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FAAH served a key membrane-anchoring and stabilizing role for NLRP3 protein independently of the endocannabinoid system

Abstract

NLRP3, the sensor protein of the NLRP3 inflammasome, plays central roles in innate immunity. Over-activation of NLRP3 inflammasome contributes to the pathogenesis of a variety of inflammatory diseases, while gain-of-function mutations of NLRP3 cause cryopyrin-associated periodic syndromes (CAPS). NLRP3 inhibitors, particularly those that inhibit inflammasome assembly and activation, are being intensively pursued, but alternative approaches for targeting NLRP3 would be highly desirable. During priming NLRP3 protein is synthesized on demand and becomes attached to the membranes of ER and mitochondria. Here, we show that fatty acid amide hydrolase (FAAH), the key integral membrane enzyme in the endocannabinoid system, unexpectedly served the critical membrane-anchoring and stabilizing role for NLRP3. The specific interaction between NLRP3 and FAAH, mediated by the NACHT and LRR domains of NLRP3 and the amidase signature sequence of FAAH, was essential for preventing CHIP- and NBR1-mediated selective autophagy of NLRP3. Heterozygous knockout of FAAH, resulting in ~50% reduction in both FAAH and NLRP3 expression, was sufficient to substantially inhibit the auto-inflammatory phenotypes of the NLRP3-R258W knock-in mice, while homozygous FAAH loss almost completely abrogates these phenotypes. Interestingly, select FAAH inhibitors, in particular URB597 and PF-04457845, disrupted NLRP3–FAAH interaction and induced autophagic NLRP3 degradation, leading to diminished inflammasome activation in mouse macrophage cells as well as in peripheral blood mononuclear cells isolated from CAPS patients. Our results unraveled a novel NLRP3-stabilizing mechanism and pinpointed NLRP3–FAAH interaction as a potential drug target for CAPS and other NLRP3-driven diseases.

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Fig. 1: Loss of FAAH induced NLRP3 degradation.
Fig. 2: FAAH knockout inhibited the auto-inflammatory phenotype of NLRP3-R258W mice.
Fig. 3: NLRP3 degradation induced by FAAH loss proceeds through selective autophagy.
Fig. 4: FAAH interacted with NLRP3 and anchored NLRP3 to mitochondria and ER membranes.
Fig. 5: FAAH interacted with CAPS-mutated NLRP3 and anchored NLRP3 to ER and mitochondria membranes.
Fig. 6: URB597 disrupted NLRP3–FAAH interaction and induced NLRP3 degradation.
Fig. 7: URB597 and PF-04457845 inhibited CAPS-associated mutant NLRP3 inflammasome activation through disrupting mutant NLRP3–FAAH interaction.

Data availability

All data are presented in the main text or Supplementary Materials. The expression plasmids reported in this paper are available upon request. The rest of the data supporting the present study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank Dr. Warren Strober for sharing the Nlrp3R258W mouse line and Prof. Zhexiong Lian for assistance on mice studies.

Funding

This work was supported by National Natural Science Foundation of China (82150118, 32171375, 32071398, T2222014), Key-Area Research and Development Program of Guangdong Province (2020B0101030006, 2020B1515120096, 2022B0202010002), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2017ZT07S054), and the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2018B030306035).

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YYZ, ZH, HWM, SQZ, YBH, SRC, YK, ZBZ, XHH, ZY, YH, XWH, MMW, and WBZ performed the experiments; LJ, and GXM designed the research; XHQ, ZMY, JNS, SY, and YCX wrote the paper; LSW, YJZ, and LPW supervised the project.

Corresponding authors

Correspondence to Liansheng Wang, Yunjiao Zhang or Longping Wen.

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All mice were housed in a specific pathogen-free facility in the Laboratory Animal Center of South China University of Technology. The experiments using patient-derived PBMCs have complied with all relevant ethical regulations with the informed consent from the patients. The experiment was ethically approved by the Department of Rheumatology and Immunology, Beijing Children’s Hospital of Capital Medical University.

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Zhu, Y., Zhang, H., Mao, H. et al. FAAH served a key membrane-anchoring and stabilizing role for NLRP3 protein independently of the endocannabinoid system. Cell Death Differ (2022). https://doi.org/10.1038/s41418-022-01054-4

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