., 2016).
References
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