14-3-3ε–dependent deubiquitination and translocation of NLRP3 activates the inflammasome during sepsis
Xingyu Li, Siqi Ming, Can Cao, Yating Xu, Jingxian Shu, Ning Tan, Xi Huang, Yongjian Wu
Xingyu Li, Siqi Ming, Can Cao, Yating Xu, Jingxian Shu, Ning Tan, Xi Huang, Yongjian Wu
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Research Article Infectious disease Inflammation

14-3-3ε–dependent deubiquitination and translocation of NLRP3 activates the inflammasome during sepsis

Abstract

The activation of the NLRP3 inflammasome is a pivotal step in hyperinflammation in sepsis; however, the regulatory mechanisms underlying its activation are not fully understood. In this study, we found that 14-3-3ε facilitates NLRP3 inflammasome activation by enhancing NLRP3 K63 deubiquitination and promoting its translocation to the mitochondria-associated ER membranes (MAMs) for full activation. Mass spectrometry revealed that 14-3-3ε binds to NLRP3 in macrophages during sepsis. Plasma 14-3-3ε levels were elevated in patients with sepsis and were positively associated with disease severity. 14-3-3ε promoted NLRP3 inflammasome activation by facilitating NLRP3 aggregation and NLRP3–ASC assembly. The interaction between 14-3-3ε and NLRP3 was dependent on phosphorylation at the S194 site of NLRP3 NACHT domain. The NLRP3–14-3-3ε interaction promoted K63 deubiquitination and enhanced the translocation of NLRP3 to MAMs, which is necessary for full activation of NLRP3 inflammasome. Furthermore, macrophage-conditional KO of 14-3-3ε or treatment with BV02, a 14-3-3 inhibitor, improved the survival rate and alleviated organ injuries in septic mice. Taken together, our data indicate that 14-3-3ε functions as a positive regulator of the NLRP3 inflammasome and could be a target for sepsis treatment.

Authors

Xingyu Li, Siqi Ming, Can Cao, Yating Xu, Jingxian Shu, Ning Tan, Xi Huang, Yongjian Wu

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Figure 6

14-3-3ε promotes the translocation of NLRP3 proteins to mitochondria-associated membranes (MAMs).

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14-3-3ε promotes the translocation of NLRP3 proteins to mitochondria-ass...
(A) iBMDMs were treated with LPS alone or co-treated with LPS and Nig. The cell lysate was used to isolate the membrane fractions via membrane flotation. The membrane markers (β-tubulin for cytosol, Cox-1 for inner mitochondrial membrane [mito], VDAC for outer mito, MFN-2 for MAMs), and calnexin for ER, 14-3-3ε, and NLRP3 in different fractions were probed with the indicated antibodies using Western blot. (B) iBMDMs were treated with LPS alone or cotreated with LPS and Nig. The cytosol, ER, MAMs, and mito were isolated using organelle isolation, and the levels of 14-3-3ε, NLRP3, and membrane markers in different fractions were detected using western blot analysis. (C) BMDMs were treated with LPS alone or cotreated with LPS and Nig, stained for FACL4 (red), and 14-3-3ε (green) and were observed by confocal microscopy. Scale bar: 5 μm. (D) The fluorescence intensity along the diagonal line from top left corner to the lower right corner of the bottom images in C were analyzed by ImageJ and were shown in the graphs. (E) BMDMs from 14-3-3εfl/fl or 14-3-3εfl/fl Lyz2Cre mice were induced and treated with LPS alone or cotreated with LPS and Nig. NLRP3 (red) and FACL4 (green) were stained and observed using confocal microscopy. Scale bar: 5 μm. (F) BMDMs from 14-3-3εfl/fl or 14-3-3εfl/fl Lyz2Cre mice were induced and treated with LPS and Nig. The cytosol, ER, MAMs, and mitochondria were isolated, and the protein levels of 14-3-3ε, NLRP3, and membrane markers were detected using western blot analysis. (G) HEK-293T cells were transfected with WT of S194A mutated Myc-tagged NLRP3 plasmids and treated with LPS and Nig. The cytosol, ER, MAMs, and mitochondria were isolated and detected using Western blot.