MARCH5‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

Abstract

The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria‐associated E3 ligase, MARCH5. Myeloid cell‐specific March5 conditional knockout (March5 cKO) mice failed to secrete IL‐1β and IL‐18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL‐1β and IL‐18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27‐linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination‐defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL‐1β production. Thus, MARCH5‐dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3‐NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.

Acetylation is required for full activation of the NLRP3 inflammasome

“…Moreover, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE), a method for detecting large protein oligomers in studying prions 19 , 20 , blue native polyacrylamide gel electrophoresis (BN-PAGE), a method for detecting large native protein complexes in the mass range of 10 KDa to 10 MDa 21 , and live cell imaging were adopted to detect the NLRP3 oligomerization. In agreement with previous studies 20 , 22 , 23 , NLRP3 formed large oligomers upon nigericin stimulation, and the oligomerization was markedly inhibited by the K24R mutation of NLRP3 (Fig. 3d ).…”

Mechanism and role of mitophagy in the development of severe infection

“…Mitochondria and their components have a risk of causing uncontrolled inflammation while activating innate immune responses to resist infection. First, mitochondria supply an effective platform for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation: MAVS and cardiolipin on the OMM directly bind NLRP3 and pro-caspase 1, and mitochondrial E3 ligase MARCH5 ubiquitinates NLRP3 on K324 and K430 to facilitate NLRP3 oligomerization [ 92 ]. VDAC oligomers, mROS, and mtDNA assist in assembling NLRP3 inflammasome and activating caspase 1 [ 93 ].…”