Molecular Mechanism for p202-Mediated Specific Inhibition of AIM2 Inflammasome Activation

Abstract

SUMMARY Mouse p202 containing two HIN domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found only HIN1 of p202 binds dsDNA, while HIN2 forms a homo-tetramer. Crystal structures of HIN1 revealed that dsDNA is bound on the opposite face to the site used in AIM2 and IFI16. The structure of HIN2 revealed a dimer of dimers, with the face analogous to the HIN1 dsDNA binding site being a dimerization interface. Electron microscopy imaging showed that HIN1 is flexibly linked to HIN2 in p202, and tetramerization provided enhanced avidity for dsDNA. Surprisingly, HIN2 of p202 interacts with AIM HIN domain. We propose this results in spatial separation of AIM2 pyrin domains, and indeed p202 prevented dsDNA-dependent clustering of ASC and AIM2 inflammasome activation. We hypothesize that while p202 was evolutionarily selected to limit AIM2-mediated inflammation in some mouse strains, the same mechanism contributes to increased interferon production and lupus susceptibility.

Deficient NLRP3 and AIM2 Inflammasome Function in Autoimmune NZB Mice

“…AIM2-dependent cell death was limited by high expression in NZB cells of the related PYHIN family member p202 (42), which has been suggested as a lupus-susceptibility factor (43). p202 binds directly to AIM2 and prevents downstream ASC oligomerization (42). In this article, we extend this observation to show defects in the AIM2 response to a viral pathogen, and surprisingly, a complete lack of NLRP3 inflammasome function in the NZB strain.…”

“…Proteins were transferred to polyvinylidene difluoride (Millipore) using a mini-trans blot system (Bio-Rad) with Tris-Glycine transfer buffer containing 10-20% methanol. Immunoblot analysis was conducted as previously described (42). Primary Abs used included rabbit polyclonals against caspase-1 p10 (M-20; Santa Cruz Biotechnology), ASC (N-15; Santa Cruz Biotechnology), fulllength caspase-3 (9662; Cell Signaling Technologies), cleaved caspase-3 (9661; Cell Signaling Technologies) and AIM2 (made to full-length recombinant mouse AIM2 and tested on Aim2 2/2 extracts), rabbit monoclonal against S6 ribosomal protein (5G10; Cell Signaling Technologies), goat polyclonals against mouse IL-1b (AF-401-NA; R&D Systems) and p202 (S-19; Santa Cruz Biotechnology), mouse monoclonals against NLRP3 (Cryo-2; Adipogen), caspase-1 p20 (Casper-1; Adipogen), S6 ribosomal protein (54D2; Cell Signaling Technologies) and a-Tubulin (B-5-1-2; Sigma-Aldrich).…”

“…Our previous work showed that NZB mouse macrophages are deficient in cell death mediated by AIM2 inflammasome recognition of cytosolic DNA (41,42). AIM2-dependent cell death was limited by high expression in NZB cells of the related PYHIN family member p202 (42), which has been suggested as a lupus-susceptibility factor (43). p202 binds directly to AIM2 and prevents downstream ASC oligomerization (42).…”

The emerging role of human PYHIN proteins in innate immunity: Implications for health and disease

“…p202 lacks a PYD but possess two DNA binding HIN domains, the first of which is capable of sequestering cytosolic dsDNA. Interestingly, the second HIN domain of p202 binds the HIN domain of AIM2, resulting in spatial separation of AIM2 PYD and the prevention of ASC clustering [53]. While no known human PYHIN possesses a similar domain architecture to mouse p202, alternatively spliced isoforms of human AIM2 have been predicted [54].…”

A Novel Flow Cytometric Method To Assess Inflammasome Formation

“…Protein extracts and immunoblot analysis were conducted, as previously described (16). AIM2 was detected using the anti-AIM2 MAb 3B10 (17), and loading was assessed with immunoblotting for tubulin using anti-atubulin, clone B-5-1-2 (Sigma-Aldrich).…”