P2X7R Modulates NEK7-NLRP3 Interaction to Exacerbate Experimental Autoimmune Prostatitis via GSDMD-mediated Prostate Epithelial Cell Pyroptosis.

Chronic prostatitis is one of the most common urologic diseases that troubles young men, with unclear etiology and ineffective treatment approach. Pyroptosis is a novel model of cell death, and its roles in chronic prostatitis are unknown. In this study, P2X7R, NEK7, and GSDMD-NT expression levels were detected in prostate tissues from benign prostate hyperplasia (BPH) patients and experiment autoimmune prostatitis (EAP) mice. P2X7R agonist, antagonist, NLRP3 inhibitor, and disulfiram were used to explore the roles of the P2X7R-NEK7-NLRP3 axis in prostate epithelial cell pyroptosis and chronic prostatitis development. We found that P2X7R, NEK7, and GSDMD-NT were highly expressed in the prostate epithelial cells of BPH patients with prostatic inflammation and EAP mice. Activation of P2X7R exacerbated prostatic inflammation and increased NLRP3 inflammasome component expressions and T helper 17 (Th17) cell proportion. Moreover, P2X7R-mediated potassium efflux promoted NEK7-NLRP3 interaction, and NLRP3 assembly and activation, which caused GSDMD-NT-mediated prostate epithelial cell pyroptosis to exacerbate EAP development. Disulfiram could effectively improve EAP by inhibiting GSDMD-NT-mediated prostate epithelial cell pyroptosis. In conclusion, the P2X7R-NEK7-NLRP3 axis could promote GSDMD-NT-mediated prostate epithelial cell pyroptosis and chronic prostatitis development, and disulfiram may be an effective drug to treat chronic prostatitis.

Investigation on the molecular mechanism of SPA interference with osteogenic differentiation of bone marrow mesenchymal stem cells.

Binding of Staphylococcus aureus protein A (SPA) to osteoblasts induces apoptosis and inhibits bone formation. Bone marrow-derived mesenchymal stem cells (BMSCs) have the ability to differentiate into bone, fat and cartilage. Therefore, it was important to analyze the molecular mechanism of SPA on osteogenic differentiation. We introduced transcript sequence data to screen out differentially expressed genes (DEGs) related to SPA-interfered BMSC. Protein-protein interaction (PPI) network of DEGs was established to screen biomarkers associated with SPA-interfered BMSC. Receiver operating characteristic (ROC) curve was plotted to evaluate the ability of biomarkers to discriminate between two groups of samples. Finally, we performed GSEA and regulatory analysis based on biomarkers. We identified 321 DEGs. Subsequently, 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap and Kif14) were identified by hubba algorithm in PPI. ROC analysis showed that six biomarkers could clearly discriminate between normal differentiated and SPA-interfered BMSC. Moreover, we found that these biomarkers were mainly enriched in the pyrimidine metabolism pathway. We also constructed '71 circRNAs-14 miRNAs-5 mRNAs' and '10 lncRNAs-5 miRNAs-2 mRNAs' networks. Kntc1 and Asf1b genes were associated with rno-miR-3571. Nek2 and Asf1b genes were associated with rno-miR-497-5p. Finally, we found significantly lower expression of six biomarkers in the SPA-interfered group compared to the normal group by RT-qPCR. Overall, we obtained 6 biomarkers (Cenpf, Kntc1, Nek2, Asf1b, Troap, and Kif14) related to SPA-interfered BMSC, which provided a theoretical basis to explore the key factors of SPA affecting osteogenic differentiation.

Genetically evaluating the causal role of peripheral immune cells in colorectal cancer: a two-sample Mendelian randomization study.

BACKGROUND: Investigating novel therapeutic strategies for colorectal cancer (CRC) is imperative. However, there is limited research on the use of drugs to target peripheral blood immune cells in this context. To address this gap, we performed a two-sample Mendelian randomization (MR) analysis to identify potential therapeutic targets for CRC. METHODS: We applied two-sample MR to identify the causal relationship between peripheral blood immune cells and CRC. GWAS data were obtained from the IEU OPEN GWAS project. Based on the implications from the MR results, we conducted a comprehensive database search and genetic analysis to explore potential underlying mechanisms. We predicted miRNAs for each gene and employed extensive research for potential therapeutic applications. RESULTS: We have identified causal associations between two peripheral immune cells and colorectal cancer. Activated & resting Treg %CD4 + cell was positively associated with the risks of CRC, while DN (CD4-CD8-) %leukocyte cell exhibited a protective role in tumor progression. NEK7 (NIMA related kinase 7) and LHX9 (LIM homeobox 9) expressed in Treg cells were positively associated with CRC risks and may play a vital role in carcinogenesis. CONCLUSIONS: This study identified causal relationship between peripheral immune cell and CRC. Treg and DN T cells were implicated to own promoting and inhibiting effects on CRC progression respectively. NEK7 and LHX9 in Treg cells were identified as potential biotarget for antitumor therapies.

In silico analysis unveiling potential biomarkers in gallbladder carcinogenesis.

Gallbladder cancer (GBC) is a rare but very aggressive most common digestive tract cancer with a high mortality rate due to delayed diagnosis at the advanced stage. Moreover, GBC progression shows asymptomatic characteristics making it impossible to detect at an early stage. In these circumstances, conventional therapy like surgery, chemotherapy, and radiotherapy becomes refractive. However, few studies reported some molecular markers like KRAS (Kirsten Rat Sarcoma) mutation, upregulation of HER2/neu, EGFR (Epidermal Growth Factor Receptor), and microRNAs in GBC. However, the absence of some specific early diagnostic and prognostic markers is the biggest hurdle for the therapy of GBC to date. The present study has been designed to identify some specific molecular markers for precise diagnosis, and prognosis, for successful treatment of the GBC. By In Silico a network-centric analysis of two microarray datasets; (GSE202479) and (GSE13222) from the Gene Expression Omnibus (GEO) database, shows 50 differentially expressed genes (DEGs) associated with GBC. Further network analysis revealed that 12 genes are highly interconnected based on the highest MCODE (Molecular Complex Detection) value, among all three genes; TRIP13 (Thyroid Receptor Interacting Protein), NEK2 (Never in Mitosis gene-A related Kinase 2), and TPX2 (Targeting Protein for Xklp2) having highest network interaction with transcription factors and miRNA suggesting critically associated with GBC. Further survival analysis data corroborate the association of these genes; TRIP13, NEK2, and TPX2 with GBC. Thus, TRIP13, NEK2, and TPX2 genes are significantly correlated with a greater risk of mortality, transforming them from mere biomarkers of the GBC for early detections and may emerge as prognostic markers for treatment.

Inhibition of NLRP3 inflammasome activation by A20 through modulation of NEK7.

The NLRP3 inflammasome, a pivotal component of innate immunity, has been implicated in various inflammatory disorders. The ubiquitin-editing enzyme A20 is well known to regulate inflammation and maintain homeostasis. However, the precise molecular mechanisms by which A20 modulates the NLRP3 inflammasome remain poorly understood. Here, our study revealed that macrophages deficient in A20 exhibit increased protein abundance and elevated mRNA level of NIMA-related kinase 7 (NEK7). Importantly, A20 directly binds with NEK7, mediating its K48-linked ubiquitination, thereby targeting NEK7 for proteasomal degradation. Our results demonstrate that A20 enhances the ubiquitination of NEK7 at K189 and K293 ubiquitinated sites, with K189 playing a crucial role in the binding of NEK7 to A20, albeit not significantly influencing the interaction between NEK7 and NLRP3. Furthermore, A20 disrupts the association of NEK7 with the NLRP3 complex, potentially through the OTU domain and/or synergistic effect of ZnF4 and ZnF7 motifs. Significantly, NEK7 deletion markedly attenuates the activation of the NLRP3 inflammasome in A20-deficient conditions, both in vitro and in vivo. This study uncovers a mechanism by which A20 inhibits the NLRP3 inflammasome.

Discovery and Development of NLRP3 Inhibitors Targeting the LRR Domain to Disrupt NLRP3-NEK7 Interaction for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease. Targeting NLRP3 inflammasome, specifically its interaction with NEK7 via the LRR domain of NLRP3, is a promising therapeutic strategy. Our research aimed to disrupt this interaction by focusing on the LRR domain. Through virtual screening, we identified five compounds with potent anti-inflammatory effects and ideal LRR binding affinity. Lead compound C878-1943 underwent structural optimization, yielding pyridoimidazole derivatives with different anti-inflammatory activities. Compound I-19 from the initial series effectively inhibited caspase-1 and IL-1ß release in an adjuvant-induced arthritis (AIA) rat model, significantly reducing joint swelling and spleen/thymus indices. To further enhance potency and extend in vivo half-life, a second series including II-8 was developed, demonstrating superior efficacy and longer half-life. Both I-19 and II-8 bind to the LRR domain, inhibiting NLRP3 inflammasome activation. These findings introduce novel small molecule inhibitors targeting the LRR domain of NLRP3 protein and disrupt NLRP3-NEK7 interaction, offering a novel approach for RA treatment.

Abnormal methylation mediated upregulation of LINC00857 boosts malignant progression of lung adenocarcinoma by modulating the miR-486-5p/NEK2 axis.

LINC00857 is frequently dysregulated in varying cancers, which in turn exerts carcinogenic effects; however, its DNA methylation status in promoter region and molecular mechanisms underlying the progression of lung adenocarcinoma (LUAD) remain rarely understood. Through bioinformatics analysis, we examined the expression state and methylation site of LINC00857 in LUAD and further investigated the properties of LINC00857 as a competitive endogenous RNA in the cancer progression. The current study revealed that the overexpression of LINC00857 in LUAD tissue and cells was mainly caused by the hypomethylation of the promoter region. LINC00857 knockdown prominently reduced cell proliferation, impeded cell migration and invasion, and restrained lymph node metastasis, with enhancing radiosensitivity. The effects of LINC00857 on tumor growth were also investigated in nude mice models. Subsequently, the downstream factors, miR-486-5p and NEK2, were screened, and the putative regulatory axis was examined. Overall, the regulatory effect of methylation-mediated LINC00857 overexpression on miR-486-5p/NEK2 axis may be a new mechanism for LUAD progression.

Hit-to-Lead Optimization of the Natural Product Oridonin as Novel NLRP3 Inflammasome Inhibitors with Potent Anti-Inflammation Activity.

Targeting NLRP3 inflammasome with inhibitors is a novel strategy for NLRP3-driven diseases. Herein, hit compound 5 possessing an attractive skeleton was identified from our in-house database of oridonin, and then a potential lead compound 32 was obtained by optimization of 5, displaying two-digit nanomolar inhibition on NLRP3. Moreover, compound 32 showed enhanced safety index (SI) relative to oridonin (IC50 = 77.2 vs 780.4 nM, SI = 40.5 vs 8.5) and functioned through blocking ASC oligomerization and interaction of NLRP3-ASC/NEK7, thereby suppressing NLRP3 inflammasome assembly and activation. Furthermore, diverse agonists-induced activations of NLRP3 could be impeded by compound 32 without altering NLRC4 or AIM2 inflammasome. Crucially, compound 32 possessed tolerable pharmaceutical properties and significant anti-inflammatory activity in MSU-induced gouty arthritis model. Therefore, this work enriched the SAR of NLRP3 inflammasome inhibitors and provided a potential candidate for the treatment of NLRP3-associated diseases.

NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1.

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.

Inhibition of METTL3 Alleviates NLRP3 Inflammasome Activation via Increasing Ubiquitination of NEK7.

N6-methyladenosine (m6A) modification, installed by METTL3-METTL14 complex, is abundant and critical in eukaryotic mRNA. However, its role in oral mucosal immunity remains ambiguous. Periodontitis is a special but prevalent infectious disease characterized as hyperinflammation of oral mucosa and bone resorption. Here, it is reported that genetic deletion of Mettl3 alleviates periodontal destruction via suppressing NLRP3 inflammasome activation. Mechanistically, the stability of TNFAIP3 (also known as A20) transcript is significantly attenuated upon m6A modification. When silencing METTL3, accumulated TNFAIP3 functioning as a ubiquitin-editing enzyme facilitates the ubiquitination of NEK7 [NIMA (never in mitosis gene a)-related kinase 7], and subsequently impairs NLRP3 inflammasome assembly. Furtherly, Coptisine chloride, a natural small-molecule, is discovered as a novel METTL3 inhibitor and performs therapeutic effect on periodontitis. The study unveils a previously unknown pathogenic mechanism of METTL3-mediated m6A modifications in periodontitis and indicates METTL3 as a potential therapeutic target.

NEK2 promotes TP53 ubiquitination to enhance the proliferation and migration of TP53 wild-type glioblastoma cells.

The most common primary malignant tumor in the adult brain is glioblastoma multiforme (GBM); however, its underlying pathogenic mechanism remains elusive. The never in mitosis (NIMA)-related kinase 2 (NEK2) has been closely associated with the prognosis of various malignancies. Nevertheless, the complete elucidation of NEK2's potential clinical value, particularly in glioma prognosis and development, remains lacking. U87MG and A172 glioblastoma cells were infected with sh-NEK2 lentivirus or oe-NEK2 plasmid to investigate the effect of NEK2 on cell proliferation, migration, and invasion. Cell viability was measured using CCK-8 and colony formation assays, while Transwell assay was utilized to assess cell migration and invasion. Protein expression levels were determined through western blot analysis. Additionally, CGGA and TCGA databases were used for bioinformatics analysis in order to examine the NEK2 expression. Through comprehensive bioinformatics analysis, we identified elevated mRNA expression levels of NEK2 in gliomas compared to normal tissues, which correlated with poor prognosis among glioma patients. Moreover, functional experiments revealed that silencing NEK2 suppressed glioma cell proliferation while overexpression of NEK2 promoted migration and invasion capabilities. Finally, our study uncovered that NEK2 regulates the malignant progression of TP53 wild-type glioblastoma by facilitating TP53 ubiquitination.

Function of NEK2 in clear cell renal cell carcinoma and its effect on the tumor microenvironment.

BACKGROUND: Previous studies have revealed the critical functions of NEK2 in controlling the cell cycle which is linked to poor prognosis in multiple tumor types, but less research has been devoted to clear cell renal cell carcinoma (ccRCC). METHODS: We downloaded clinical data from the gene expression omnibus (GEO) and TCGA databases together with transcriptional and mutational datasets. Strongly coexpressed genes with NEK2 were extracted from TCGA-KIRC cohort, and were submitted to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analyses. According to NEK2 levels, the survival status, mutational characteristics, response to immunotherapy and sensitivity to drugs of the patients were studied. The potential correlations between NEK2 levels and immune cell state as well as immune cell infiltration were examined using the GEPIA, TIMER and TISIDB databases. Double immunofluorescence (IF) was performed to identify the NEK2 overexpression and relationship with CD8 in ccRCC. RESULTS: The NEK2 gene was overexpressed and would enhance the nuclear division and cell cycle activities in ccRCC. ccRCC patients with high NEK2 expression had worse clinical outcomes, higher mutation burden and better therapeutic response. Moreover, NEK2 gene overexpression was positively related to various immune cell marker sets, which was also proved by validation cohort, and more infiltration of various immune cells. CONCLUSION: ccRCC patients with NEK2 high expression have a poorer prognosis than those with NEK2 low expression, resulting from its function of promoting proliferation, accompanied by increased infiltration of CD8 + T cells and Tregs and T-cell exhaustion and will respond better to proper treatments.

ß-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells.

ß-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular ß-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of ß-catenin, suggestive of a Nek10 role in the control of ß-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for ß-catenin degradation, where it phosphorylates ß-catenin at Tyr30, located within the regulatory region governing ß-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of ß-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.

New treatment alternatives for primary and metastatic colorectal cancer by an integrated transcriptome and network analyses.

Metastatic colorectal cancer (CRC) is still in need of effective treatments. This study applies a holistic approach to propose new targets for treatment of primary and liver metastatic CRC and investigates their therapeutic potential in-vitro. An integrative analysis of primary and metastatic CRC samples was implemented for alternative target and treatment proposals. Integrated microarray samples were grouped based on a co-expression network analysis. Significant gene modules correlated with primary CRC and metastatic phenotypes were identified. Network clustering and pathway enrichments were applied to gene modules to prioritize potential targets, which were shortlisted by independent validation. Finally, drug-target interaction search led to three agents for primary and liver metastatic CRC phenotypes. Hesperadin and BAY-1217389 suppress colony formation over a 14-day period, with Hesperadin showing additional efficacy in reducing cell viability within 48 h. As both candidates target the G2/M phase proteins NEK2 or TTK, we confirmed their anti-proliferative properties by Ki-67 staining. Hesperadinin particular arrested the cell cycle at the G2/M phase. IL-29A treatment reduced migration and invasion capacities of TGF-ß induced metastatic cell lines. In addition, this anti-metastatic treatment attenuated TGF-ß dependent mesenchymal transition. Network analysis suggests IL-29A induces the JAK/STAT pathway in a preventive manner.

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1ß (IL-1ß) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Inhibition of NEK2 Promotes Chemosensitivity and Reduces KSHV-positive Primary Effusion Lymphoma Burden.

Non-Hodgkin lymphoma (NHL) is a common cancer in both men and women and represents a significant cancer burden worldwide. Primary effusion lymphoma (PEL) is a subtype of NHL infected with Kaposi sarcoma-associated herpesvirus (KSHV). PEL is an aggressive and lethal cancer with no current standard of care, owing largely to its propensity to develop resistance to current chemotherapeutic regimens. Here, we report a reliance of KSHV-positive PEL on the mitotic kinase, NEK2, for survival. Inhibition of NEK2 with the inhibitor, JH295, resulted in caspase 3-mediated apoptotic cell death of PEL. Furthermore, NEK2 inhibition significantly prolonged survival and reduced tumor burden in a PEL mouse model. We also demonstrate that the ABC transporter proteins, MDR1 and MRP, are most active in PEL and that inhibition of NEK2 in PEL reduced the expression and activity of these ABC transporter proteins, which are known to mediate drug resistance in cancer. Finally, we report that JH295 treatment sensitized lymphomas to other chemotherapeutic agents such as rapamycin, resulting in enhanced cancer cell death. Overall, these data offer important insight into the mechanisms underlying PEL survival and drug resistance, and suggest that NEK2 is a viable therapeutic target for PEL. SIGNIFICANCE: The mitotic kinase, NEK2, is important for the survival of KSHV-positive PEL. NEK2 inhibition resulted in PEL apoptosis and reduced tumor burden in a mouse model. NEK2 inhibition also reduced drug resistance.

The mesenchymal morphology of cells expressing the EML4-ALK V3 oncogene is dependent on phosphorylation of Eg5 by NEK7.

Echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) oncogenic fusion proteins are found in approximately 5% of non-small cell lung cancers. Different EML4-ALK fusion variants exist with variant 3 (V3) being associated with a significantly higher risk than other common variants, such as variant 1 (V1). Patients with V3 respond less well to targeted ALK inhibitors, have accelerated rates of metastasis, and have poorer overall survival. A pathway has been described downstream of EML4-ALK V3 that is independent of ALK catalytic activity but dependent on the NEK9 and NEK7 kinases. It has been proposed that assembly of an EML4-ALK V3-NEK9-NEK7 complex on microtubules leads to cells developing a mesenchymal-like morphology and exhibiting enhanced migration. However, downstream targets of this complex remain unknown. Here, we show that the microtubule-based kinesin, Eg5, is recruited to interphase microtubules in cells expressing EML4-ALK V3, whereas chemical inhibition of Eg5 reverses the mesenchymal morphology of cells. Furthermore, we show that depletion of NEK7 interferes with Eg5 recruitment to microtubules in cells expressing EML4-ALK V3 and cell length is reduced, but this is reversed by coexpression of a phosphomimetic mutant of Eg5, in a site, S1033, phosphorylated by NEK7. Intriguingly, we also found that expression of Eg5-S1033D led to cells expressing EML4-ALK V1 adopting a more mesenchymal-like morphology. Together, we propose that Eg5 acts as a substrate of NEK7 in cells expressing EML4-ALK V3 and Eg5 phosphorylation promotes the mesenchymal morphology typical of these cells.

The Mitochondrial Connection: The Nek Kinases' New Functional Axis in Mitochondrial Homeostasis.

Mitochondria provide energy for all cellular processes, including reactions associated with cell cycle progression, DNA damage repair, and cilia formation. Moreover, mitochondria participate in cell fate decisions between death and survival. Nek family members have already been implicated in DNA damage response, cilia formation, cell death, and cell cycle control. Here, we discuss the role of several Nek family members, namely Nek1, Nek4, Nek5, Nek6, and Nek10, which are not exclusively dedicated to cell cycle-related functions, in controlling mitochondrial functions. Specifically, we review the function of these Neks in mitochondrial respiration and dynamics, mtDNA maintenance, stress response, and cell death. Finally, we discuss the interplay of other cell cycle kinases in mitochondrial function and vice versa. Nek1, Nek5, and Nek6 are connected to the stress response, including ROS control, mtDNA repair, autophagy, and apoptosis. Nek4, in turn, seems to be related to mitochondrial dynamics, while Nek10 is involved with mitochondrial metabolism. Here, we propose that the participation of Neks in mitochondrial roles is a new functional axis for the Nek family.

Silencing of METTL3 inhibits m6A methylation of NEK7 to suppress pyrolysis in an HT-22 cell-based model of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) induces severe neurological damage, and its progression is driven by METTL3. This study aimed to investigate the role of METTL3 in ICH via in vitro experiments. For this purpose, HT-22 cells were treated with hemin to mimic ICH in vitro, followed by evaluating cell pyroptosis using flow cytometry, lactic dehydrogenase release analysis, enzyme-linked immunosorbent assay, and western blotting. Moreover, N6-methyl adenosine (m6A) methylation of NEK7 was assessed using methylated RNA immunoprecipitation, RNA immunoprecipitation, dual-luciferase reporter assay, and quantitative real-time polymerase chain reaction. Results indicated that knockdown of METTL3 inhibited hemin-induced pyroptosis and suppressed m6A methylation of NEK7 due to METTL3 downregulation, reducing NEK7 mRNA stability. The effects on METTL3-induced cell pyroptosis were abrogated by overexpressing NEK7, while IGF2BP2 increased NEK7 expression. Similarly, IGF2BP2 silence downregulated NEK7 expression mediated by METTL3. In conclusion, silencing of METTL3 inhibited hemin-induced HT-22 cell pyroptosis by suppressing m6A methylation of NEK7, which was recognized by IGF2BP2. These findings are envisaged to identify a novel therapeutic strategy for ICH.

Structural basis for the oligomerization-facilitated NLRP3 activation.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer's interfaces reduce IL-1ß signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.