Figure 7. MARCH5‐mediated ubiquitination of NLRP3 is essential for inflammasome activation.

• A
  HEK293T cells were transfected with NLRP3 WT or indicated Lys mutants and ASC. Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin for 30 min. The percentage of cells with an ASC speck was quantified after Confocal microscopy. At least 100 cells were analyzed. Values are the mean ± SD. Values, **P < 0.01, ***P < 0.001 (two‐tailed Student's t‐test). Bar, 10 μm.
• B
  HEK293T cells were transfected with ASC and NLRP3 (WT or individual NLRP3 mutants). Cells were treated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cell lysates were incubated with 2 mM DSS for cross‐linking. Triton X‐100 insoluble pellets and soluble fraction lysates were detected by immunoblotting with the indicated antibodies.
• C
  NLRP3 inflammasome‐reconstituted HEK293T cells with NLRP3 WT or indicated mutants were stimulated for 4 h with 200 ng/ml LPS and 30 min with 15 μM nigericin. Harvested cells were detected by immunoblotting with the indicated antibodies.
• D
  Culture supernatants were obtained from (C) and subjected to ELISA to quantify secreted IL‐1β. Representative data are shown from independent experiments that were repeated at least three times. Values are the mean ± SD. *P < 0.05, ***P < 0.001 (two‐tailed Student's t‐test)
• E
  Schematic diagram of NLRP3 inflammasome activation by MARCH5. Upon activation by a priming signal, NLRP3 associates with MAVS on mitochondria ①. MARCH5 recognizes and ubiquitinates two lysine sites, K324 and K430, on the NLRP3 NACHT domain via K27‐linked polyubiquitination ②. NLRP3 ubiquitination by MARCH5 promotes the recruitment of NEK7 and enables self‐oligomerization ③. As a result, ASC and pro‐caspase‐1 are recruited to oligomerized NLRP3 ④ for NLRP3 inflammasome assembly ⑤ and trigger the activation of caspase‐1 and pro‐cytokine cleavage ⑥.

Source data are available online for this figure.