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The NLRP3 inflammasome can be activated by stimuli that include nigericin, uric acid crystals, amyloid-β fibrils and extracellular ATP. The mitotic kinase NEK7 licenses the assembly and activation of the NLRP3 inflammasome in interphase. Here we report a cryo-electron microscopy structure of inactive human NLRP3 in complex with NEK7, at a resolution of 3 .8 Å. The earring-shaped NLRP3 consists of curved leucine-rich-repeat and globular NACHT domains, and the C-terminal lobe of NEK7 nestles against both NLRP3 domains. Structural recognition between NLRP3 and NEK7 is confirmed by mutagenesis both in vitro and in cells. Modelling of an active NLRP3–NEK7 conformation based on the NLRC4 inflammasome predicts an additional contact between an NLRP3-bound NEK7 and a neighbouring NLRP3. Mutations to this interface abolish the ability of NEK7 or NLRP3 to rescue NLRP3 activation in NEK7-knockout or NLRP3-knockout cells. These data suggest that NEK7 bridges adjacent NLRP3 subunits with bipartite interactions to mediate the activation of the NLRP3 inflammasome. A cryo-electron microscopy structure of human NLRP3 in complex with the mitotic kinase NEK7 provides insights into the interactions that mediate the activation of the NLRP3 inflammasome.

The molecular codes underpinning the functions of plant NLR immune receptors are poorly understood. We used in vitro Mu transposition to generate a random truncation library and identify the minimal functional region of NLRs. We applied this method to NRC4—a helper NLR that functions with multiple sensor NLRs within a Solanaceae receptor network. This revealed that the NRC4 N-terminal 29 amino acids are sufficient to induce hypersensitive cell death. This region is defined by the consensus MADAxVSFxVxKLxxLLxxEx (MADA motif) that is conserved at the N-termini of NRC family proteins and ~20% of coiled-coil (CC)-type plant NLRs. The MADA motif matches the N-terminal α1 helix of Arabidopsis NLR protein ZAR1, which undergoes a conformational switch during resistosome activation. Immunoassays revealed that the MADA motif is functionally conserved across NLRs from distantly related plant species. NRC-dependent sensor NLRs lack MADA sequences indicating that this motif has degenerated in sensor NLRs over evolutionary time. Just like humans, plants get sick. They can be infected by parasites as diverse as fungi, bacteria, viruses, nematode worms and insects. But, also like humans, plants have an immune system that helps them defend against disease. Their first line of defence are disease resistance genes. Many of these genes encode so-called immune receptors, which are proteins that detect parasites and kick-off the immune response. Plant genomes may encode anywhere between 50 and 1000 immune receptors; some of which work solo as singletons, while others operate in pairs or as complex networks. Understanding how immune receptor genes have evolved would give fundamental knowledge about how they work, which in turn would set the stage for researchers to be able to use them to protect agricultural crops from disease. One driving force behind the evolution of many genes is gene duplication. Genes duplicate and afterwards the two copies can evolve in different ways. The original immune receptors are multi-tasking proteins that both detect parasites and trigger the immune response. Yet, following gene duplication, evolution has led to some immune receptors becoming dedicated to detection and losing the ability to trigger a defence response on their own. Now, Adachi et al. have discovered a molecular signature – named the MADA motif – that defines the subset of immune receptors that can trigger the immune response in plants. This motif is made of just 21 amino acids (the building blocks of proteins) at one end of the receptor and, remarkably, a short fragment of the protein containing this motif is enough to trigger a defence response when produced in plants. In contrast, the immune receptors that have specialized to only detect parasites have lost this molecular signature throughout evolution, presumably because they do not need it as they rely on their receptor partners to trigger defences instead. Every year, billions of dollars’ worth of food is lost to plant diseases. These new findings will enable the research community to classify disease resistance genes into categories to help deduce the network architecture of the plant immune system. A better understanding of this, and how networks of plant immune receptor evolve, should set the stage for breeding crop plants that are more able to resist diseases.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis 1 . NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer 2 – 6 . However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated 4 , 7 , 8 . Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system. Structural and mutational studies and cellular assays show that the inflammasome sensor NLRP1 forms a complex with thioredoxin, which acts as a negative regulator of inflammasome activity.

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors 1 , 2 . Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity 3 , 4 . Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation 5 . The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 ( Sl NRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of Sl NRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester Sl NRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana . The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP 6 ) or pentakisphosphate (IP 5 ) bound to the inner surface of the C-terminal leucine-rich repeat domain of Sl NRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of Sl NRC2 and pathogen-induced Sl NRC2-mediated cell death in N. benthamiana . Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs. Cryo-electron microscopy reveals that the tomato immune receptor NRC2 forms oligomers to stabilize its inactive state and sequester it from activation, with inositol phosphates acting as immunoregulatory cofactors.

The nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) responds to a vast variety of stimuli, and activated NLRP3 forms an inflammasome, which in turn is associated with conditions such as atherosclerosis, Alzheimer’s disease, and diabetes. A multilayered regulatory mechanism ensures proper NLRP3 inflammasome activation, although the structural basis for this process remains unclear. This study aimed to investigate the cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950. The inhibitor binds to the cavity distinct from the nucleotide binding site in the NACHT domain and stabilizes the closed conformation of NLRP3. The barrel-shaped dodecamer composed of the inactive form of NLRP3 is formed mainly through LRR–LRR interactions on the lateral side, and the highly positively charged top and bottom sides composed of NACHT domains provide a scaffold for membrane association. The cryo-electron microscopy structure suggests that oligomerization of NLRP3 is necessary for its membrane association; it is subsequently disrupted for activation, hence serving as a key player in controlling the spatiotemporal NLRP3 inflammasome activation. These findings are expected to contribute to the development of drugs targeting NLRP3 in future.

The NLRP3 inflammasome is a multiprotein complex that mediates caspase-1 activation and the release of proinflammatory cytokines, including interleukin (IL)-1β and IL-18. Gain-of-function variants in the gene encoding NLRP3 (also called cryopyrin) lead to constitutive inflammasome activation and excessive IL-1β production in cryopyrin-associated periodic syndromes (CAPS). Here we present functional screening and automated analysis of 534 NLRP3 variants from the international INFEVERS registry and the ClinVar database. This resource captures the effect of NLRP3 variants on ASC speck formation spontaneously, at low temperature, after inflammasome stimulation and with the specific NLRP3 inhibitor MCC950. Most notably, our analysis facilitated the updated classification of NLRP3 variants in INFEVERS. Structural analysis suggested multiple mechanisms by which CAPS variants activate NLRP3, including enhanced ATP binding, stabilizing the active NLRP3 conformation, destabilizing the inactive NLRP3 complex and promoting oligomerization of the pyrin domain. Furthermore, we identified pathogenic variants that can hypersensitize the activation of NLRP3 in response to nigericin and cold temperature exposure. We also found that most CAPS-related NLRP3 variants can be inhibited by MCC950; however, NLRP3 variants with changes to proline affecting helices near the inhibitor binding site are resistant to MCC950, as are variants in the pyrin domain, which likely trigger activation directly with the pyrin domain of ASC. Our findings could help stratify the CAPS population for NLRP3 inhibitor clinical trials and our automated methodologies can be implemented for molecules with a different mechanism of activation and in laboratories worldwide that are interested in adding new functionally validated NLRP3 variants to the resource. Overall, our study provides improved diagnosis for patients with CAPS, mechanistic insight into the activation of NLRP3 and stratification of patients for the future application of targeted therapeutics. Gain-of-function variants in the gene encoding NLRP3 lead to constitutive inflammasome activation and excessive IL-1β production. In this resource, authors perform functional screening of clinically relevant NLRP3 variants. Structural analysis suggested multiple mechanisms by which variants activate NLRP3 and the identification of pathogenic variants that can sensitize the activation of NLRP3 in response to nigericin and cold temperature exposure.

Plant nucleotide-binding and leucine-rich repeat (NLR) receptors recognize avirulence effectors directly through their integrated domains (IDs) or indirectly via the effector-targeted proteins. Previous studies have succeeded in generating designer NLR receptors with new recognition profiles by engineering IDs or targeted proteins based on prior knowledge of their interactions with the effectors. However, it is yet a challenge to design a new plant receptor capable of recognizing effectors that function by unknown mechanisms. Several rice NLR immune receptors, including RGA5, possess an integrated heavy metal–associated (HMA) domain that recognizes corresponding Magnaporthe oryzae Avrs and ToxB-like (MAX) effectors in the rice blast fungus. Here, we report a designer rice NLR receptor RGA5HMA2 carrying an engineered, integrated HMA domain (RGA5-HMA2) that can recognize the noncorresponding MAX effector AvrPib and confers the RGA4-dependent resistance to the M. oryzae isolates expressing AvrPib, which originally triggers the Pib-mediated blast resistance via unknown mechanisms. The RGA5-HMA2 domain is contrived based on the high structural similarity of AvrPib with two MAX effectors, AVR-Pia and AVR1-CO39, recognized by cognate RGA5-HMA, the binding interface between AVR1-CO39 and RGA5-HMA, and the distinct surface charge of AvrPib and RAG5-HMA. This work demonstrates that rice NLR receptors with the HMA domain can be engineered to confer resistance to the M. oryzae isolates noncorresponding but structurally similar MAX effectors, which manifest cognate NLR receptor–mediated resistance with unknown mechanisms. Our study also provides a practical approach for developing rice multilines and broad race spectrum–resistant cultivars by introducing a series of engineered NLR receptors.

This first‐in‐human study evaluated the safety, tolerability, single‐ and multiple‐dose pharmacokinetic profiles with dietary influence, and pharmacodynamics (PD) of DFV890, an oral NLRP3 inhibitor, in healthy participants. In total, 122 participants were enrolled into a three‐part trial including single and 2‐week multiple ascending oral doses (SAD and MAD, respectively) of DFV890, and were randomized (3:1) to DFV890 or placebo (SAD [3–600 mg] and MAD [fasted: 10–200 mg, once‐daily or fed: 25 and 50 mg, twice‐daily]). DFV890 was generally well‐tolerated. Neither deaths nor serious adverse events were reported. A less than dose‐proportional increase in exposure was observed with the initially used crystalline suspension (3–300 mg); however, an adjusted suspension formulation using spray‐dried dispersion (SDD; 100–600 mg) confirmed dose‐proportional increase in exposure. Relative bioavailability between crystalline suspension and tablets, and food effect were evaluated at 100 mg. Under fasting conditions, Cmax of the tablet yielded 78% compared with the crystalline suspension, and both formulations showed comparable AUC. The fed condition led to a 2.05‐ and 1.49‐fold increase in Cmax and AUC0–last compared with the fasting condition. The median IC50 and IC90 for ex‐vivo lipopolysaccharide‐stimulated interleukin IL‐1β release inhibition (PD) were 61 (90% CI: 50, 70) and 1340 ng/mL (90% CI: 1190, 1490). Crystalline tablets of 100 mg once‐daily or 25 mg twice‐daily were sufficient to maintain ~90% of the IL‐1β release inhibition over 24 h at steady state. Data support dose and formulation selection for further development in diseases, in which an overactivated NLRP3 represents the underlying pathophysiology.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone which is essential in eukaryotes. It is required for the activation and stabilization of a wide variety of client proteins and many of them are involved in important cellular pathways. Since Hsp90 affects numerous physiological processes such as signal transduction, intracellular transport, and protein degradation, it became an interesting target for cancer therapy. Structurally, Hsp90 is a flexible dimeric protein composed of three different domains which adopt structurally distinct conformations. ATP binding triggers directionality in these conformational changes and leads to a more compact state. To achieve its function, Hsp90 works together with a large group of cofactors, termed co-chaperones. Co-chaperones form defined binary or ternary complexes with Hsp90, which facilitate the maturation of client proteins. In addition, posttranslational modifications of Hsp90, such as phosphorylation and acetylation, provide another level of regulation. They influence the conformational cycle, co-chaperone interaction, and inter-domain communications. In this review, we discuss the recent progress made in understanding the Hsp90 machinery.