Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Characterization of TRAF6 mediated ubiquitination of presenilins and γ-secretase substrates

Post-translational modification of the γ-secretase protease complexes and their substrates has an important role in controlling receptor-initiated signalling events, which are critically important in the pathogenesis of cancer, inflammatory and Alzheimer’s disease. Our lab has previously characterised an interaction between TRAF6 and presenilin-1, which lead to the identification of interleukin-1 (IL-1) receptor type 1 (IL-1R1) and Toll-like receptor-4 (TLR4) as novel γ-secretase substrates. Subsequently our group showed that TRAF6 promoted ubiquitination and γ-secretase cleavage of IL-1R1. The aim of this project is to study the association between TRAF6 and the presenilins, the critical γ-secretase complex components, and to determine the functional importance of TRAF6-mediated ubiquitination of γ-secretase substrates. Firstly, we show that the full-length presenilins are novel substrates of TRAF6-mediated Lysine-63-linked polyubiquitination. Secondly, we show that co-expression of TRAF6 and the presenilins increases the stability and alters the turnover of the presenilins. Thirdly, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate the full-length presenilin functions such as ER Ca2+ signalling. Previously, we have reported IL-1R1 as a novel substrate of TRAF6-mediated ubiquitination. In this study, we identified five lysine residues in the IL-1R1 intracellular domain targeted by TRAF6-mediated polyubiquitination. Furthermore, mutagenesis of these five lysine residues led to decreased IL-1R1 cell surface expression, precluded the ectodomain shedding and attenuated the responsiveness to IL-1β stimulation, demonstrating the critical role of TRAF6 in IL-1R1 trafficking.

Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor κ B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-κB, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

Genetic Variation and Clinical Manifestations in Inflammatory Bowel Disease

Crohn s disease (CD) and ulcerative colitis (UC), collectively known as inflammatory bowel disease (IBD), are characterised by chronic inflammation of the gastrointestinal tract. IBD prevalence in Finland is approximately 3-4 per 1000 inhabitants with a peak incidence in adolescence. The symptoms of IBD include diarrhoea, abdominal pain, fever, and weight loss. The precise aetiology of IBD is unknown but interplay of environmental risk factors and immunologic changes trigger the disease in a genetically susceptible individual. Twin and family studies have provided strong evidence for genetic factors in IBD susceptibility, and genetic factors may be more prominent in CD than UC. The first CD susceptibility gene was identified in 2001. Three common mutations R702W, G908R, and 1007fs of the CARD15/NOD2 gene are shown to associate independently with CD but the magnitude of association varies between different populations. The present study aimed at identifying mutations and genetic variations in IBD susceptibility and candidate genes. In addition, correlation to phenotype was also assessed. One of the main objectives of this study was to evaluate the role of CARD15 in a Finnish CD cohort. 271 CD patients were studied for the three common mutations and the results showed a lower mutation frequency than in other Caucasian populations. Only 16% of the patients carried one of the three mutations. Ileal location as well as stricturing and penetrating behaviour of the disease were associated with occurrence of the mutations. The whole protein coding region of CARD15 was screened for possible Finnish founder mutations. In addition to several sequence variants, five novel mutations (R38M, W355X, P727L, W907R, and R1019X) were identified in five patients. Functional consequences of these novel variants were studied in vitro, and these studies demonstrated a profound impairment of MDP response. Investigation of CARD15 mutation frequency in healthy people across three continents showed a large geographic fluctuation. No simple correlation between mutation frequency and disease incidence was seen in populations studied. The occurrence of double mutant carriers in healthy controls suggested that the penetrance of risk alleles is low. Other main objectives aimed at identifying other genetic variations that are involved in the susceptibility to IBD. We investigated the most plausible IBD candidate genes including TRAF6, SLC22A4, SLC22A5, DLG5, TLR4, TNFRSF1A, ABCB1/MDR1, IL23R, and ATG16L1. The marker for a chromosome 5 risk haplotype and the rare HLA-DRB1*0103 allele were also studied. The study cohort consisted of 699 IBD patients (240 CD and 459 UC), of which 23% had a first-degree relative with IBD. Of the several candidate genes studied, IL23R was associated with CD susceptibility, and TNFRSF1A as well as the HLA-DRB1*0103 allele with UC susceptibility. IL23R variants also showed association with the stricturing phenotype and longer disease duration in CD patients. In addition, TNFRSF1A variants were more common among familial UC and ileocolonic CD. In conclusion, the common CARD15 mutations were shown to account for 16% of CD cases in Finland. Novel CARD15 variants identified in the present study are most likely disease-causing mutations, as judged by the results of in vitro studies. The present study also confirms the IL23R association with CD susceptibility and, in addition, TNFRSF1A and HLA-DRB1*0103 allele association with UC of specific clinical phenotypes.

Function of microRNA-146a and NF-κB in physiologic and pathologic hematopoiesis

During inflammation and infection, hematopoietic stem and progenitor cells (HSPCs) are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of the gene encoding miR-146a—expressed in all blood cell types—produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to exhaustion of the HSCs and hematopoietic neoplasms. Six-week-old deleted mice are normal, with no effect on cell numbers, but by 4 months bone marrow hypercellularity can be seen, and by 8 months marrow exhaustion is becoming evident. The ability of HSCs to replenish the entire hematopoietic repertoire in a myelo-ablated mouse also declines precipitously as miR-146a-deficient mice age. In the absence of miR-146a, LPS-mediated serial inflammatory stimulation accelerates the effects of aging. This chronic inflammatory stress on HSCs in deleted mice involves a molecular axis consisting of upregulation of the signaling protein TRAF6 leading to excessive activity of the transcription factor NF-κB and overproduction of the cytokine IL-6. At the cellular level, transplant studies show that the defects are attributable to both an intrinsic problem in the miR-146a-deficient HSCs and extrinsic effects of miR-146a-deficient lymphocytes and non-hematopoietic cells. This study has identified a microRNA, miR-146a, to be a critical regulator of HSC homeostasis during chronic inflammatory challenge in mice and has provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms. This may have implications for human hematopoietic malignancies, such as myelodysplastic syndrome, which frequently displays downregulated miR-146a expression.

Zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) mediates multiple intracellular signaling pathways

Insect PGRPs can function as bacterial recognition molecules triggering proteolytic and/or signal transduction pathways, with the resultant production of antimicrobial peptides. To explore if zebrafish peptidoglycan recognition protein SC (zfPGRP-SC) has such effects, RNA interference (siRNA) and high-density oligonucleotide microarray analysis were used to identify differentially expressed genes regulated by zfPGRP-SC. The mRNA levels for a set of genes involved in Toll-like receptor signaling pathway, such as TLRs, SARM, MyD88, TRAF6 and nuclear factor (NF)-kappa B2 (p100/p52), were examined by quantitative RT-PCR (QT-PCR). The results from the arrays and QT-PCR showed that the expression of 133 genes was involved in signal transduction pathways, which included Toll-like receptor signaling, Wnt signaling, BMP signaling, insulin receptor signaling, TGF-beta signaling, GPCR signaling, small GTPase signaling, second-messenger-mediated signaling, MAPK signaling, JAK/STAT signaling, apoptosis and anti-apoptosis signaling and other signaling cascades. These signaling pathways may connect with each other to form a complex network to regulate not just immune responses but also other processes such as development and apoptosis. When transiently over-expressed in HEK293T cells, zfPGRP-SC inhibited NF-kappa B activity with and without lipopolysacharide (LPS) stimulation. (C) 2008 Elsevier Ltd. All rights reserved.

栉孔扇贝(Chlamys farreri)Toll样受体及其信号传递相关基因的克隆与表达

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的，其主要原因是养殖环境恶化、扇贝种质衰退和抗病力下降。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 Toll样受体（TLRs）家族是新近发现的模式识别受体（PRRs），参与识别病原体相关的分子模式（PAMPs），在天然免疫系统中起着非常重要的作用。哺乳动物中Toll样受体信号通路还参与诱导树枝状细胞成熟、参与免疫耐受、参与凋亡发生发展、介导非感染性因素的识别等，被视为联系天然免疫和获得性免疫的桥梁。同时果蝇的Toll信号通路也是不具备获得性免疫的果蝇赖以抵御病毒、细菌和真菌感染，介导天然免疫反应的重要信号通路。 本研究采用大规模EST测序方法，结合Genome Walker库的构建和cDNA末端快速扩增技术，从栉孔扇贝克隆得到CfToll-1、CfMyd88、CfTRAF6和CfCactus这四个Toll样受体信号通路基因的全长cDNA，同时用荧光实时定量PCR技术检测了这些基因的组织分布及在脂多糖（LPS）和肽聚糖（PGN）刺激下的表达规律。 栉孔扇贝Toll样受体（CfToll-1）的cDNA序列全长4308 bp，包含5’非翻译区（UTR）211 bp，3597 bp的开放阅读框，500 bp的3’UTR，最后为18个腺嘌呤的ploy A 尾巴。开放阅读框编码1198个氨基酸的多肽，该多肽的估计分子量为137.41kd，估计的等电点为5.62，该多肽有信号肽，具有一个预测的跨膜区，因此是一种跨膜蛋白。经BLAST比对，CfToll-1基因与节肢动物多种Toll蛋白高度的相似性。SMART（Simple Modular Architecture Research Tool）软件分析，CfToll-1包含典型的Toll样受体的结构：富含亮氨酸的重复序列的胞外区（leucine-rich repeats, LRR），一段跨膜结构域，以及胞内区的TIR结构域（Toll/IL-1 receptor homologous region）。利用Real-time RT-PCR发现CfToll-1mRNA在扇贝体内普遍存在于血细胞、肌肉、外套膜、心、性腺和鳃组织中。利用体外培养的原代血细胞系研究不同浓度LPS刺激后CfToll-1的表达变化，结果显示低剂量（100ng.mL-1 ）LPS 使CfToll-1 mRNA表达量减小，该变化在1.5h、3h 和9h组差异显著，虽然在6h组表达量稍有恢复，但尚未达到对照水平；用1μg.mL-1LPS处理细胞时， 6h组CfToll-1表达量明显上调，约为对照水平的2倍。证实细菌结构脂多糖对CfToll-1基因的表达有影响，且这种影响有剂量依赖效应。 栉孔扇贝Myd88同源基因（CfMyd88）的cDNA序列全长1554bp，包含5’UTR 427 bp，1101bp的开放阅读框，最后为18个腺嘌呤的ploy A 尾。CfMyd88的开放阅读框可编码367个氨基酸的多肽，该多肽的估计分子量为42.37kD，估计的等电点为5.71。利用SMART程序分析发现CfMyd88编码了Death和TIR结构域, 这两个结构域是Myd88特征结构。BLAST程序发现扇贝的序列与数据库哺乳动物的Myd88基因高度同源。原代培养的扇贝血细胞在受到PGN刺激后，CfMyd88 mRNA表达在1.5小时开始下调，直到9小时下调至对照表达量的1/10，证实肽聚糖结构对CfMyd88基因的表达有影响。 栉孔扇贝TRAF6同源基因（CfTRAF6）的cDNA序列全长2510bp，包含5’UTR 337 bp，1965bp的开放阅读框，3’UTR 208bp，最后为21 个腺嘌呤的ploy A 尾巴。CfTRAF6开放阅读框编码655个氨基酸的多肽，该多肽的估计分子量为74.09kD，估计的等电点为6.01。InterPro Scan在线分析发现CfTRAF6有典型的TRAF蛋白家族的特征结构，包括的一个指环结构，两个锌指结构，一个MATH （the meprin and TRAF homology）结构域以及Coiled-coil区域。CfTRAF6的序列与数据库多物种的TRAF6高度同源，同源性最高的是乌贼序列（Identity＝68）和鼠类（Identity＝45%）。利用Real-time RT-PCR，发现CfTRAF6在各组织普遍存在，在性腺中的表达最高。原代培养的扇贝血细胞在受到不同浓度PGN刺激后，与CfMyd88的情况一样，CfTRAF6的表达量变化减少，且这种变化随剂量的增加更加明显。 栉孔扇贝Cactus同源基因（CfCactus）的cDNA序列全长2488bp，包含5’UTR 181 bp，840bp的开放阅读框， 3’UTR 1467bp，最后为19个腺嘌呤的ploy A 尾巴。CfCactus的开放阅读框编码279个氨基酸的多肽，该多肽的估计分子量为31.37 kD；估计的等电点为4.74，与果蝇的Cactus基因的等电点相近（4.5）。利用SMART程序分析发现CfCactus主要编码了ANK结构域（ankyrin repeats）。Cactus基因为哺乳动物NF-κB抑制蛋白IκB的同源分子，BLAST 程序发现扇贝的序列与数据库多物种的Cactus或IκB基因高度同源。同源性最高的是太平洋牡蛎（Identity=35%）和圆尾鲎（Identities = 44%）。对CfTCactus mRNA在扇贝的血细胞、性腺、 肠的组织表达进行分析，并同时与CfTRAF6和CfMyd88的表达量进行了对比，发现CfCactus的表达水平明显高于这两个基因，而且CfTRAF6的基因表达量也高于CfMyd88，表现出级联放大效应。正常情况下，三个基因在性腺的表达量最高，推测这条通路可能和发育等功能密切相关。 通过本研究我们首次在双壳类软体动物找得到与果蝇Toll蛋白家族高度同源的CfToll-1基因，同时发现其他三个在Toll样受体信号传递过程中起重要作用的基因，其中包括在软体动物中获得的第一个Toll样受体的接头分子－CfMyd88基因，该结果直接证明软体动物具有与哺乳动物和节肢动物高度类似Myd88依赖的Toll样受体信号通路。同时通过这些基因组织分布的研究以及细菌结构LPS和PGN对这条通路上基因表达的影响，证明扇贝Toll信号通路可能与在果蝇中一样，参与扇贝的发育和免疫防御等多种功能。

Investigation of p75 neurotrophin receptor and the role of its post-translational modifications in tumour cell motility and invasiveness

The p75 neurotrophin receptor (p75NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners - including the TNF receptor associated factor 6 (TRAF6) E3 ubiquitin ligase - to produce cellular responses such as apoptosis, survival, and inhibition of neurite outgrowth. Recently,p75NTR was also shown to undergo γ-secretase-mediated regulated intramembrane proteolysis, and the receptor ICD was found to migrate to the nucleus where it regulates gene transcription. Moreover, γ-secretase-mediated proteolysis was shown to be involved in glioblastoma cell migration and invasion. In this study we report that TRAF6-mediated K63-linked polyubiquitination at multiple or alternative lysine residues influences p75NTR-ICD stability in vitro. In addition, we found that TRAF6-mediated ubiquitination of p75NTR is not influenced by inhibition of dynamin. Moreover, we report beta-transducin repeats-containing protein (β-TrCP) as a novel E3- ligase that ubiquitinates p75NTR, which is independent of serine phosphorylation of the p75NTR destruction motif. In contrast to its influence on other substrates, co-expression of β-TrCP did not reduce p75NTR stability. We created U87-MG glioblastoma cell lines stably expressing wild type, γ-secretaseresistant and constitutively cleaved receptor, as well as the ICD-stabilized mutant K301R. Interestingly, only wild-type p75NTR induces increased glioblastoma cell migration, which could be reversed by application of γ-secretase inhibitor. Microarray and qRT-PCR analysis of mRNA transcripts in these cell lines yielded several promising genes that might be involved in glioblastoma cell migration and invasion, such as cadherin 11 and matrix metalloproteinase 12. Analysis of potential transcription factor binding sites revealed that transcription of these genes might be regulated by well known p75NTR signalling cascades such as NF-κB or JNK signalling, which are independent of γ-secretase-mediated cleavage of the receptor. In contrast, while p75NTR overexpression was confirmed in melanoma cell lines and a patient sample of melanoma metastasis to the brain, inhibition of γ-secretase did not influence melanoma cell migration. Collectively, this study provides several avenues to better understand the physiological importance of posttranslational modifications of p75NTR and the significance of the receptor in glioblastoma cell migration and invasion.

Expression of the NF-κB inhibitor A20 is altered in the Cystic Fibrosis epithelium

Research Question: A20 is an LPS-inducible, cytoplasmic zinc finger protein, that inhibits TLR-activated NF-?B signalling by deubiquitinating TRAF6. A20 action is facilitated by complex formation with RNF11, Itch and TAX1BP1. This study investigates if the expression of A20 is altered in the chronically inflamed Cystic Fibrosis (CF) airway epithelium.

Methods: Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-?B subunits was analysed. Formation of the A20 ubiquitin editing complex was also investigated.

Results: In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-.

Conclusion: he expression of A20 is significantly altered in CF and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-?B-driven inflammation.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3-and viral-induced expression of type i interferons

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression. © 2012 Nature America, Inc. All rights reserved.

Orphan receptor IL-17RD tunes IL-17A signalling and is required for neutrophilia

Interleukin-17A, the prototypical member of the interleukin-17 cytokine family, coordinates local tissue inflammation by recruiting neutrophils to sites of infection. Dysregulation of interleukin-17 signalling has been linked to the pathogenesis of inflammatory diseases and autoimmunity. The interleukin-17 receptor family members (A-E) have a broad range of functional effects in immune signalling yet no known role has been described for the remaining orphan receptor, interleukin-17 receptor D, in regulating interleukin-17A-induced signalling pathways. Here we demonstrate that interleukin-17 receptor D can differentially regulate the various pathways employed by interleukin-17A. Neutrophil recruitment, in response to in vivo administration of interleukin-17A, is abolished in interleukin-17 receptor D-deficient mice, correlating with reduced interleukin-17A-induced activation of p38 mitogen-activated protein kinase and expression of the neutrophil chemokine MIP-2. In contrast, interleukin-17 receptor D deficiency results in enhanced interleukin-17A-induced activation of nuclear factor-kappa B and interleukin-6 and keratinocyte chemoattractant expression. Interleukin-17 receptor D disrupts the interaction of Act1 and TRAF6 causing differential regulation of nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling pathways. © 2012 Macmillan Publishers Limited. All rights reserved.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Cystic Fibrosis from Laboratory to Bedside: The Role of A20 in NF-κB-Mediated Inflammation

Cystic fibrosis (CF) is a lifelong, inflammatory multi-organ disease and the most common lethal, genetic condition in Caucasian populations, with a median survival rate of 41.5 years. Pulmonary disease, characterized by infective exacerbations, bronchiectasis and increasing airway insufficiency is the most serious manifestation of this disease process, currently responsible for over 80% of CF deaths. Chronic dysregulation of the innate immune and host inflammatory response has been proposed as a mechanism central to this genetic condition, primarily driven by the nuclear factor κB (NF-κB) pathway. Chronic activation of this transcription factor complex leads to the production of pro-inflammatory cytokines and mediators such as IL-6, IL-8 and TNF-α. A20 has been described as a central and inducible negative regulator of NF-κB. This intracellular molecule negatively regulates NF-κB-driven pro-inflammatory signalling upon toll-like receptor activation at the level of TRAF6 activation. Silencing of A20 increases cellular levels of p65 and induces a pro-inflammatory state. We have previously shown that A20 expression positively correlates with lung function (FEV1%) in CF. Despite improvement in survival rates in recent years, advancements in available therapies have been incremental. We demonstrate that the experimental use of naturally occurring plant diterpenes such as gibberellin on lipopolysaccharide-stimulated cell lines reduces IL-8 release in an A20-dependent manner. We discuss how the use of a novel bio-informatics gene expression connectivity-mapping technique to identify small molecule compounds that similarly mimic the action of A20 may lead to the development of new therapeutic approaches capable of reducing chronic airway inflammation in CF. 

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.