Oligomerization Domain Research Articles

Strategic Mutations in Designer Native Peptides Combat NLRP3 Inflammasome Activation in Neurodegenerative Disorders.

Nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain (PYD)-containing protein 3 (NLRP3) form an inflammasome by assembling with apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1 that plays a pivotal role in various neurodegenerative diseases such as Alzheimer's and Parkinson diseases. We designed native peptides derived from the PYDs of NLRP3 and ASC based on their interfacial interaction to inhibit NLRP3 inflammasome formation. Screening revealed that NP3, derived from NLRP3, inhibits inflammasome activation. Furthermore, a strategic mutation (F → L) in native peptide NP3 results in MNP2 that selectively binds to PYD of ASC with nanomolar affinity, inhibiting NLRP3 inflammasome formation, interleukin-1β (IL-1β) release, and caspase-1 maturation. MNP2 also reduced potassium (K) and chloride (Cl) ion efflux, key signals in NLRP3 activation, and prevented mitochondrial damage and reactive oxygen species (ROS) production. MNP2 significantly reduced NLRP3 inflammasome formation in neurodegenerative conditions triggered by amyloid-β (Aβ), Tau, and α-Synuclein (α-Syn), suggesting a promising therapeutic strategy for NLRP3-related inflammatory diseases, including neurodegenerative disorders.

PRRSV-2 nsp2 Ignites NLRP3 inflammasome through IKKβ-dependent dispersed trans-Golgi network translocation.

The NLRP3 inflammasome is a fundamental component of the innate immune system, yet its excessive activation is intricately associated with viral pathogenesis. Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), belonging to the family Arteriviridae, triggers dysregulated cytokine release and interstitial pneumonia, which can quickly escalate to acute respiratory distress and death. However, a mechanistic understanding of PRRSV-2 progression remains unclear. Here, we screen that PRRSV-2 nsp2 activates the NLRP3 inflammasome, thereby instigating a state of hyperinflammation. Mechanistically, PRRSV-2 nsp2 interacts with the nucleotide-binding and oligomerization (NACHT) domain of NLRP3, augmenting IKKβ recruitment to driving NLRP3 translocation to the dispersed trans-Golgi network (dTGN) for oligomerization. This process facilitates ASC polymerization, culminating in the activation of the NLRP3 inflammasome. In addition, the IKKβ-dependent NLRP3 translocation to the dTGN is pivotal for pseudorabies virus (PRV) and encephalomyocarditis virus (EMCV)-induced inflammatory responses. Collectively, these results elucidate a novel mechanism of NLRP3 inflammasome activation during PRRSV-2 infection, providing valuable insights into PRRSV-2 pathogenesis.

In ovo delivery of carvacrol triggers expression of chemotactic factors, antimicrobial peptides and pro-inflammatory pathways in the yolk sac of broiler chicken embryos

BackgroundBroiler chickens are most vulnerable immediately after hatching due to their immature immune systems, making them susceptible to infectious diseases. The yolk plays an important role in early immune defence by showing relevant antioxidant and passive immunity capabilities during broiler embryonic development. The immunomodulatory effects of phytogenic compound carvacrol have been widely reported. After in ovo delivery in the amniotic fluid during embryonic development carvacrol is known to migrate to the yolk sac. However, it is unknown whether carvacrol in the yolk could enhance defence responsiveness in the yolk sac. Therefore, the aim of this study was to improve early immune function in chicken embryos, and it was hypothesized that in ovo delivery of carvacrol would result in immunomodulatory effects in the yolk sac, potentially improving post-hatch resilience.MethodsOn embryonic day (E)17.5, either a saline (control) or carvacrol solution was injected into the amniotic fluid. Yolk sac tissue samples were collected at E19.5, and transcriptomic analyses using RNA sequencing were performed, following functional enrichment analyses comparing the control (saline) and carvacrol-injected groups.ResultsThe results showed that 268 genes were upregulated and 174 downregulated in the carvacrol group compared to the control (P < 0.05; logFC < −0.5 or log FC > 0.5). Functional analyses of these differentially expressed genes, using KEGG, REACTOME, and Gene Ontology databases, showed enrichment of several immune-related pathways. This included the pathways ‘Antimicrobial peptides’ (P = 0.001) and ‘Chemoattractant activity’ (P = 0.004), amongst others. Moreover, the ‘NOD-like receptor signaling’ pathway was enriched (P = 0.002). Antimicrobial peptides are part of the innate immune defence and are amongst the molecules produced after the nucleotide oligomerization domain (NOD)-like receptor pathway activation. While these responses may be associated with an inflammatory reaction to an exogenous threat, they could also indicate that in ovo delivery of carvacrol could prepare the newly hatched chick against bacterial pathogens by potentially promoting antimicrobial peptide production through activation of NOD-like receptor signaling in the yolk sac.ConclusionIn conclusion, these findings suggest that in ovo delivery of carvacrol has the potential to enhance anti-pathogenic and pro-inflammatory responses in the yolk sac via upregulation of antimicrobial peptides, and NOD-like receptor pathways.

BCL6 Alleviates Hepatic Ischemia/Reperfusion Injury Via Recruiting SIRT1 to Repress the NF-κB/NLRP3 Pathway.

Hepatic ischemia/reperfusion (I/R) injury (HIRI) is an intrinsic phenomenon observed in the process of various liver surgeries. Unfortunately, there are currently few options available to prevent HIRI. Accordingly, we aim to explore the role and key downstream effects of B-cell lymphoma 6 (BCL6) in hepatic I/R (HIR). BCL6 expression levels were measured in I/R liver tissue and primary hepatocytes stimulated by hypoxia/reoxygenation (H/R). Moreover, we ascertained the BCL6 effect on HIR in vivo using liver-specific BCL6 knockout mice and adenovirus-BCL6-infected mice. RNA-sequencing, luciferase, chromatin immunoprecipitation, and interactome analysis were combined to identify the direct target and corresponding molecular events contributing to BCL6 function. DNA pull-down was applied to identify upstream of BCL6 in the H/R challenge. HIR represses BCL6 expression in vivo and in vitro. Hepatic BCL6 overexpression attenuates inflammation and apoptosis after I/R injury, whereas BCL6 deficiency aggravates I/R-induced liver injury. RNA-sequencing showed that BCL6 modulated nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 inflammasome signaling in HIRI. Mechanistically, BCL6 deacetylated nuclear factor kappa-B p65 lysine 310 by recruiting sirtuin 1 (SIRT1), thereby inhibiting the nuclear factor kappa-B/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 pathway. Moreover, overexpression of SIRT1 blocked the detrimental effects of BCL6 depletion. Moreover, EX 527, a SIRT1 inhibitor, vanished protection from BCL6 overexpression. Furthermore, transcription factor 7 was found to mediate the transcription regulation of BCL6 on H/R challenge. Our results provide the first evidence supporting BCL6 as an important protective agent of HIR. This suggests a potential therapeutic approach for HIR.

Inhibition of RIPK1-driven necroptosis ameliorates inflammatory hyperalgesia caused by lipopolysaccharide: involvement of TLR-, NLRP3-, and caspase-11-mediated signaling pathways.

Increasing evidence suggests that inhibition of receptor-interacting serine/threonine-protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain-like pseudokinase (MLKL) necrosome has protective effects in vivo models of painful conditions seen in humans associated with inflammation and demyelination in the central nervous system. However, the contribution of RIPK1-driven necroptosis to inflammatory pain remains unknown. Therefore, this study aims to determine the effect of necrostatin (Nec) -1s, a selective RIPK1 inhibitor, on lipopolysaccharide (LPS)-induced inflammatory pain and related underlying mechanisms. In the saline-, LPS-, and/or Nec-1s-injected male mice, thermal hyperalgesia was evaluated by hot plate test. Alterations in the expression of proteins involved in the RIPK1, toll-like receptor (TLR) 4, myeloid differentiation factor (MyD) 88/toll-interleukin (IL)-1 receptor domain-containing adapter-inducing interferon-β (TRIF)/nuclear factor (NF)-kB, nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing (NLRP) 3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/pro-caspase-1, and caspase-11/gasdermin D (GSDMD) signaling pathways, as well as proteins related to demyelination and remyelination in the brain and spinal cord were determined by the immunoblotting method. The LPS-induced alleviation of thermal hyperalgesia was prevented by necrostatin-1s. Necrostatin-1s reversed (1) increased activity of RIPK1, RIPK3, MLKL, and NF-kB p65, (2) enhanced expression of TLR4, MyD88, TRIF, NF-kB p65, HMGB1, NLRP3, ASC, caspase-1 p20, IL-1β, caspase-11 p20, p30-GSDMD, and semaphorin 3A, and (3) diminished myelin PLP expression induced by LPS. These findings suggest that the use of RIPK1 inhibitors could be a therapeutic approach in the management of inflammatory pain associated with necroptosis, pyroptosis, and demyelination.

High expression of nucleotide-binding oligomerization domain protein 1 correlates with poor prognosis and immune cell infiltration in Glioblastoma Multiforme patients

Nucleotide-binding oligomerization domain protein 1 (NOD1) is one of the innate immune receptors that has been associated with tumorigenesis and abnormally expressed in various cancers. However, the role of NOD1 in Glioblastoma Multiforme (GBM) has not been investigated. We used the Tumor Immune Estimate Resource (TIMER) database to compare the differential expression of NOD1 in various tumors. NOD1 expression in GBM was further validated in the GEO database, and the survival of NOD1 was assessed by the Kaplan–Meier method. Clinical samples were collected to validate NOD1 expression. GSEA was carried out to expound on NOD1-related pathways involved in GBM. NOD1 co-expression and enrichment analysis were performed using the Linked Omics database and R software. The relationship between immune infiltrates and NOD1 expression was assessed by TIMER. Besides, the correlation between NOD1 and immune signatures (immunomodulators and chemokine) was evaluated by TISIDB. We found that NOD1 expression was significantly upregulated in GBM patients, and higher expression of NOD1 was associated with a poor prognosis. GSEA and enrichment analysis revealed that NOD1 might play a vital role in immune response and GBM progression. TIMER analysis showed a positive correlation between NOD1 expression and 17 types of tumor-infiltrating immune cells. Moreover, NOD1 expression was positively correlated with the expression of chemokine and immunomodulators in GBM. Overall, our findings suggest that NOD1 is a promising prognostic biomarker and is associated with immune cell infiltration in GBM, making it a potential diagnostic biomarker for this aggressive brain cancer.

Molecular mechanisms underlying allosteric behavior of Escherichia coli DgoR, a GntR/FadR family transcriptional regulator.

GntR/FadR family featuring an N-terminal winged helix-turn-helix DNA-binding domain and a C-terminal α-helical effector-binding and oligomerization domain constitutes one of the largest families of transcriptional regulators. Several GntR/FadR regulators govern the metabolism of sugar acids, carbon sources implicated in bacterial-host interactions. Although effectors are known for a few sugar acid regulators, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using DgoR, a transcriptional repressor of d-galactonate metabolism in Escherichia coli, as a model, and its superrepressor alleles, we probed allostery in a GntR/FadR family sugar acid regulator. Genetic and biochemical studies established compromised response to d-galactonate as the reason for the superrepressor behavior of the mutants: T180I does not bind d-galactonate, and while A97V, S171L and M188I bind d-galactonate, effector binding does not induce a conformational change required for derepression, suggesting altered allostery. For mechanistic insights into allosteric communication, we performed simulations of the modeled DgoR structure in different allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We finally validated the allosteric communication model by testing in silico predictions with experimental data.

Structural analysis of ExaC, an NAD+-dependent aldehyde dehydrogenase, from Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa (Pa) utilizes ethanol as an energy source, however, ethanol metabolism generates acetaldehyde, a toxic byproduct. To mitigate this toxicity, P. aeruginosa employs aldehyde dehydrogenases (ALDHs) to oxidize acetaldehyde into less harmful compounds. ExaC, an NAD+-dependent ALDH from P. aeruginosa (PaExaC) and a member of group X ALDHs, plays a critical role in this detoxification by oxidizing both aldehydes and hydrazones. In this study, we determined the crystal structures of PaExaC in its apo and NAD+-bound forms. PaExaC functions as a homodimer, with three distinct domains: an NAD+ binding domain, a catalytic domain, and an oligomerization domain. Structural analyses revealed that PaExaC’s substrate entry channel (SEC) is optimized for size-selective aldehyde metabolism, with Leu120, Tyr462, and Thr302. Comparative structural and docking analyses with other ALDHs further validated PaExaC’s preference for small aliphatic aldehydes and hydrazones. These findings highlight PaExaC’s role in aldehyde detoxification, facilitating P. aeruginosa survival in diverse environments, and provide structural insights for developing targeted inhibitors to help treat infections.

Role of NLRP3 inflammasome in nanoparticle adjuvant-mediated immune response.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome is pivotal in orchestrating the immune response induced by nanoparticle adjuvants. Understanding the intricate mechanisms underlying the activation of NLRP3 inflammasome by these adjuvants is crucial for deciphering their immunomodulatory properties. This review explores the involvement of the NLRP3 inflammasome in mediating immune responses triggered by nanoparticle adjuvants. It delves into the signaling pathways and cellular mechanisms involved in NLRP3 activation, highlighting its significance in modulating the efficacy and safety of nanoparticle-based adjuvants. A comprehensive grasp of the interplay between NLRP3 inflammasome and nanoparticle adjuvants holds promise for optimizing vaccine design and advancing immunotherapeutic strategies.

Network Pharmacology Suggests Mechanisms for Therapeutic Effects of Caulis Sinomenii on Avian Gout.

Avian gout (AG) is detrimental to the survival and production performance of poultry and effective drugs are lacking. Caulis sinomenii has shown clinical efficacy against arthritis and may have potential value in AG prevention and treatment. In the present study, the components and targets of C. sinomenii and AG-related targets were identified using relevant databases. The common targets, target interactions, and signaling pathways involved in the prevention and treatment of AG by C. sinomenii were determined using software to explore the potential mechanisms of action. Sixteen components of C. sinomenii, eight of which were active ingredients with 351 targets and 2993 AG-related targets, were identified using several databases. A total of 156 common targets were associated with 202 biological processes and 34 pathways. Toll-like receptor 4 (TLR4) and prostaglandin endoperoxide synthase 2 were core targets. These targets may exert therapeutic effects on AG through four pathways: the nucleotide-binding oligomerization domain (NOD)-like receptor, mammalian target of rapamycin, TLR, and mitogen-activated protein kinase signaling pathways. In summary, C. sinomenii has potential therapeutic efficacy against AG through multicomponent, multi-target, and multi-pathway mechanisms.

Mechanism of action of the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome and its regulation in liver injury

Abstract Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) is a cytosolic pattern recognition receptor that recognizes multiple pathogen-associated molecular patterns and damage-associated molecular patterns. It is a cytoplasmic immune factor that responds to cellular stress signals, and it is usually activated after infection or inflammation, forming an NLRP3 inflammasome to protect the body. Aberrant NLRP3 inflammasome activation is reportedly associated with some inflammatory diseases and metabolic diseases. Recently, there have been mounting indications that NLRP3 inflammasomes play an important role in liver injuries caused by a variety of diseases, specifically hepatic ischemia/reperfusion injury, hepatitis, and liver failure. Herein, we summarize new research pertaining to NLRP3 inflammasomes in hepatic injury, hepatitis, and liver failure. The review addresses the potential mechanisms of action of the NLRP3 inflammasome, and its regulation in these liver diseases.

Targeting NLRP3 signaling with a novel sulfonylurea compound for the treatment of vascular cognitive impairment and dementia.

As a key inflammatory factor, the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in neuroinflammation and the progression of neurodegenerative diseases. Dysregulation of NLRP3 signaling can trigger various inflammatory responses in the brain, contributing to the development of neurodegenerative diseases such as ischemic stroke, vascular dementia (VaD), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Therefore, the NLRP3 signaling pathway is a promising therapeutic target for the treatment of neurodegenerative diseases, including VaD. In this study, we investigated the therapeutic effects of a synthetic sulfonylurea NLRP3 inhibitor, AMS-17, in a VaD mouse model using bilateral common carotid artery stenosis (BCAS) and elucidated the underlying mechanisms. All mice were randomly divided into three groups: Sham, VaD + Vehicle, and VaD + AMS-17. Cognitive function was assessed using the Y-maze and Morris water maze (MWM) on the 50th day after BCAS. Brain sections and blood serum samples were collected for biomarker analysis and immunohistochemistry. Neurodegeneration, expressions of the molecules involved in the NLRP3 signaling pathways, tight junction proteins, and myelination were assessed using western blotting and immunofluorescence (IF). The levels of Interleukin-1 beta (IL-1β), Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-4 (IL-4) in the blood were measured using ELISA. AMS-17 treatment improved cognitive function, enhanced blood-brain barrier (BBB) integrity, and promoted remyelination in VaD mice. Additionally, AMS-17 reduced neurodegeneration and decreased the expression of NLRP3 and its associated proteins, Apoptosis-associated speck-like protein (ASC), and cleaved caspase-1 in the brain. It also lowered pro-inflammatory TNF-α and IL-1β levels, while increasing the anti-inflammatory IL-4 level in the blood. The findings of this study provide the first promising evidence for the use of AMS-17 in VaD treatment in mice. This study introduces AMS-17 as a novel chemical scaffold with NLRP3 inhibitory activity, which can be further developed for the treatment of VaD in humans.

DARPins as a novel tool to detect and degrade p73

The concept of Targeted Protein Degradation (TPD) has been introduced as an attractive alternative to the development of classical inhibitors. TPD can extend the range of proteins that can be pharmacologically targeted beyond the classical targets for small molecule inhibitors, as a binding pocket is required but its occupancy does not need to lead to inhibition. The method is based on either small molecules that simultaneously bind to a protein of interest and to a cellular E3 ligase and bring them in close proximity (molecular glue) or a bi-functional molecule synthesized from the chemical linkage of a target protein-specific small molecule and one that binds to an E3 ligase (Proteolysis Targeting Chimeras (PROTAC)). The further extension of this approach to bioPROTACs, in which a small protein-based binding module is fused directly to an E3 ligase or an E3 ligase adaptor protein, makes virtually all proteins amenable to targeted degradation, as this method eliminates the requirement for binding pockets for small molecules. Designed Ankyrin Repeat Proteins (DARPins) represent a very attractive class of small protein-based binding modules that can be used for the development of bioPTOTACS. Here we describe the characterization of two DARPins generated against the oligomerization domain and the SAM domain of the transcription factor p73, a member of the p53 protein family. The DARPins can be used for (isoform-)selective pulldown experiments both in cell culture as well as primary tissue lysates. We also demonstrate that they can be used for staining in cell culture experiments. Fusing them to the speckle type POZ protein (SPOP), an adaptor protein for cullin-3 E3 ligase complexes, yields highly selective and effective degraders. We demonstrate that selective degradation of the ΔNp73α isoform reactivates p53.

Nucleotide-binding oligomerization domain 1 (NOD1) regulates microglial activation in pseudorabies virus infection

The primary cause of viral encephalitis (VE) is invasion of the central nervous system (CNS) by the virus, which leads to neuroinflammation and poses a significant threat to global public health. Microglia, as CNS-resident macrophages, play a crucial role in neuroinflammation and are often identified as the preferred target for the prevention or treatment of VE. In this study, we used pseudorabies virus (PRV)-induced VE in mice and pigs as a model to investigate the regulation of microglial responses during viral encephalitis and explored the mechanism of microglial activation. Cellular experiments revealed that microglial activation was accompanied by cell migration, characteristic morphological changes, phagocytosis, inflammatory cytokine production, and antigen presentation. Transcriptome analysis revealed that genes related to inflammation in PRV-infected BV2 cells were significantly enriched. The expression of the NOD1 gene in BV2 cells was significantly increased during PRV infection, after which NOD1 in BV2 cells was silenced by siRNA and overexpressed via a plasmid. NOD1 was found to be involved in the secretion of cytokines in BV2 cells by regulating the MAPK/NF-κB signalling pathway. Mouse and pig experiments have shown that NOD1 is involved in the secretion of cytokines by microglia by regulating the MAPK/NF-κB signalling pathway during PRV infection.

Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation.

Rickettsia spp. cause life-threatening diseases in humans. The fundamental pathophysiological changes in fatal rickettsial diseases are disrupted endothelial barrier and increased microvascular permeability. However, it remains largely unclear how rickettsiae induce microvascular endothelial injury. In the present study, we demonstrated that Rickettsia conorii infection disrupts the continuous immunofluorescence expression of the interendothelial tight junction protein, zonula occludens-1 (ZO-1), in infected monolayers of microvascular endothelial cells (MVECs), accompanied by significantly diminished total expression levels of ZO-1. Interestingly, R. conorii activated inflammasome in MVECs, as evidenced by cleaved caspase-1 and IL-1β in the cell lysates in association with significantly elevated expression levels of nucleotide binding and oligomerization domain, leucine-rich repeat, and pyrin containing protein 3 (NLRP3). Furthermore, selective inhibition of NLRP3 by MCC950 significantly suppressed the activation and cleavage of caspase-1 induced by R. conorii in endothelial cells, which further prevented the disruption of interendothelial junctions and reduction of ZO-1 expression. Of note, pharmaceutical inhibition of NLRP3 mitigated the disrupted endothelial integrity caused by R. conorii, measured by fluorescein isothiocyanate-dextran passage in a Transwell assay, independent of bacterial growth and cellular cytotoxicity. Taken together, our results suggest that R. conorii affected microvascular endothelial junction integrity likely via diminishing and interrupting the junctional protein ZO-1 in association with activating NLRP3 inflammasome. These data not only highlight the potential of ZO-1 as a biomarker for Rickettsia-induced microvascular injury but also provide insight into targeting NLRP3 inflammasome/ZO-1 signaling as a potentially adjunctive therapeutic approach for severe rickettsioses.

Extracellular Vesicles From LPS-Treated PDLSCs Induce NLRP3 Inflammasome Activation in Periodontitis.

This study aimed to investigate the effects of lipopolysaccharide (LPS)-pretreated primary periodontal ligament stem cell (PDLSC)-derived extracellular vesicles (EVs) (L-PDLSC-EVs) on periodontitis. PDLSCs were obtained from mouse periodontal ligaments via enzymatic digestion. An invitro inflammatory microenvironment for PDLSCs was established using LPS, and L-PDLSC-EVs were isolated through ultracentrifugation and identified. EVs from different treatments were co-incubated with RAW264.7 macrophages (Mφs) or periodontal ligament fibroblasts (PLFs) and their co-cultures, whereafter the biological behaviors in Mφs and PLFs were evaluated. Periodontitis mouse models were established to verify the role of L-PDLSC-EVs and the mechanisms involved. There were no significant changes in the characteristics of L-PDLSC-EVs compared with control EVs. L-PDLSC-EVs promoted M1-type Mφ polarization and activated the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome. Furthermore, L-PDLSC-EVs promoted PLF cytotoxicity and apoptosis by enhancing the M1 polarization of Mφs. In periodontitis mouse models, L-PDLSC-EVs facilitated alveolar bone loss, PLF injury, and inflammatory responses, accompanied by an increased proportion of M1-type Mφs and reinforced NLRP3 inflammasome activation. L-PDLSC-EVs promoted PLF injury and exacerbated periodontitis through activating the NLRP3 inflammasome and promoting the polarization of M1-type Mφs, providing novel insights for the periodontitis progression.

NLRP3 inflammasome in expressed prostatic secretions as a potential biomarker of chronic prostatitis/chronic pelvic pain syndrome.

Pyroptosis has been implicated in the progression of chronic prostatitis (CP)/chronic pelvic pain syndrome (CPPS). The present study was performed to explore the diagnostic value of the levels of the pyroptosis-related protein nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome in the expressed prostatic secretions (EPS) of patients with CP. A total of 167 CP patients, including 85 National Institutes of Health (NIH)-IIIA CP patients and 82 NIH-IIIB CP patients, as well as 80 benign prostatic hyperplasia (BPH) patients and 80 healthy controls, were enrolled. The levels of NLRP3, interleukin 1 beta (IL-1β), and interleukin 18 (IL-18) in EPS were detected using an enzyme-linked immunosorbent assay (ELISA). Disease severity was assessed using the Bergman CP scale. Differences in EPS NLRP3 inflammasome levels between the groups were analyzed, and receiver operating characteristic (ROC) curves were used to investigate the clinical value of the NLRP3 inflammasome in the diagnosis of CP. The numerical rating scale (NRS), the National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) and the Danish Prostatic Symptom Score (DAN-PSS-1) were applied to evaluate symptom severity. The cutoff value of NLRP3 expression was calculated using R language. NLRP3 inflammasome levels in EPS were significantly higher in CP patients of NIH-IIIA and NIH-IIIB compared to the BPH patients and controls. NLRP3 levels in EPS were positively associated with Bergman grade. In addition, NRS levels were in a positive relationship with NIH-CPSI and DAN-PSS-1. The ROC curve analysis demonstrated that NLRP3 in EPS may act as a decent indicator for the diagnosis of CP/CPPS. The cutoff value of EPS NLRP3 expression was ≥55.25 ng/mL. NLRP3 levels in EPS were significantly higher in NIH-IIIA and NIH-IIIB patients compared to BPH patients and healthy controls. NLRP3 inflammasome levels in EPS may be valuable as diagnostic indicators, and targeting chemokines may present a promising approach to treatment for those suffering from CPPS.

Cryo-EM structure of Nipah virus L-P polymerase complex

Nipah virus (NiV) is a non-segmented, negative-strand (NNS) RNA virus, belonging to Paramyxoviridae. The RNA polymerase complex, composed of large (L) protein and tetrameric phosphoprotein (P), is responsible for genome transcription and replication by catalyzing NTP polymerization, mRNA capping and cap methylation. Here, we determine the cryo-electron microscopy (cryo-EM) structure of fully bioactive NiV L-P polymerase complex at a resolution of 3.19 Å. The L-P complex displays a conserved architecture like other NNS RNA virus polymerases and L interacts with the oligomerization domain and the extreme C-terminus region of P tetramer. Moreover, we elucidate that NiV is naturally resistant to the allosteric L-targeting inhibitor GHP-88309 due to the conformational change in the drug binding site. We also find that the non-nucleotide drug suramin can inhibit the NiV L-P polymerase activity at both the enzymatic and cellular levels. Our findings have greatly enhanced the molecular understanding of NiV genome replication and transcription and provided the rationale for broad-spectrum polymerase-targeted drug design.

Antiviral peptide targeting P protein oligomerization: proof of concept for mononegaviruses.

In Mononegavirales, phosphoproteins (P) are essential polymerase cofactors, forming oligomers and interacting with viral components to facilitate replication. Previous studies have demonstrated that a P-derived peptide (PFr) from the respiratory syncytial virus (RSV), containing the oligomerization domain (OD) and C-terminal domain (CTD), effectively inhibits RSV replication. Here, we extend this approach to paramyxoviruses, including HPIV3, MeV and MuV. Customized PFrs exhibited potent inhibitory effects against their respective viruses, with IC50 values below 100 nM, while showing minimal cytotoxicity. These findings highlight the potential of targeting P oligomerization as a broad-spectrum antiviral strategy for paramyxoviruses and other mononegaviruses.

The role of inflammasomes in hepatocellular carcinoma: Mechanisms and therapeutic insights.

Hepatocellular carcinoma is among the most frequent forms of primary liver cancer and develops within a context of chronic inflammation, frequently associated with a multitude of risk factors, including viral infections, metabolic dysfunction-associated fatty liver disease, metabolic dysfunction-associated steatohepatitis and liver fibrosis. The tumor microenvironment is crucial for the progression of HCC, as immune cells, tumor-associated fibroblasts and hepatic stellate cells interact to promote chronic inflammation and tumor spread. Inflammasomes, the multiprotein complexes that launch the innate immune response, emerge as important mediators in the pathogenesis of HCC. Among others, the inflammasome Nucleotide-binding oligomerization domain, Leucine rich Repeat (NLR) and Pyrin (NLRP) 3 (NLRP3), and absent in melanoma 2 (AIM2), exhibit a dual role in HCC background. It has been reported that they can exert oncosuppressive functions by triggering the inflammatory death of cancer cells. Vice versa, chronic activation contributes to the development of a pro-tumorigenic environment, thus supporting tumor growth. In addition, other inflammasomes such as Nucleotide-binding oligomerization domain, Leucine rich Repeat (NLR) and Pyrin (NLRP) 6 and 12 (NLRP6 and NLRP12, respectively) regulate HCC onset and progression, although more experimental evidence is required. This review focuses on the molecular mechanisms underpinning the inflammasome's contribution to the onset, progression and spread of HCC. Moreover, we will explore the potential therapeutic approaches currently under investigation, which aim to improve the efficacy and reduce the side effects of the treatments currently available. Targeting inflammasomes may be a promising therapeutic strategy for the treatment of HCC, offering new opportunities to improve patient prognosis.