Mitochondrial DNA release into the cytosol is a critical event in innate immune activation, often acting as a damage-associated molecular pattern (DAMP) that triggers inflammasome assembly. Here, we demonstrate that NLRP3 is involved in the release of D-loop mtDNA into the cytosol. We further show that NLRP3 interacts with NLRP10. NLRP10-mediated oxidized DNA cleavage involves a Schiff base intermediate and is inhibited by small molecules known to inhibit glycosylases. These findings support a model where NLRP10 interaction with oxidized DNA may contribute to long-term senescence secretory phenotype and modulate inflammasome activation. Our study highlights a novel mechanism by which NLRP10 can respond to mitochondrial stress signals to influence innate immunity and suggests therapeutic potential for targeting these interactions in inflammatory diseases. The cytosolic innate immune sensor NLRP10 interacts with NLRP3 and mediates the cleavage of oxidized mitochondrial DNA

Metabolism is known to influence cell identity, but the underlying mechanisms remain unclear. Here we reveal spatiotemporal dynamics of phosphofructokinase 1 (PFK1), a key glycolytic enzyme, within the skeletal muscle lineage. The expression of PFKM (the muscle isoform of PFK1) is low in muscle stem cells and increases during differentiation. Mechanistically, Wnt signalling rapidly induces lysosomal degradation of PFKM through a methyl arginine degron motif, which gets selectively methylated by the protein arginine methyltransferase (PRMT1) and delivered to lysosomes through microautophagy. PFKM degradation shifts glucose metabolism from glycolysis to the pentose phosphate pathway. PFKM overexpression increases glycolysis and promotes differentiation into terminally differentiated myofibres. On the other hand, PFKM knockdown blunts differentiation, which can be rescued by supplementation with the downstream glycolytic intermediate 3-phosphoglycerate. In sum, our findings highlight the importance of compartmentalized metabolism in cell fate decisions.

Abstract Aberrant activation of the NLRP3 inflammasome contributes to a wide range of chronic inflammatory disorders. Here, we investigate small-molecule inhibitors originally developed to target the DNA repair enzyme hOGG1 and demonstrate their ability to inhibit NLRP3 activation in human cells. These compounds, including TH5487 (IC50 1.62 µM in human PBMCs), reduce IL-1β secretion while increasing type I interferon responses. Cryo-EM reveals direct association between NLRP3 and mitochondrial DNA, while structural modeling predicts interaction with oxDNA. Notably, inhibitors of the DNA repair glycosylase hOGG1 remain effective in L353P mutant PBMCs from FCAS patients and L351P in mice, at doses where the canonical NLRP3 inhibitor MCC950 is ineffective. Our findings uncover an additional druggable mechanism for inflammasome regulation via interference with oxidized DNA sensing, offering innovative therapeutic opportunities for autoinflammatory disease.

The NLRP3 inflammasome plays a central role in innate immunity and is activated in response to mitochondrial dysfunction and oxidized DNA. Here, we demonstrate that repurposed small-molecule inhibitors originally developed for DNA glycosylases, TH5487 and SU0268, potently inhibit NLRP3 activation ex vivo in human Peripheral Blood Mononuclear Cells (PBMCs) with IC 50 of 1.62 µM and 3.24 µM, respectively. We show that these inhibitors prevent mitochondrial localization of NLRP3 and directly block inflammasome assembly. They also reshape the immune landscape decreasing IL-1β, while increasing IFN-β. Structural and biophysical analyses reveal a two-site DNA binding model in which NLRP3 engages oxidized DNA with a KD1 of 0.268 nM and KD2 3.02 nM. Importantly, these inhibitors block IL-1β secretion in L353P Familial Cold Autoinflammatory Syndrome (FCAS) patient PBMCs where MCC950 fails, demonstrating the therapeutic potential for inflammasome-driven diseases. Together, our findings reveal a novel druggable mechanism of inflammasome inhibition through interference with oxidized DNA sensing and localization, offering new opportunities for treatment of chronic inflammatory disorders.

Staphylococcus aureus α-hemolysin (Hla) is a major virulence factor that utilizes cell surface ADAM10 to oligomerize and form a functional heptameric pore. We show here that Hla from strain USA300 is required to induce IL-1β secretion by neutrophils and to cause severe corneal disease in mice. We also demonstrate that in contrast to USA300 and other clonal complex 8 (CC8) methicillin resistant S. aureus (MRSA) isolated from the skin, CC5 Hla from corneas of infected patients have single nucleotide polymorphisms (SNP) that result in two amino acid substitutions, D208E (Asp-Glu) and I275T (Ile-Thr). Structural modeling predicts CC5 Hla self-assembly and altered binding to ADAM10 that is distinct from CC8 Hla. The ADAM10 inhibitor GI254023X blocked neutrophil IL-1β secretion induced by Hla-expressing CC8, but not by CC5 conditioned media, indicating that these Hla polymorphisms play an important role in Hla receptor binding and neutrophil IL-1β secretion, and affect corneal disease severity.

The NLRP3 inflammasome enables release of mitochondrial DNA to circulation. Circulating oxidized mitochondrial DNA generated in response to NLRP3 inflammasome activation functions as an alarmin that contributes to the maintenance of systemic inflammation. The discovery that NLRP3 could cleave oxidized mtDNA led to repurposed chemical inhibitors that dually target NLRP3 and DNA glycosylase OGG1, resulting in pro-survival type-1 interferon. Using molecular dynamics and immunology we show that RM1mAb, a full-length monoclonal antibody targeting the NLRP3 pyrin domain, can prevent NLRP3 from interacting with mitochondrial DNA. We further illustrate RM1mAb can exploit FCγRs for cell entry and avoid destruction by the lysosomal pathway to inhibit IL-1β secretion in peripheral mononuclear blood cells (PBMCs) isolated from patients with Familial Cold Autoinflammatory Syndrome harboring NLRP3 L353P gain of function mutation. We show RM1mAb and repurposed inhibitor TH5487 synergistically inhibit inflammasome activation. These findings illustrate the promise of exploiting FCγRs as a means of IgG entry to target cytosolic proteins and improve human health. One-Sentence Summary:RM1mAb and TH5487 synergistically inhibit inflammasome activation in human FCAS PBMCs.

Recent discoveries have emphasized the critical role of oxidized DNA (ox-DNA) in inflammation and immune regulation. Produced during oxidative stress from infection or tissue damage, ox-DNA activates signaling pathways that drive the release of proinflammatory cytokines, specifically engaging the NLRP3 inflammasome, a key player in cytokine maturation and host defense. NLRP3 is increasingly implicated in inflammatory and autoimmune diseases, with ox-DNA recognized as a central activator of this inflammasome. This review examines the role of ox-DNA in inflammasome activation, its broader impact on inflammatory processes, and promising therapeutic approaches targeting ox-DNA through both immunological and structural lenses. These insights highlight ox-DNA’s relevance in inflammation and offer potential avenues for the treatment of a range of immune-related disorders.

Mitochondrial DNA (mtDNA) release into the cytosol is a critical event in innate immune activation, often acting as a damage-associated molecular pattern (DAMP) that triggers inflammasome assembly. Here, we demonstrate that NLRP3 plays a direct role in cleaving and facilitating the release of D-loop mtDNA into the cytosol. We further show that NLRP3 interacts with NLRP10. NLRP10-mediated ox-DNA cleavage involves a Schiff base intermediate and is inhibited by small molecules known to inhibit glycosylases. These findings support a model where NLRP10 interaction with oxidized DNA may contribute to long-term senescence secretory phenotype and modulate inflammasome activation. Our study highlights a novel mechanism by which NLRP10 can respond to mitochondrial stress signals to influence innate immunity and suggests therapeutic potential for targeting these interactions in inflammatory diseases.

The NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is a megadalton complex implicated in numerous inflammation-driven diseases including COVID-19, Alzheimer's disease, and gout. Although past efforts have focused on inhibiting IL-1β downstream of NLRP3 activation using drugs such as canakinumab, no FDA-approved NLRP3-targeted inhibitors are currently available. MCC950, a direct NLRP3 inhibitor, showed promise but exhibited off-target effects. Recent research has focused on optimizing the sulfonylurea-based MCC950 scaffold by leveraging recent structural and medicinal chemistry insights into the NLRP3 nucleotide-binding and oligomerization (NACHT) domain to improve solubility and clinical efficacy. In addition, oxidized DNA (oxDNA) has emerged as a key inflammasome trigger, and molecules targeting the pyrin domain have shown promise in inhibiting NLRP3 activation. This review discusses the role of NLRP3 in inflammation-related diseases, the status of ongoing clinical trials, and emerging small-molecule therapeutics targeting NLRP3.

Concatemeric viral DNA is packaged into bacteriophage P22 procapsids via a headful packaging mechanism mediated by a molecular machine consisting of small (gp3) and large (gp2) terminase subunits. Although a negative stain reconstruction exists for the terminase holoenzyme, it is not clear how this complex binds the dodecameric portal protein located at a 5-fold mismatch vertex. Herein, we describe new assemblies for the holoenzyme. Both native mass spectrometry and transmission electron microscopy reveal that the P22 terminase complex adopts three main assemblies, which include a nonameric S-terminase bound to two L-terminase 1(gp3)9:2(gp2), two nonameric S-terminase bound to five L-terminase 2(gp3)9:5(gp2), and three nonameric S-terminase bound to seven L-terminase 3(gp3)9:7(gp2). Native agarose gel electrophoresis shows that the terminase complex interacts with procapsids with mild crosslinking. These results herein illustrate the P22 terminase complex can adopt a variety of conformations and assembly states.

The NLRP3 inflammasome plays a pivotal role in orchestrating the innate immune response during sterile tissue injury and is implicated in various chronic and acute diseases. Diverse physical and chemical agents including oxidized DNA can activate the NLRP3 inflammasome. We use cryo-EM and other biophysical techniques to elucidate the molecular mechanism of NLRP3 stimulation by oxidized DNA. In this study, we investigate the distinctive binding of NLRP3 to non-oxidized (non-ox) and oxidized (ox) mitochondrial DNA (mtDNA).

Interactions between proteins and small molecules or nucleic acids play a pivotal role in numerous biological processes critical for human health and are fundamental for advancing our understanding of biological systems. Proteins are the workhorses of the cell, executing various functions ranging from catalyzing biochemical reactions to transmitting signals within the body. Small molecules, including drugs and metabolites, can modulate protein activity, thereby impacting cellular processes and disease pathways. Similarly, nucleic acids, such as DNA and RNA, regulate protein synthesis and function through intricate interactions. Understanding these interactions is crucial for drug discovery and development and can shed light on gene regulation, transcriptional control, and RNA processing, providing insights into genetic diseases and developmental disorders. Moreover, studying protein-small molecule and protein-nucleic acid interactions enhances our comprehension of fundamental biological mechanisms. A wide array of methods to study these interactions range in cost, sensitivity, materials usage, throughput, and complexity. Notably in the last decade, new techniques have been developed that enhance our understanding of these interactions. In this review, we aim to summarize the new state-of-the-art methods for detecting interactions between proteins and small molecules or nucleic acids, as well as discuss older methods that still hold value today. (c) 2024 Wiley Periodicals LLC.

Despite recent advances in the mechanism of oxidized DNA activating NLRP3, the molecular mechanism and consequence of oxidized DNA associating with NLRP3 remains unknown. Cytosolic NLRP3 binds oxidized DNA which has been released from the mitochondria, which subsequently triggers inflammasome activation. Human glycosylase (hOGG1) repairs oxidized DNA damage which inhibits inflammasome activation. The fold of NLRP3 pyrin domain contains amino acids and a protein fold similar to hOGG1. Amino acids that enable hOGG1 to bind and cleave oxidized DNA are conserved in NLRP3. We found NLRP3 could bind and cleave oxidized guanine within mitochondrial DNA. The binding of oxidized DNA to NLRP3 was prevented by small molecule drugs which also inhibit hOGG1. These same drugs also inhibited inflammasome activation. Elucidating this mechanism will enable the design of drug memetics that treat inflammasome pathologies, illustrated herein by NLRP3 pyrin domain inhibitors which suppressed interleukin-1β (IL-1β) production in macrophages.