Nucleotide-binding Domain And Leucine-rich Repeat-containing Research Articles

Chromofungin mitigates free fatty acids-induced endothelial inflammation via inhibition of NOD-like receptor thermal protein domain-associated protein 3 mediated by adenosine 5'-monophosphate-activated protein kinase.

Free fatty acids (FFAs) have emerged as significant risk factors for atherosclerosis (AS). Prolonged exposure to FFAs induces vascular endothelial injury, including inflammatory responses and oxidative stress, which are central events in AS. Chromofungin (CHR), a peptide derived from chromogranin A (CGA), has been implicated in various biological functions. However, its physiological roles in endothelial biology and its involvement in the pathological development of AS have not been previously reported. In the present study, we investigated the underlying mechanisms through which CHR exerts its beneficial effects on FFA-challenged human aortic endothelial cells (HAECs). We found that treatment with CHR ameliorated the FFA-induced reduction in cell viability and increase in lactate dehydrogenase (LDH) release. Additionally, CHR mitigated oxidative stress by reducing mitochondrial reactive oxygen species (ROS) levels and increasing superoxide dismutase (SOD) activity. Furthermore, exposure to FFAs increased NADPH oxidase (NOX) 4 expression at both the mRNA and protein levels, which were attenuated by CHR in a dose-dependent manner. Notably, CHR reduced the levels of nucleotide-binding domain and leucine-rich repeat-containing (NLR) family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and cleaved caspase-1 (p10), key components of the NLRP3 inflammasome complex, as well as interleukin 1β (IL-1β) and interleukin-18 (IL-18) expression. Mechanistically, it was demonstrated that FFAs reduced the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), which were rescued by CHR in a dose-dependent manner. Conversely, inhibition of AMPK with its specific inhibitor compound C abolished the protective effects of CHR against FFA-induced activation of the NLRP3 inflammasome in HAECs. Based on these findings, we conclude that CHR may serve as a promising agent for maintaining normal endothelial cell function and treating AS.

NRC immune receptor networks show diversified hierarchical genetic architecture across plant lineages.

Plants' complex immune systems include nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, which help recognize invading pathogens. In solanaceous plants, the NRC (NLR required for cell death) family includes helper NLRs that form a complex genetic network with multiple sensor NLRs to provide resistance against pathogens. However, the evolution and function of NRC networks outside solanaceous plants are currently unclear. Here, we conducted phylogenomic and macroevolutionary analyses comparing NLRs identified from different asterid lineages and found that NRC networks expanded significantly in most lamiids but not in Ericales and campanulids. Using transient expression assays in Nicotiana benthamiana, we showed that NRC networks are simple in Ericales and campanulids, but have high complexity in lamiids. Phylogenetic analyses grouped the NRC helper NLRs into three NRC0 subclades that are conserved, and several family-specific NRC subclades of lamiids that show signatures of diversifying selection. Functional analyses revealed that members of the NRC0 subclades are partially interchangeable, whereas family-specific NRC members in lamiids lack interchangeability. Our findings highlight the distinctive evolutionary patterns of the NRC networks in asterids and provide potential insights into transferring disease resistance across plant lineages.

An atypical NLR protein modulates the NRC immune receptor network in Nicotiana benthamiana.

The NRC immune receptor network has evolved in asterid plants from a pair of linked genes into a genetically dispersed and phylogenetically structured network of sensor and helper NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins. In some species, such as the model plant Nicotiana benthamiana and other Solanaceae, the NRC (NLR-REQUIRED FOR CELL DEATH) network forms up to half of the NLRome, and NRCs are scattered throughout the genome in gene clusters of varying complexities. Here, we describe NRCX, an atypical member of the NRC family that lacks canonical features of these NLR helper proteins, such as a functional N-terminal MADA motif and the capacity to trigger autoimmunity. In contrast to other NRCs, systemic gene silencing of NRCX in N. benthamiana markedly impairs plant growth resulting in a dwarf phenotype. Remarkably, dwarfism of NRCX silenced plants is partially dependent on NRCX paralogs NRC2 and NRC3, but not NRC4. Despite its negative impact on plant growth when silenced systemically, spot gene silencing of NRCX in mature N. benthamiana leaves doesn't result in visible cell death phenotypes. However, alteration of NRCX expression modulates the hypersensitive response mediated by NRC2 and NRC3 in a manner consistent with a negative role for NRCX in the NRC network. We conclude that NRCX is an atypical member of the NRC network that has evolved to contribute to the homeostasis of this genetically unlinked NLR network.

Effects of quercetin on apoptosis and extracellular matrix degradation of chondrocytes induced by oxidative stress-mediated pyroptosis.

Quercetin has been preliminarily proven to serve as a potential agent for the treatment of osteoarthritis (OA). However, its effects and potential mechanisms on the pathological process of OA are not very clear. This study aimed to study the protective effect of quercetin on OA. Lipopolysaccharide (LPS)-treated chondrocytes (C28/I2 cell) and anterior cruciate ligament transection with partial medial meniscectomy-treated Wistar rats were used as models of OA in vitro and in vivo. Cell counting kit-8 (CCK-8 kit), flow cytometry, enzyme-linked immunosorbent assay (ELISA) kit, western blot, dichlorodihydrofluorescein diacetate (DCFH-DA) kit, thiobarbituric acid (TBA) test, toluidine blue staining, Hematoxylin eosin (HE) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining were used to evaluate cell viability, cell apoptosis, inflammatory cytokines level, protein expression, reactive oxygen species (ROS) level, malondialdehyde (MDA) content, morphological changes, and chondrocyte apoptosis of cartilage, respectively. Results showed that quercetin could reduce LPS-induced C28/I2 cell apoptosis, extracellular matrix (ECM) degradation, and cell pyroptosis, and overexpression of nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) revealed that quercetin reduced chondrocyte apoptosis and ECM degradation by inhibiting NLRP3-mediated pyroptosis. Furthermore, quercetin could reduce chondrocyte apoptosis and ECM degradation, and inhibit NLRP3-mediated pyroptosis through blocking oxidative stress. It was further confirmed in the rat OA model that quercetin alleviated OA by blocking oxidative stress, reduces chondrocyte pyroptosis, apoptosis, and ECM degradation. In conclusion, quercetin inhibited OA via blocking oxidative stress-induced chondrocyte pyroptosis in models of OA in vitro and in vivo.

A bacterial effector protein prevents MAPK-mediated phosphorylation of SGT1 to suppress plant immunity.

Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.

PALs: Emerging Key Players in Broad-Spectrum Disease Resistance.

Phenylalanine ammonia lyases (PALs) are involved in resistance to plant pathogens, but the mechanism by which they contribute to plant immunity is unclear. Two recent studies provide direct evidence for PALs' contributions to rice broad-spectrum resistance (BSR) mediated by nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins and RNA-binding proteins (Zhai et al., Mol. Cell, 2019; Zhou et al., Proc. Natl. Acad. Sci. U. S. A., 2018).

Molecular cloning and functional characterisation of NLRX1 in grass carp (Ctenopharyngodon idella)

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. In the present study, the NLR family gene NLRX1 from grass carp (Ctenopharyngodon idella) was cloned and characterised. NLRX1 was widely expressed in all tissues examined, albeit at varying levels. After exposure to the grass carp reovirus (GCRV), NLRX1 mRNA expression levels were altered in immune organs, and dramatically altered in liver. Subcellular localisation indicated that NLRX1 protein co-localised with the mitochondria in the transfected cells. Additionally, the bimolecular fluorescence complementation (BiFC) system was introduced to detect the interaction between tumour necrosis factor (TNF) receptor associated factor 6 (TRAF6) and NLRX1. Moreover, deficient of NLRX1 in CIK cells with small interference RNA (siRNA) promoted polyinosinic:polycytidylic acid (poly (I:C))-induced IFN-related genes production, including IRF3, IRF7, and IFN-I, which reveals that NLRX1 is a negative regulator of IFN. Taken together, our results demonstrate that NLRX1 gene plays an important role in innate immune regulation and provide new insights into understanding the functional characteristics of the NLRX1 in teleosts.

An NB-LRR gene, TYNBS1, is responsible for resistance mediated by the Ty-2 Begomovirus resistance locus of tomato

An NB-LRR gene, TYNBS1, was isolated from Begomovirus-resistance locus Ty-2. Transgenic plant analysis revealed that TYNBS1 is a functional resistance gene. TYNBS1 is considered to be synonymous with Ty-2. Tomato yellow leaf curl disease caused by Tomato yellow leaf curl virus (TYLCV) is a serious threat to tomato (Solanum lycopersicum L.) production worldwide. A Begomovirus resistance gene, Ty-2, was introduced into cultivated tomato from Solanum habrochaites by interspecific crossing. To identify the Ty-2 gene, we performed genetic analysis. Identification of recombinant line 3701 confirmed the occurrence of a chromosome inversion in the Ty-2 region of the resistant haplotype. Genetic analysis revealed that the Ty-2 gene is linked to an introgression encompassing two markers, SL11_25_54277 and repeat A (approximately 200kb). Genomic sequences of the upper and lower border of the inversion section of susceptible and resistant haplotypes were determined. Two nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR) genes, TYNBS1 and TYNBS2, were identified around the upper and lower ends of the inversion section, respectively. TYNBS1 strictly co-segregated with TYLCV resistance, whereas TYNBS2 did not. Genetic introduction of genomic fragments containing the TYNBS1 gene into susceptible tomato plants conferred TYLCV resistance. These results demonstrate that TYNBS1 is a functional resistance gene for TYLCV, and is synonymous with the Ty-2 gene.

NLR network mediates immunity to diverse plant pathogens

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that "sensor" NLR proteins are paired with "helper" NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.

Pathogen perception by NLRs in plants and animals: Parallel worlds.

Intracellular NLR (Nucleotide-binding domain and Leucine-rich Repeat-containing) receptors are sensitive monitors that detect pathogen invasion of both plant and animal cells. NLRs confer recognition of diverse molecules associated with pathogen invasion. NLRs must exhibit strict intramolecular controls to avoid harmful ectopic activation in the absence of pathogens. Recent discoveries have elucidated the assembly and structure of oligomeric NLR signalling complexes in animals, and provided insights into how these complexes act as scaffolds for signal transduction. In plants, recent advances have provided novel insights into signalling-competent NLRs, and into the myriad strategies that diverse plant NLRs use to recognise pathogens. Here, we review recent insights into the NLR biology of both animals and plants. By assessing commonalities and differences between kingdoms, we are able to develop a more complete understanding of NLR function.

Plant TRAF Proteins Regulate NLR Immune Receptor Turnover.

In animals, Tumor necrosis factor receptor-associated factor (TRAF) proteins are molecular adaptors that regulate innate and adaptive immunity, development, and abiotic stress responses. Although gene families encoding TRAF domain-containing proteins exhibit enriched diversity in higher plants, their biological roles are poorly defined. Here, we report the identification of two redundant TRAF proteins, Mutant, snc1-enhancing 13 (MUSE13) and MUSE14, that contribute to the turnover of nucleotide-binding domain and leucine-rich repeat-containing (NLR) immune receptors SNC1 and RPS2. Loss of both MUSE13 and MUSE14 leads to enhanced pathogen resistance, NLR accumulation, and autoimmunity, while MUSE13 overexpression results in reduced NLR levels and activity. In planta, MUSE13 associates with SNC1, RPS2, and the E3 ubiquitin ligase SCF(CPR1). Taken together, we speculate that MUSE13 and MUSE14 associate with the SCF E3 ligase complex to form a plant-type TRAFasome, which modulates ubiquitination and subsequent degradation of NLR immune sensors to maintain their homeostasis.

Effector Recognition and Activation of the Arabidopsis thaliana NLR Innate Immune Receptors

Plant immunity to coevolved pathogens relies on the perception of pathogenic effectors by nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins-sophisticated intracellular receptors that have both perception and signaling roles in activating defenses. Given the conserved domain architecture of NLRs, a structural biology perspective is particularly relevant to understanding mechanisms of their activation. Here, we summarize our recent findings on the Arabidopsis resistance protein RPP1, a member of the TIR-NBS-LRR family of plant NLRs that specifically recognizes the cognate effector protein ATR1. To study the basis of RPP1 activation, we have taken advantage of a series of RPP1 and ATR1 alleles that differentially condition resistance. In planta association between the LRR domain of RPP1 and ATR1 only occurs in a resistance-activating combination of alleles, suggesting that a direct interaction between RPP1 and ATR1 protein leads to activation of the NLR (Krasileva et al. 2010). Given critical amino acid residues' locations on the solved ATR1 crystal structure, and variable amino acid residues on the predicted LRR structure of RPP1, we hypothesize that specific "hotspots" of the horseshoe-like LRR fold mediate binding to the ATR1 ligand and that polymorphisms mapping to these surfaces condition differences in allelic recognition specificity. We present docking models of a possible co-complex between RPP1 and ATR1, and we propose that ATR1 binding relieves autoinhibition of RPP1 resistance signaling. This is consistent with current models of activation for both TIR- and CC-type plant NLRs, where conformational changes could lead to NLR oligomerization, nucleotide binding, translocation, and other critical downstream events in triggering immunity.

NLRX1 Protein Attenuates Inflammatory Responses to Infection by Interfering with the RIG-I-MAVS and TRAF6-NF-κB Signaling Pathways

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-β, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction withRNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells.Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.

Activation of a Rac GTPase by the NLR Family Disease Resistance Protein Pit Plays a Critical Role in Rice Innate Immunity

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

The HSP90-SGT1 Chaperone Complex for NLR Immune Sensors

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as immune sensors in both plants and animals. NLR proteins recognize, directly or indirectly, pathogen-derived molecules and trigger immune responses. To function as a sensor, NLR proteins must be correctly folded and maintained in a recognition-competent state in the appropriate cellular location. Upon pathogen recognition, conformational changes and/or translocation of the sensors would activate the downstream immunity signaling pathways. Misfolded or used sensors are a threat to the cell and must be immediately inactivated and discarded to avoid inappropriate activation of downstream pathways. Such maintenance of NLR-type sensors requires the SGT1-HSP90 pair, a chaperone complex that is structurally and functionally conserved in eukaryotes. Deciphering how the chaperone machinery works would facilitate an understanding of the mechanisms of pathogen recognition and signal transduction by NLR proteins in both plants and animals.

The NLRP3 Inflammasome Mediates In Vivo Innate Immunity to Influenza A Virus through Recognition of Viral RNA

The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) family of pattern-recognition molecules mediate host immunity to various pathogenic stimuli. However, in vivo evidence for the involvement of NLR proteins in viral sensing has not been widely investigated and remains controversial. As a test of the physiologic role of the NLR molecule NLRP3 during RNA viral infection, we explored the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, Pycard, or caspase-1, but not Nlrc4, exhibited dramatically increased mortality and a reduced immune response after exposure to the influenza virus. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrated that an NLRP3-mediated response could be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by the influenza virus was dependent on lysosomal maturation and reactive oxygen species (ROS). Inhibition of ROS induction eliminated IL-1beta production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.