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Published on: May 21, 2018
The hydrophobicity of the CARD8 N-terminus tunes inflammasome activation
Lydia P Tsamouri1, Jeffrey C Hsiao1, Qinghui Wang2
1Pharmacology Program of the Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Insights
Hydrophobic modifications of CARD8 inflammasome by antioxidants directly activate it. These modifications destabilize the CARD8 N-terminal region, promoting inflammasome activation and indicating cysteines
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Proteotoxic stress is a known activator of the CARD8 inflammasome.
- The precise mechanisms and signals controlling CARD8 inflammasome activation remain incompletely understood.
- Hydrophobic radical-trapping antioxidants (RTAs) were previously found to potentiate CARD8 inflammasome activation via an unknown pathway.
Purpose of the Study:
- To elucidate the mechanism by which hydrophobic RTAs potentiate CARD8 inflammasome activation.
- To identify the specific molecular targets and modifications involved in RTA-mediated CARD8 activation.
- To investigate the role of cysteine residues in CARD8 regulation and response to proteotoxic stress.
Main Methods:
- Chemical modification assays to detect alkylation of CARD8 by RTAs.
- Western blotting and proteasome inhibition assays to assess CARD8 protein stability and degradation.
- Inflammasome activation assays (e.g., IL-1β release) in response to RTAs, electrophiles, and CARD8 mutants.
- Site-directed mutagenesis to replace CARD8 cysteine residues with isoleucines.
Main Results:
- Hydrophobic RTAs, such as JSH-23, directly alkylate cysteine residues in the N-terminal disordered region of CARD8.
- This alkylation destabilizes the repressive N-terminal fragment of CARD8, leading to its proteasomal degradation.
- The degradation releases the CARD8 C-terminal fragment from autoinhibition, thereby activating the inflammasome.
- Unrelated hydrophobic electrophiles and mutation of CARD8 cysteines to isoleucines mimicked the RTA-induced potentiation of inflammasome activation.
Conclusions:
- The N-terminal cysteines of CARD8 are critical targets for hydrophobic modifications that regulate inflammasome activation.
- Hydrophobic modifications and subsequent degradation of the CARD8 N-terminus represent a novel mechanism for CARD8 inflammasome activation, linked to protein folding stress.
- These findings highlight the role of CARD8 N-terminal cysteines in sensing and responding to proteotoxic stress, offering new insights into inflammasome regulation.
Abstract:
Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.
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