A Single Amino Acid Residue Defines the Difference in NLRP3 Inflammasome Activation between NEK7 and NEK6.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a critical component of the innate immune system that is activated by microbial infections and cellular stress signals. The molecular mechanism of NLRP3 inflammasome activation remains not fully understood. As an NLRP3-interacting partner, NEK7 has emerged as a critical mediator for NLRP3 inflammasome activation. In contrast to NEK7, NEK6, the closely related member of the NEK family, does not support NLRP3 inflammasome activation. In this study, we show that the mouse NEK7 catalytic domain, which shares high sequence identity with the counterpart of NEK6, mediates its interaction with NLRP3 and inflammasome activation in mouse macrophages. Within their catalytic domains, a single amino acid residue at a corresponding position (R121NEK7, Q132NEK6) differentiates their function in NLRP3 inflammasome activation. Surprisingly, substitution of the glutamine residue to an arginine residue at position 132 confers NEK6 the ability of NLRP3 binding and inflammasome activation in mouse macrophages. Furthermore, our results suggest a structural pocket surrounding the residue R121 of NEK7 that is essential for NLRP3 binding and inflammasome activation.

The leucine-rich repeat (LRR) domain of NLRP3 is required for NLRP3 inflammasome activation in macrophages.

The NLRP3 inflammasome is a critical component of innate immunity that defends the host from microbial infections. However, its aberrant activation contributes to the pathogenesis of several inflammatory diseases. Activation of the NLRP3 inflammasome induces the secretion of proinflammatory cytokines IL-1ß and IL-18 and pyroptotic cell death. NLRP3 contains a leucine-rich repeat (LRR) domain at its C terminus. Although posttranslational modifications in this LRR domain have been shown to regulate NLRP3 inflammasome activation, the role of the entire LRR domain in NLRP3 inflammasome activation remains controversial. Here, we generated mouse macrophages that express an endogenous NLRP3 mutant lacking the LRR domain. Deletion of the LRR domain diminished NLRP3 inflammasome activation in macrophages. Furthermore, using NLRP3-deficient macrophages that are reconstituted with NLRP3 mutants lacking the LRR domain, we found that deletion of the LRR domain inhibited NLRP3 inflammasome activation. Mechanistically, deletion of the LRR domain inhibited NLRP3 self-association, oligomerization, and interaction with the essential regulator NEK7. Our results demonstrate a critical role for the LRR domain in NLRP3 inflammasome activation.

The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation.

The NLRP3 inflammasome is a critical component of the innate immune system that mediates caspase-1 activation and the secretion of proinflammatory cytokines IL-1ß/IL-18 in response to microbial infection and cellular damage. However, the aberrant activation of the NLRP3 inflammasome has been linked with several inflammatory disorders, which include cryopyrin-associated periodic syndromes, Alzheimer's disease, diabetes, and atherosclerosis. The NLRP3 inflammasome is activated by diverse stimuli, and multiple molecular and cellular events, including ionic flux, mitochondrial dysfunction, and the production of reactive oxygen species, and lysosomal damage have been shown to trigger its activation. How NLRP3 responds to those signaling events and initiates the assembly of the NLRP3 inflammasome is not fully understood. In this review, we summarize our current understanding of the mechanisms of NLRP3 inflammasome activation by multiple signaling events, and its regulation by post-translational modifications and interacting partners of NLRP3.

Assessment of NLRP3 Inflammasome Activation and NLRP3-NEK7 Complex Assembly.

The NLRP3 inflammasome is a critical component of innate immunity that activates caspase-1 to induce inflammation in response to a wide spectrum of endogenous and exogenous stimuli. NLRP3 inflammasome activation has been shown by assays for the cleavage of caspase-1 and gasdermin D, the maturation of IL-1ß and IL-18, and ASC speck formation in innate immune cells such as macrophages and monocytes. Recently, NEK7 has been revealed as an essential regulator for NLRP3 inflammasome activation by forming high-molecular-weight complexes with NLRP3. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used to study multi-protein complexes in many experimental systems. Here, we provide a detailed protocol to detect NLRP3 inflammasome activation and NLRP3-NEK7 complex assembly in mouse macrophages by Western blot and BN-PAGE.