The NLRP3 (Nod-like receptor protein 3) inflammasome is a key sensor of tissue damage and pathogen infection. Exposure of immune cells to any of a plethora of toxicants causes mitochondrial dysfunction, which leads to inflammasome activation. Studies have connected release of oxidized DNA from the mitochondria to NLRP3 inflammasome activation. Indeed, oxidized DNA does co-precipitate with NLRP3 upon immune cell stimulation. NLRP3 inflammasome activation is supposed to be protective, but inability of the host to clear the infection leaves NLRP3 active, where its over-production of IL-1β and activation of gasdermin D leads to pyroptosis. This establishes a cytokine storm which can lead to septic shock and ultimately organ failure in severe cases. Humans harboring one of about a hundred NLRP3 point mutations, collectively called cryopyrin associated periodic syndrome (CAPS), have variably active NLRP3 which contributes to increased pathogenesis. Patients with CAPS are more susceptible to NLRP3 activators. For example, NLRP3 is activated in diets containing excessive high-fructose and aspirin. This leads to non-alcoholic steatohepatitis (NASH) and fatty liver disease (NAFLD) which can progress to hepatocellular carcinoma.

 Human glycosylase (hOGG1) serves to excise 8-oxo-deoxyguanine from oxidized DNA. Since reactive oxygen species lead to NLRP3 inflammasome activation, amelioration of oxidized DNA via hOGG1 inhibits inflammasome activation. Instead of repairing each oxidized base, macrophages treat their mitochondrial as expendable and degrade them, so-called mitophagy. Mitophagy inhibits inflammasome activation.

 The full-length structures of oligomerized NLRP3 has been recently determined. Human NLRP3 assembles as a decamer, formed by two back-to-back pentamers. There is also a cryo-EM structure of NLRP3 lacking its pyrin domain, with the inhibitor MCC950 bound to the NACHT domain. There are also crystal structures of human glycosylase bound to oxidized DNA and 8-oxodG nucleoside.

 In our current work, we purified NLRP3 protein and tested if it could bind oxidized and non-oxidized mitochondrial D-Loop DNA. We developed an assay in which Streptavidin Dyna beads were first bound to biotinylated oxidized or non-oxidized DNA. Purified NLRP3 was incubated and bound to DNA on the beads. Next, label-free competitor DNA of the same sequence was added to elute protein from the beads, generating a new protein-DNA complex. We analyzed the bound fraction remaining on the beads and found we could elute NLRP3 with increasing concentrations of oxidized or non-oxidized DNA. Interestingly, wildtype NLRP3 had a preference for non-oxidized DNA over oxidized DNA. The neonatal onset multisystem inflammatory disease (NOMID) mutant preferred oxidized DNA. Given that NOMID is the most severe CAPS mutant, it is logical that it responds more to oxidized DNA, which activates the NLRP3 inflammasome.

 Sequence comparison of NLRP3 to both bacterial and human glycosylase illustrate a small stretch of sequence conservation. The bacterial glycosylase maps to the NACHT domain while the human glycosylase maps almost entirely to the pyrin domain. We show that amino acids that are required for the glycosylase to interact with oxidized DNA are either exactly the same or very similar within NLRP3. We then mapped the DNA-binding sequence homology to the 3D structures of the proteins and superposed NLRP3 pyrin domain with that of hOGG1 using ChimeraX Matchmaker. We show that the human glycosylase helix structure and position in 3D space very similar to NLRP3. This indicates the proteins share a protein fold.

We also examined the interaction between NLRP3 and oxidized DNA using electromobility shift assays (EMSA’s). Although we could see a DNA shift, we could not see the protein on a western using a monoclonal antibody. We empirically increased DNA concentration while maintaining a high protein concentration. We found that NLRP3 protein was visible only at low concentrations of DNA. High concentrations of DNA created a shadow where the DNA shift was, only allowing us to see protein not bound to DNA. This showed our monoclonal antibody which targets the pyrin domain could not bind when DNA was present. Analysis using an antibody against the NACHT domain restored protein intensity.

 To better understand the portion of NLRP3 that is interacting with oxidized DNA, we performed site-directed mutagenesis to generate two constructs: one lacking the pyrin domain (NACHT-LRR) and one with isolated pryin_(1-93) domain. We found NLRP3 lacking the pyrin domain could still bind either oxidized or non-oxidized DNA.  The NACHT domain does not share 3D structural homology with glycosylase but does have sequence homology with bacterial glycosylase. Moreover, analysis of the electrostatic surface potential for NLRP3 showed positive charge spanning from the linker region to the back shoulder of the NACHT domain, which could bind negatively charged DNA. The NLRP3 pyrin _(1-93) domain construct showed a strong preference for oxidized DNA.

On the basis of the data shown herein, we propose NLRP3 has two sites capable of interacting with oxidized DNA, one on the NACHT domain and one on the pyrin domain. The protein fold for 3 helices of human glycosylase hOGG1 resembles NLRP3 pyrin. Data supports that the pyrin domain is likely to interact with oxidized DNA in a similar way as human glycosylase.

 References:

1. Radom, C. T., Banerjee, A. & Verdine, G. L. Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations. J Biol Chem 282, 9182-9194, doi:10.1074/jbc.M608989200 (2007).
   
   
2. Xian, H. et al. Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling. Immunity 55, 1370-1385 e1378, doi:10.1016/j.immuni.2022.06.007 (2022).
   
   
3. Hochheiser, I. V. et al. Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3. Nature 604, 184-189, doi:10.1038/s41586-022-04467-w (2022).