NLR Family Apoptosis Inhibitory Protein Research Articles

Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.

The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.

Structural basis for flagellin-induced NAIP5 activation.

The NAIP (NLR family apoptosis inhibitory protein)/NLRC4 (NLR family CARD containing protein 4) inflammasome senses Gram-negative bacterial ligand. In the ligand-bound state, the winged helix domain of NAIP forms a steric clash with NLRC4 to open it up. However, how ligand binding activates NAIP is less clear. Here, we investigated the dynamics of the ligand-binding region of inactive NAIP5 and solved the cryo-EM structure of NAIP5 in complex with its specific ligand, FliC from flagellin, at 2.9-Å resolution. The structure revealed a "trap and lock" mechanism in FliC recognition, whereby FliC-D0C is first trapped by the hydrophobic pocket of NAIP5, then locked in the binding site by ID (insertion domain) and C-terminal tail of NAIP5. The FliC-D0N domain further inserts into ID to stabilize the complex. According to this mechanism, FliC triggers the conformational change of NAIP5 by bringing multiple flexible domains together.

Changes in expression levels of Nod-like receptors in the spleen of ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) have critical effects on interfaces of the immune and reproductive systems, and the spleen plays a key role in both innate and adaptive immune functions. It is hypothesized that NLR family participates in maternal splenic immune regulation during early pregnancy in sheep. In this study, maternal spleens were collected on day 16 of the estrous cycle, and days 13, 16 and 25 of gestation (n = 6 for each group) in ewes. Expression of NLR family, including NOD1, NOD2, class II transactivator (CIITA), NLR family apoptosis inhibitory protein (NAIP), nucleotide-binding oligomerization domain, Leucine rich repeat and Pyrin domain containing 1 (NLRP1), NLRP3 and NLRP7, was analyzed using quantitative real-time PCR, Western blot and immunohistochemistry analysis. The results revealed that expression levels of NOD1, NOD2, CIITA and NLRP3 were downregulated at days 13 and 16 of pregnancy, but expression of NLRP3 was increased at day 25 of pregnancy. In addition, expression values of NAIP and NLRP7 mRNA and proteins were improved at days 16 and 25 of pregnancy, and NLRP1 was peaked at days 13 and 16 of pregnancy in the maternal spleen. Furthermore, NOD2 and NLRP7 proteins were limited to the capsule, trabeculae and splenic cords. In summary, early pregnancy changes expression of NLR family in the maternal spleen, which may be related with the maternal splenic immunomodulation during early pregnancy in sheep.

Modulation of Nod-like Receptor Expression in the Thymus during Early Pregnancy in Ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep.

AIM2 Inflammasome's First Decade of Discovery: Focus on Oral Diseases.

A common feature of many acute and chronic oral diseases is microbial-induced inflammation. Innate immune responses are the first line of defense against pathogenic microorganisms and are initiated by pattern recognition receptors (PRRs) that specifically recognize pathogen-associated molecular patterns and danger-associated molecular patterns. The activation of certain PRRs can lead to the assembly of macromolecular oligomers termed inflammasomes, which are responsible for pro-inflammatory cytokine maturation and secretion and thus activate host inflammatory responses. About 10 years ago, the absent in melanoma 2 (AIM2) was independently discovered by four research groups, and among the “canonical” inflammasomes [including AIM2, NLR family pyrin domain (NLRP)1, NLRP3, NLR family apoptosis inhibitory protein (NAIP)/NLR family, caspase activation and recruitment domain (CARD) containing (NLRC)4, and pyrin], AIM2 so far is the only one that simultaneously acts as a cytosolic DNA sensor due to its DNA-binding ability. Undoubtedly, such a double-faceted role gives AIM2 greater mission and more potential in the mediation of innate immune responses. Therefore, AIM2 has garnered much attention from the broad scientific community during its first 10 years of discovery (2009–2019). How the AIM2 inflammasome is related to oral diseases has aroused debate over the past few years and is under active investigation. AIM2 inflammasome may potentially be a key link between oral diseases and innate immunity. In this review, we highlight the current knowledge of the AIM2 inflammasome and its critical role in the pathogenesis of various oral diseases, which might offer future possibilities for disease prevention and targeted therapy utilizing this continued understanding.

AK002210 promotes the proliferation, migration and invasion of trophoblast cell through regulating miR-590/NAIP signal axis

Preeclampsia (PE) is a pregnancy-related syndrome and has become the leading cause of maternal and neonatal morbidity and mortality. LncRNA has been elucidated to play critical roles in the phenotype of trophoblast cells. However, the effect of AK002210 has not been reported. We aim to investigate the effect of AK002210 on the phenotype of trophoblast cells. Quantitative reverse transcription PCR was used to assess the gene expression. CCK-8 assay was used to evaluate the cell proliferation. Transwell assay was performed to detect the migration and invasion of trophoblast cells. Luciferase assay and rescue experiment were carried out to verify the interaction between miR-590–3p and AK002210 as well as NLR family apoptosis inhibitory protein (NAIP). The results revealed that AK002210 promoted the proliferation, migration and invasion of trophoblast cell while AK002210 knockdown inhibited that. Mechanically, we found that AK002210 was targeted by miR-590–3p. Moreover, miR-590–3p also directly targets NAIP which served as a ceRNA of AK002210. Rescue experiment showed that miR-590–3p reversed the effect of AK002210 which further confirmed their interaction. Moreover, AK002210 was proved to participated in the regulation of ERK/MMP-2 signal axis. In conclusion, we found that AK002210 knockdown may play a critical role in the progression of PE via miR-590–3p/NAIP and ERK/MMP signaling. It has potential to be a novel prognostic or therapeutic marker of PE.

Filamin B extensively regulates transcription and alternative splicing, and is associated with apoptosis in HeLa cells.

Post-transcriptional mechanisms are an important approach in the treatment of cancer, and may also be hijacked by tumor cells to help adapt to the local microenvironment. Filamin B (FLNB), an actin-binding protein that provides crucial scaffolds for cell motility and signaling, has also been identified as an RNA-binding protein. Recent studies demonstrated that FLNB might play an important role, not only in skeletal development, but also in regulating tumorigenesis; however, the effects of dysregulated expression of FLNB at the molecular level are not clear. In the present study, RNA-sequencing was performed to analyze changes in overall transcriptional and alternative splicing between the knocked-down FLNB and the control in HeLa cells. Decreased FLNB levels resulted in significantly lower apoptosis compared with control cells. FLNB knockdown extensively regulated the expression of genes in cell apoptosis, tumorigenesis, metastases, transmembrane transport and cartilage development. Moreover, FLNB regulated alternative splicing of a large number of genes involved in ‘cell death’ and the ‘apoptotic process’. Some genes and alternative splicing related to skeletal development were enriched and regulated by FLNB. Reverse transcription-quantitative-PCR identified FLNB-regulated transcription and alternative splicing of genes, such as NLR family apoptosis inhibitory protein, interleukin 23 subunit α, metastasis associated lung adenocarcinoma transcript 1, phosphofurin acidic cluster sorting protein 2, bone morphogenetic protein 7, matrix metallopeptidase 13, collagen type II α 1 chain, fibroblast growth factor receptor 2 and vitamin D receptor. The present study is the first study, to the best of the authors’ knowledge, to provide transcriptome-wide analysis of differential gene expression and alternative splicing upon FLNB silencing. The present results suggested that FLNB may play an important regulatory role in cervical cancer cell apoptosis via regulation of transcription and alternative splicing, which provide insight for the current understanding of the mechanisms of FLNB-mediated gene regulation.

Combined Use of Three Machine Learning Modeling Methods to Develop a Ten-Gene Signature for the Diagnosis of Ventilator-Associated Pneumonia.

BackgroundThis study aimed to use three modeling methods, logistic regression analysis, random forest analysis, and fully-connected neural network analysis, to develop a diagnostic gene signature for the diagnosis of ventilator-associated pneumonia (VAP).Material/MethodsGSE30385 from the Gene Expression Omnibus (GEO) database identified differentially expressed genes (DEGs) associated with patients with VAP. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment identified the molecular functions of the DEGs. The least absolute shrinkage and selection operator (LASSO) regression analysis algorithm was used to select key genes. Three modeling methods, including logistic regression analysis, random forest analysis, and fully-connected neural network analysis, also known as also known as the feed-forward multi-layer perceptron (MLP), were used to identify the diagnostic gene signature for patients with VAP.ResultsSixty-six DEGs were identified for patients who had VAP (VAP+) and who did not have VAP (VAP−). Ten essential or feature genes were identified. Upregulated genes included matrix metallopeptidase 8 (MMP8), arginase 1 (ARG1), haptoglobin (HP), interleukin 18 receptor 1 (IL18R1), and NLR family apoptosis inhibitory protein (NAIP). Down-regulated genes included complement factor D (CFD), pleckstrin homology-like domain family A member 2 (PHLDA2), plasminogen activator, urokinase (PLAU), laminin subunit beta 3 (LAMB3), and dual-specificity phosphatase 2 (DUSP2). Logistic regression, random forest, and MLP analysis showed receiver operating characteristic (ROC) curve area under the curve (AUC) values of 0.85, 0.86, and 0.87, respectively.ConclusionsLogistic regression analysis, random forest analysis, and MLP analysis identified a ten-gene signature for the diagnosis of VAP.

Retracted: MicroRNA‐4328 promotes lens epithelial cell apoptosis by targeting NLR family, apoptosis inhibitory protein in age‐related cataract

Cataract is the leading cause of blindness in the world while the molecular mechanisms of cataracts pathogenesis are not well elucidated. Dysregulated microRNA (miRNA) expressions have been implicated in cataract. However, the precise role of miR-4328 in cataract is still unknown. We compared the expression level of total miR-4328 between clinical samples from healthy people and cataract patient and between UV-irradiated and control lens epithelial cells. We determined the effect of miR-4328 on lens epithelial cell proliferation and apoptosis by suppressing miR-4328. We further predicted NLR Family Apoptosis Inhibitory Protein (NAIP) as potential target of miR-4328 and continued to evaluate the effects of NAIP on cell proliferation and apoptosis. MiR-4328 was up-regulated in cataract sample and in UV-irradiated lens epithelial cells. Suppressing MiR-4328 promoted cell proliferation and inhibited apoptosis. MiR-4328 targeted NAIP and suppressed its expression. Knocking down NAIP abolished the effects of miR-4328 on cell proliferation and inhibited apoptosis. MiR-4328 promotes lens epithelial cell apoptosis by targeting NAIP in age-related cataract. SIGNIFICANCE OF THE STUDY: MiR-4328 targeted NAIP and suppressed its expression. Knocking down NAIP abolished the effects of miR-4328 on cell proliferation and inhibited apoptosis. MiR-4328 promotes lens epithelial cell apoptosis by targeting NAIP in age-related cataract.

Downregulation of microRNA-1 and microRNA-145 contributes synergistically to the development of colon cancer.

The aim of the present study was to identify the differentially expressed microRNAs (miRNAs or miRs) in patients with colon cancer and examine their roles in the pathogenesis of the disease. The expression profiles of miRNAs were examined in tumor tissue samples collected from 20 patients with histologically confirmed colon cancer and in the adjacent non-cancerous control tissues using microarray analysis. We found that numerous miRNAs were differentially expressed in the colon cancer tissues compared with the control tissues. Following functional analysis, we noted that three miRNAs which were significantly downregulated, miR-145, miR-451 and miR-1, shared the same target gene, [NLR family, apoptosis inhibitory protein (NAIP)], which has previously been reported to be involved in the development of colon cancer. We then confirmed that NAIP is a target gene of miR-145 and miR-1, but not of miR-451, using a luceriferase assay, and we confirmed the expression patterns of these miRNAs and NAIP in the tumor tissue, as well as in the adjacent non-cancerous control tissues. Additionally, in order to examine the function of miR-145 and miR-1 in human colon cancer, we transiently transfected a human colon cancer cell line with miR-145 and miR-1 mimics or inhibitors, and the results of MTT assay revealed that the re-expression of miR-145 and miR-1 inhibited the survival of colon cancer cells, which may be attributed to the inhibition of the anti-apoptotic activity of NAIP. Our findings demonstrated that miR-145 and miR-1 play a negative regulatory role in the proliferation of colon cancer by targeting NAIP; thus, miR-145 and miR-1 may prove to be novel therapeutic targets in the treatment of colon cancer.

Joint effect of the SMN2 and SERF1A genes on childhood-onset types of spinal muscular atrophy in Serbian patients.

Spinal muscular atrophy (SMA) is caused by functional loss of the survival of motor neuron 1 (SMN1) gene. Despite genetic homogeneity, phenotypic variability indicates the involvement of disease modifiers. SMN1 is located in 5q13.2 segmental duplication, enriched in genes and prone to unequal rearrangements, which results in copy number polymorphism (CNP). We examined the influence of CNP of 5q13.2 genes and their joint effect on childhood-onset SMA phenotype. Multiplex ligation-dependent probe amplification (MLPA) was used to construct 5q13.2 alleles and assess copy number of the SMN2, small EDRK-rich factor 1A (SERF1A) and NLR family apoptosis inhibitory protein (NAIP) genes in 99 Serbian patients with SMN1 homozygous absence (23-type I, 37-type II and 39-mild type III) and 122 patients' parents. Spearman rank test was performed to test correlation of individual genes and SMA type. Generalized linear models and backward selection were performed to obtain a model explaining phenotypic variation with the smallest set of variables. 5q13.2 alleles most commonly associated with type I harbored large-scale deletions, while those detected in types II and III originated from conversion of SMN1 to SMN2. Inverse correlation was observed between SMN2, SERF1A and NAIP CNP and SMA type (P=2.2e-16, P=4.264e-10, P=2.722e-8, respectively). The best minimal model describing phenotypic variability included SMN2 (P<2e-16), SERF1A (P<2e-16) and their interaction (P=0.02628). SMN2 and SERF1A have a joint modifying effect on childhood-onset SMA phenotype.

Sulforaphane inhibits multiple inflammasomes through an Nrf2-independent mechanism.

The inflammasomes are intracellular complexes that have an important role in cytosolic innate immune sensing and pathogen defense. Inflammasome sensors detect a diversity of intracellular microbial ligands and endogenous danger signals and activate caspase-1, thus initiating maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18. These events, although crucial to the innate immune response, have also been linked to the pathology of several inflammatory and autoimmune disorders. The natural isothiocyanate sulforaphane, present in broccoli sprouts and available as a dietary supplement, has gained attention for its antioxidant, anti-inflammatory, and chemopreventive properties. We discovered that sulforaphane inhibits caspase-1 autoproteolytic activation and interleukin-1β maturation and secretion downstream of the nucleotide-binding oligomerization domain-like receptor leucine-rich repeat proteins NLRP1 and NLRP3, NLR family apoptosis inhibitory protein 5/NLR family caspase-1 recruitment domain-containing protein 4 (NAIP5/NLRC4), and absent in melanoma 2 (AIM2) inflammasome receptors. Sulforaphane does not inhibit the inflammasome by direct modification of active caspase-1 and its mechanism is not dependent on protein degradation by the proteasome or de novo protein synthesis. Furthermore, sulforaphane-mediated inhibition of the inflammasomes is independent of the transcription factor nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and the antioxidant response-element pathway, to which many of the antioxidant and anti-inflammatory effects of sulforaphane have been attributed. Sulforaphane was also found to inhibit cell recruitment to the peritoneum and interleukin-1β secretion in an in vivo peritonitis model of acute gout and to reverse NLRP1-mediated murine resistance to Bacillus anthracis spore infection. These findings demonstrate that sulforaphane inhibits the inflammasomes through a novel mechanism and contributes to our understanding of the beneficial effects of sulforaphane.

The NLRP1 inflammasomes.

Inflammasomes are cytosolic protein complexes that serve as platforms for the recruitment and activation of the pro-inflammatory CASPASE-1 protease. CASPASE-1 activation leads to processing and maturation of the cytokines interleukin-1β and interleukin-18 and a lytic form of cell death termed pyroptosis. Inflammasome assembly is initiated by cytosolic sensors in response to microbial infections. Many of these sensors, including NLRP1 (NLR family, pyrin domain containing 1), are described to form an inflammasome, but until recently, the mechanism of inflammasome activation and its physiological functions in host defense have remained unclear. In the last few years, important advances in our understanding of NLRP1 biology have been achieved. In this review, we discuss the activation of NLRP1 by various stimuli, including Bacillus anthracis lethal toxin, Toxoplasma gondii, muramyl dipeptide, and host intracellular ATP depletion. The role NLRP1 plays in pathogen recognition and resistance during infection is also discussed, as is the regulation of NLRP1 by host and viral proteins. We conclude by discussing the unexpected differences in the mechanism of NLRP1 inflammasome activation, as compared to the activation of other inflammasomes, such as the NAIP (NLR family, apoptosis inhibitory protein)/NLRC4 inflammasomes.

Abstract 816: Functional analysis of miRNA in chemotherapy resistant neuroblastoma

Abstract Neuroblastoma is a paediatric cancer of the sympathetic nervous system, accounting for 15 % of childhood cancer related deaths. Treatment of neuroblastoma (NB) patients involves intensive rounds of multimodal chemotherapy, with initial responses often being positive, but with a high likelihood of relapse and development of therapy resistance. The aim of this project is to identify miRNAs associated with resistance to chemotherapeutics in NB, and elucidate the molecular mechanisms involved. In order to understand miRNA involvement in NB drug resistance, two MYCN amplified (KellyCis83, CHP-212Cis100) and one MYCN single copy cell line (SK-N-ASCis24) resistant to cisplatin (CDDP) were developed using ‘drug pulse selection’. An increase in resistance to CDDP was confirmed by examination of IC50s before and after selection. Expression of 770 miRNAs was analyzed using Taqman arrays and drug resistant profiles were compared to those of parental lines. 41, 51 and 72 miRNAs were differentially expressed in KellyCis83, SKNASCis24 and CHP-212Cis100 respectively. A total of 8 miRNA exhibited the same pattern of differential expression in both KellyCis83 and SKNASCis24, 6 miRNA in KellyCis83 and CHP-212Cis100, and 8 miRNA in SKNASCis24 and CHP-212Cis100. 2 miRNA were commonly differentially expressed across all three cell lines. Assessment of cellular proliferation revealed resistant variants KellyCis83 and CHP-212Cis100 grown in the presence of low-dose CDDP consistently outgrew cells grown in normal media. This increase in growth is not observed in the parental cell lines. MiRNA expression profiles of cells challenged with 0.2 μM CDDP were obtained and 31 miRNAs differentially expressed in KellyCis83 and CHP-212Cis100 identified. Many miRNAs identified have been previously associated with drug resistance, confirming the efficacy of this approach. For example, in the panel of 8 miRNA common to KellyCis83 and SK-N-ASCis24, all miRNA are predicted to target at least one of two key copper transporters involved in CDDP transport; hCtr1 and ATP7A. Each cell line was also analyzed using aCGH to identify DNA copy number alterations. A region of focal gain containing NAIP (NLR family, apoptosis inhibitory protein) was identified in SKNASCis24, and a 15-fold increase of NAIP mRNA was confirmed using qPCR. NAIP inhibits caspases-3, 7 and 9 resulting in increased cell survival. This discovery was of interest because 13 of the 33 miRNAs down-regulated in SK-N-ASCis24 are predicted to target NAIP. SiRNA depletion of NAIP mRNA led to increased sensitivity to etoposide, but not CDDP in SK-N-ASCis24. However, both drugs have different mechanisms of action and it is possible that NAIP has a greater influence over the function of etoposide in our model. In conclusion, we have identified miRNA associated with drug resistance in NB, and are now working to elucidate the mechanism of miRNA involvement in the process of multidrug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 816. doi:1538-7445.AM2012-816