Neuronal apoptosis inhibitory protein (NAIP) localizes to the cytokinetic machinery during cell division

The neuronal apoptosis inhibitory protein (NAIP) is a constituent of the inflammasome and a key component of the innate immune system. Here we use immunofluorescence to position NAIP within the cytokinetic apparatus, contiguous to chromosomal passenger complex (CPC), Centralspindlin, PRC1 and KIF4A. During metaphase, NAIP accumulates in the mitotic spindle poles and is shown in spindle microtubules; in anaphase NAIP is detected in the middle of the central spindle. At the end of cytokinesis, NAIP is localized in the outlying region of the stem body, the center of the intercellular bridge formed between daughter cells prior to cellular abscission. We also describe the sustained presence of NAIP mRNA and protein throughout the cell cycle with a significant increase observed in the G2/M phase. Consistent with a role for NAIP in cytokinesis, NAIP overexpression in HeLa cells promotes the acquisition of a multinuclear phenotype. Conversely, NAIP siRNA gene silencing results in an apoptotic lethal phenotype. Our confocal and super resolution stimulated-emission-depletion (STED) examination of mammalian cell cytokinesis demonstrate a potential new role for NAIP in addition to anti-apoptotic and innate immunology functions.This work was supported by operating grants from FightSMA, The SMA Foundation, The Canadian Gene Cure Foundation, CIHR and Families of SMA to A.M

METTL1 promotes tumorigenesis through tRNA-derived fragment biogenesis in prostate cancer

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N-7-methylguanosine (m(7)G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m(7)G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m(7)G tRNA methylation in cancer cell translation control and tumour biology

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.This work was supported by Universidad de Granada, Plan Propio 2015;#P3B: FAM, VMC (http://investigacion.ugr.es/pages/planpropio/2015/ resoluciones/p3b_def_28072015); Universidad de Granada CEI BioTic;#CAEP2-84: VMC (http:// biotic.ugr.es/pages/resolucionprovisional enseaanzapractica22demayo/!); and Canadian nstitutes of Health Research;#231421, #318176, #361847: STB, ECL, RK (http://www.cihr-irsc.gc. ca/e/193.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Inhibitor of apoptosis proteins (IAPs) expression in monocyte to macrophage differentiation and M1/M2 polarization

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens and fulfill other essential roles in tissue homeostasis. The ability of monocytes to differentiate into macrophages constitutes an important asset in inflammatory situations because it allows for the quick recruitment of immune cells from the bloodstream to the site of infection. Furthermore, macrophages are highly plastic and can respond differently to changes in their environment and acquire a more cytotoxic and pro-inflammatory phenotype (M1) or a more healing and anti-inflammatory one (M2) and perform different roles during inflammation and resolution of inflammation. This polarization capacity enables the innate immune system to quickly respond to threatening situations faster than tissue specific differentiation or adaptive immune responses. Both processes, differentiation and polarization of macrophages, are important in inflammation resolution and their deregulation promotes the development of several pathologies, including cancer. On the other hand, macrophage activation syndrome (MAS), is a life-threatening complication of some rheumatic diseases, it is caused by an excessive activation and expansion of macrophages and T-lymphocytes. The inhibitor of apoptosis protein family (IAPs) has important roles in the regulation of several cellular processes and in innate and adaptive immune responses, specifically in many macrophage functions and almost every signaling pathway leading to NF-κB activation, which is implicated in pro-inflammatory cytokine production. In this thesis we wanted to characterize the expression profile of IAPs in the differentiation of monocytes to macrophages and in polarization into M1/M2 states, study the effect of IAPs pharmacological inhibition in macrophage differentiation and polarization and analyze the possibility of IAPs targeting as a therapeutic approach for macro- phage activation syndrome.En esta tesis queríamos evaluar la implicación de las proteínas inhibidoras de la apoptosis (IAPs, en inglés) en la diferenciación y polarización de macrófagos y estudiar la posibilidad de usar los IAPs como dianas terapéuticas en el tratamiento del síndrome de activación macrofágica.Tesis Univ. Granada. Programa Oficial de Doctorado en Biomedicin

A therapy PUSh for GBM

Pseudouridine is the most abundant RNA modification, but its biological role remains poorly understood. A study now finds dysregulated pseudouridine synthase PUS7 in glioblastoma and demonstrates that pharmacological inhibition of PUS7 leads to reduced tumorigenesis, which underpins the therapeutic potential of targeting epitranscriptomic regulators in cancer.Peer reviewe

Adamdec1, Ednrb and Ptgs1/Cox1, inflammation genes upregulated in the intestinal mucosa of obese rats, are downregulated by three probiotic strains.

We have previously reported that administration of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 to obese Zucker-Lepr fa/fa rats attenuates liver steatosis and exerts anti-inflammatory effects. The goal of the present work was to investigate the modulation of gene expression in intestinal mucosa samples of obese Zucker-Lepr fa/fa rats fed the probiotic strains using a DNA microarray and postgenomic techniques. We also measured secretory IgA content in the gut and lipopolysaccharide (LPS)-binding protein (LBP) in serum. Expression of three genes (Adamdec1, Ednrb and Ptgs1/Cox1) was up-regulated in the intestinal mucosa of the obese rats compared with that in the rats when they were still lean. Probiotic administration down-regulated expression of Adamdec1 and Ednrb at the mRNA and protein levels and that of Ptgs1/Cox1 at the mRNA level, and this effect was in part mediated by a decrease in both macrophage and dendritic cell populations. Probiotic treatment also increased secretory IgA content and diminished the LBP concentration. Based on results reported in this work and else where, we propose a possible mechanism of action for these bacterial strains

NAIP expression increases in a rat model of liver mass restoration

The neuronal apoptosis inhibitory protein (NAIP) is a constituent of the NLRC4 inflammasome, which plays a key role in innate immunity, and an antiapoptotic protein. Recently, we reported the previously undescribed role of NAIP in cell division. The liver is one of the body’s most actively regenerative organs. Given the novel mitotic role of NAIP, we examined its expression in hepatic mass restoration. The major liver lobe of Wistar rats was removed, and samples from both newly formed liver tissue, assessed by positive Ki67 immunostaining, and the remnant, intact liver lobes from hepatectomized rats were taken 3 and 7 days after surgery. Naip5 and Naip6 mRNA levels were significantly higher in regenerating hepatic tissue than in intact liver lobe tissue, and this increase was also observed at the protein level. Naip5 and Naip6 mRNA in situ hybridization showed that this increase occurred in the hepatic parenchyma. The histology of the regenerated liver tissue was normal, with the exception of a noticeable deficiency of hepatic lobule central veins. The results of this study suggest the involvement of NAIP in liver mass restoration following partial hepatectomy.This work was supported by the “Visiting Scholars P22-2017 Program, Plan Propio de Investigación de la Universidad de Granada” (to FAM), and by Grant #PI-0538-2017 from Junta de Andalucía, Spain (to LF)

NAIP expression in monocytes, M0-, M1- and M2-macrophages.

<p>A, B: The protein level of NAIP was assessed by western blotting of cell extracts from undifferentiated monocytes and M0 or polarized M1/M2 THP-1 macrophages. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of NAIP protein abundance in western blots normalized to HSC-70 of three independent experiments. C: NAIP mRNA expression of NAIP was was analysed by RT-qPCR using primers that span exon 4 or between exons 16 and 17 of NAIP. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).</p

NAIP expression in polarized primary human macrophages.

<p>A: NAIP mRNA levels from PBMC-derived monocytes or M1 and M2 macrophages were analyzed by RT-qPCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. B: Protein levels of NAIP in the indicated cell populations were assessed by western blotting. Representative western blot from samples of one of the donors. Graph represents the quantification of the NAIP protein, normalized to HSC-70 of experiments from four different healthy donors. C: Representative confocal microscopy images of monocytes, M1 and M2 macrophages from volunteer 1. Note the amoeboid-like shape in M1 and M2 macrophages. Bar, 10<i>μ</i>m. D: Mean fluorescence intensity in monocytes and M1 and M2 macrophages from two healthy donors. 10 randomly selected cells in each case were analyzed for their mean fluorescence intensity per cell area using the <i>ImageJ</i> program. *Significant difference (P = 0.0163), **significant difference (P = 0.0037), ***significant difference (P = 0.0005).</p

cIAP1 and cIAP2 expression in unpolarized (THP-1/monocytes and M0-) and polarized M1- and M2-macrophages.

<p>A, B: Polarized and unpolarized THP-1 cell proteins were extracted and cIAP1/2 protein abundance was assessed by western blotting using the RIAP1 antibody. HSC-70 was used as loading control. A: Representative western blot of one of three experiments with similar results. B: Quantification of cIAP1 and cIAP2 protein abundance in western blots normalized to HSC-70 of three independent experiments. C: Total RNA was extracted, reverse transcribed and cIAP1 and cIAP2 mRNA were analysed by RTq-PCR. Values are normalized to internal controls (HPRT1 and GAPDH) and presented as mean values ± s.d. of fold-change of three independent experiments. *P<0.05 ***P< 0.001 (ANOVA with Bonferroni post hoc).</p