Host Cell Redox Alterations Promote Latent HIV-1 Reactivation through Atypical Transcription Factor Cooperativity.

Immune cell state alterations rewire HIV-1 gene expression, thereby influencing viral latency and reactivation, but the mechanisms are still unfolding. Here, using a screen approach on CD4+ T cell models of HIV-1 latency, we revealed Small Molecule Reactivators (SMOREs) with unique chemistries altering the CD4+ T cell state and consequently promoting latent HIV-1 transcription and reactivation through an unprecedented mechanism of action. SMOREs triggered rapid oxidative stress and activated a redox-responsive program composed of cell-signaling kinases (MEK-ERK axis) and atypical transcription factor (AP-1 and HIF-1α) cooperativity. SMOREs induced an unusual AP-1 phosphorylation signature to promote AP-1/HIF-1α binding to the latent HIV-1 proviral genome for its activation. Consistently, latent HIV-1 reactivation was compromised with pharmacologic inhibition of oxidative stress sensing or of cell-signaling kinases, and transcription factor's loss of expression, thus functionally linking the host redox-responsive program to viral transcriptional rewiring. Notably, SMOREs induced the redox program in primary CD4+ T cells and reactivated latent HIV-1 in aviremic patient samples alone and in combination with known latency-reversing agents, thus providing physiological relevance. Our findings suggest that manipulation of redox-sensitive pathways could be exploited to alter the course of HIV-1 latency, thus rendering host cells responsive to help achieve a sterilizing cure.

Antigen presentation capability and AP-1 activation accompany methotrexate-induced colon cancer cell senescence in the context of aberrant ß-catenin signaling.

Reversible cellular senescence was demonstrated previously to constitute colon cancer cell response to methotrexate. The current study presents a comparison of two senescent states of colon cancer cells, arrested and reversing, resulting from respectively, 120 h exposure to the drug, and 48 h exposure followed by 96 h regrowth in drug-free media. The upregulation of immunoproteasome subunit-coding genes and the increase in human leukocyte antigen HLA-A/B/C membrane level indicated MHC-I-restricted antigen presentation as common to both senescent states. Nuclear factor NF-κB p65 level decreased and activating protein AP-1: c-Jun, Fra2 and JunB nuclear levels increased in both senescent cell populations. Notably, the increase in AP-1- dependent transcription occurred after 48 h exposure to methotrexate. ß-catenin nuclear level increased after 48 h exposure to the drug and remained as such only in senescence-arrested cells. ß-catenin level was found uncoupled from the protein phosphorylation status indicating the deregulation of ß-catenin signaling in colon cancer cells employed in the study. These findings carry implications for both, a general mechanism of senescence establishment and putative advantages for colon cancer treatment.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

The Clusters of Transcription Factors NFATC2, STAT5, GATA2, AP1, RUNX1 and EGR2 Binding Sites at the Induced Il13 Enhancers Mediate Il13 Gene Transcription in Response to Antigenic Stimulation.

IL-13 plays a critical role in mediating many biological processes responsible for allergic inflammation. Mast cells express Il13 mRNA and produce IL-13 protein in response to antigenic stimulation. Enhancers are essential in promoting gene transcription and are thought to activate transcription by delivering essential accessory cofactors to the promoter to potentiate gene transcription. However, enhancers mediating Il13 have not been identified. Furthermore, which Il13 enhancers detect signals triggered by antigenic stimulation have not yet been defined. In this study, we identified potential mouse Il13 enhancers using histone modification monomethylation at lysine residue 4 on histone 3 (H3K4me1) chromatin immunoprecipitation sequencing and acetylation at lysine residue 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing. We used Omni-assay for transposase-accessible chromatin sequencing to determine which accessible regions within the potential Il13 enhancers that responded to IgE receptor crosslinking. We also demonstrated that the transcription factor cluster consisting of the NFATC2, STAT5, GATA2, AP1, and RUNX1 binding sites at the proximal Il13 enhancer and the transcription factor cluster consisting of the EGR2 binding site at the distal Il13 E+6.5 enhancer are critical in sensing the signals triggered by antigenic stimulation. Those enhancers, which are responsive to antigenic stimulation and are constitutively active, cooperate to generate greater transcriptional outputs. Our study reveals a novel mechanism underlying how antigenic stimulation induces robust Il13 mRNA expression in mouse mast cells.

Fish IKKα from Japanese eel (Anguilla japonica) can activate NF-κB, AP1, and type I IFN signaling pathways.

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays a pivotal role in the activation of nuclear factor kappa-B (NF-κB) pathway in response to pathogens infections in mammals, but the information about IKKα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKKα homologue, AjIKKα, was cloned by 5' and 3' SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKKα GFP fusion protein and the induction of AjIKKα in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKKα has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKKα is similar to that of human IKKα. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKKα in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKKα expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKKα in the spleen. In vitro, the AjIKKα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 × 106 cfu/mL, 1 × 107 cfu/mL, and 1 × 108 cfu/mL). Luciferase assays demonstrated that AjIKKα expression could significantly induce NF-κB, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKKα was evenly distributed in the cytoplasm in the natural state, but AjIKKα was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKKα plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1and type I IFN signaling pathway.

Isodeoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber Linn., inhibits pro-inflammatory mediators' production through both NF-κB and AP-1 pathways in LPS-activated macrophages.

Isodeoxyelephantopin (IDET) has been identified as an anti-tumor natural constituent whose anti-tumor activity and mechanism have been widely investigated. Since the occurrence and development of cancer usually accompany with inflammation, and tumor signaling shares many components with inflammation signaling, the agents with anti-tumor activity are likely to possess anti-inflammation potential. Thus, the current study aims to demonstrate the anti-inflammatory activity along with the underlying mechanism of IDET in lipopolysaccharide (LPS)-primed macrophages. By using Griess method and ELISA, we found that in both bone marrow derived macrophages and alveolar macrophage cell line, IDET, at relatively low concentrations (0.75, 1.5 and 3 µM), could inhibit LPS-induced expression of various pro-inflammatory mediators including nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS), interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) and IL-1ß. Meanwhile, in activated MH-S cells, the inhibitory action of IDET on mRNA expression levels of these cytokines was also detected using qPCR. Mechanistically, the effects of IDET on two key inflammatory signalings, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) pathways, were determined in LPS-activated MH-S cells by reporter gene along with western blot assays. On the one hand, IDET suppressed NF-κB signaling via down-regulating phosphorylation and degradation of inhibitor of NF-κB (IκB)-α and the subsequent p65 translocation. On the other hand, IDET dampened AP-1 signaling through attenuating phosphorylation of both c-jun N-terminal kinase 1/2 (JNK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Our study indicates that IDET might be a promising constituent from the anti-inflammatory herb Elephantopus scaber Linn. in mitigating inflammatory conditions, especially respiratory inflammation.

Positive and negative regulatory effects of transcription factor activator protein 1 (AP1) on the expression of antimicrobial peptides in Macrobrachium nipponense.

Transcription factor activator protein 1 (AP1) plays an irreplaceable role in the response to a variety of external stimulants, such as cellar stress, bacterial and viral infections, and inflammatory cytokines. In this study, we identified a novel AP1 gene from Macrobrachium nipponense and named it MnAP1, which has a full length of 1747 bp contains an 882 bp open reading frame, and encodes a protein with 293 amino acids. The MnAP1 protein contains Pfam and bZIP domains. MnAP1 is widely distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestinal tissues. The expression levels of MnAP1 in the gills and stomach were significantly upregulated after Vibrio parahaemolyticus and Staphylococcus aureus attacks. We studied the relationship between MnAP1 and the transcripts of antimicrobial peptides (AMPs) in gills through RNA interference. Interestingly, the regulatory effects of MnAP1 on the expression of different AMPs were different. We found that the expression levels of crustins, including Cru1, Cru3, and Cru4 in the gills were evidently decreased, whereas the synthesis of Cru5 and anti-lipopolysaccharide factors (ALF3 and ALF4) were obviously increased. We further explored the effect of MnAP1 on the expression of transcription factor relish from M. nipponense. The result showed that the knockdown of MnAP1 can remarkably upregulate the expression of MnRelish. Relish as a member of the nuclear factor κB family that regulates the expression of AMPs in the innate immunity of crustacean. Hence, we also detected the expression levels of Cru5, ALF3, and ALF4 in the gills of MnRelish-silenced prawns. The Data showed that the expression levels of these three AMPs were evidently reduced after MnRelish silencing. Our results indicated that MnAP1 plays a positive role in regulating the expression of AMPs, promotes the JNK/AP1 signaling pathway, and exerts a negative regulatory effect on the synthesis of AMPs by inhibiting the transcription of NF-κB factor in the innate immunity of M. nipponense.

AP-1 activity induced by co-stimulation is required for chromatin opening during T cell activation.

Activation of T cells is dependent on the organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 h of activation. These newly opened regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at >70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced the expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1-dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease, and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.

c-Jun overexpression in CAR T cells induces exhaustion resistance.

Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.

Swine-Derived Probiotic Lactobacillus plantarum Modulates Porcine Intestinal Endogenous Host Defense Peptide Synthesis Through TLR2/MAPK/AP-1 Signaling Pathway.

Host defense peptides (HDPs) have antimicrobial and immunoregulatory activities and are involved in epithelial innate immune defense. Dietary modulation of endogenous HDP synthesis is an effective way to boost the host innate immune system. This study aimed to investigate the role of the probiotic Lactobacillus plantarum strain ZLP001 in porcine HDP induction and the underlying mechanism. To this end, we evaluated the stimulatory effect of L. plantarum ZLP001 on HDP expression in piglet intestinal tissue in vivo and porcine IPEC-J2 cells and 3D4/31 cells in vitro, and we examined the underlying intracellular signaling pathway in IPEC-J2 cells. L. plantarum ZLP001 treatment increased the mRNA expression of jejunal and ileal HDPs in weaned piglets. In IPEC-J2 and 3D4/31 cells, L. plantarum ZLP001 stimulated HDP expression, but different HDP induction patterns were observed, with the various HDPs exhibiting different relative mRNA levels in each cell line. L. plantarum ZLP001 induced porcine HDP expression through toll-like receptor (TLR)2 recognition as evidenced by the fact that HDP expression was suppressed in TLR2-knockdown IPEC-J2 cells. Furthermore, we found that L. plantarum ZLP001 activated the extracellular signal-regulated kinase (ERK)1/2 and c-jun N-terminal kinase (JNK) signaling pathways, as indicated by enhanced phosphorylation of both ERK1/2 and JNK and the fact that HDP expression was suppressed upon inhibition of ERK1/2 and JNK. Furthermore, L. plantarum ZLP001 activated c-fos and c-jun transcription factor phosphorylation and activity. We conclude that L. plantarum ZLP001 induces porcine HDP expression in vivo and in vitro, and the induction seems to be regulated via TLR2 as well as the ERK1/2/JNK and c-jun/c-fos signaling pathways. Modulation of endogenous HDPs mediated by L. plantarum ZLP001 might be a promising approach to improving intestinal health and enhancing diarrhea resistance in weaning piglets.

Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef-AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells.

Current combined antiretroviral therapy (cART) mainly targets 3 of the 15 HIV proteins leaving many potential viral vulnerabilities unexploited. To purge the HIV-1 latent reservoir, various strategies including "shock and kill" have been developed. A key question is how to restore impaired immune surveillance. HIV-1 protein Nef has long been known to mediate the downregulation of cell-surface MHC-I and assist HIV-1 to evade the immune system. Through high throughput screening of Food and Drug Administration (FDA) approved drugs, we identified lovastatin, a statin drug, to significantly antagonize Nef to downregulate MHC-I, CD4, and SERINC5, and inhibit the intrinsic infectivity of virions. In addition, lovastatin boosted autologous CTLs to eradicate the infected cells and effectively inhibit the subsequent viral rebound in CD4+ T-lymphocytes isolated from HIV-1-infected individuals receiving suppressive cART. Furthermore, we found that lovastatin inhibits Nef-induced MHC-I downregulation by directly binding with Nef and disrupting the Nef-AP-1 complex. These results demonstrate that lovastatin is a promising agent for counteracting Nef-mediated downregulation of MHC-I, CD4, and SERINC5. Lovastatin could potentially be used in the clinic to enhance anti-HIV-1 immune surveillance.

Insight into the molecular function and transcriptional regulation of activator protein 1 (AP-1) components c-Jun/c-Fos ortholog in red lip mullet (Liza haematocheila).

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Porcine reproductive and respiratory syndrome virus induces interleukin-1ß through MyD88/ERK/AP-1 and NLRP3 inflammasome in microglia.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection which caused severe reproductive failure and respiratory disorders in swine is accompanied with severe nervous symptoms. Our previous studies demonstrated that microglia, the resident innate immune cells in central nervous system (CNS), could support PRRSV infection and replication in vitro. And PRRSV infection led to the increased expressions of large amounts of proinflammatory cytokines and chemokines which contributed to neuropathogenesis of PRRSV. Interleukin-1ß (IL-1ß) is one of the increased proinflammatory cytokines, which possesses diverse functions in immune response upon virus infection, including activation of innate immune and modulation of adaptive immune responses. Importantly, considerable evidences indicated that 1L-1ß is involved in neuronal injury. Here, we demonstrated that PRRSV infection up-regulated IL-1ß expression at both the mRNA and protein levels in microglia in a dose-dependent manner. Myeloid differentiation primary response gene 88 (MyD88), extracellular signal-regulated kinase1/2 (ERK) and activator protein 1 (AP-1) were involved in PRRSV induced IL-1ß production in microglia. Moreover, NOD-like receptor protein 3 (NLRP3) inflammasome is activated by PRRSV in microglia, which is required for IL-1ß secretion. Taken together, our data indicated that PRRSV infection could induce IL-1ß up-regulation, which was likely mediated by MyD88/ERK/AP-1 and NLRP3 inflammasome. These findings will provide new insights into the molecular mechanisms of IL-1ß production and some implications for neuropathogenesis of PRRSV.

Holding All the CARDs: How MALT1 Controls CARMA/CARD-Dependent Signaling.

The scaffold proteins CARMA1-3 (encoded by the genes CARD11, -14 and -10) and CARD9 play major roles in signaling downstream of receptors with immunoreceptor tyrosine activation motifs (ITAMs), G-protein coupled receptors (GPCR) and receptor tyrosine kinases (RTK). These receptors trigger the formation of oligomeric CARMA/CARD-BCL10-MALT1 (CBM) complexes via kinases of the PKC family. The CBM in turn regulates gene expression by the activation of NF-κB and AP-1 transcription factors and controls transcript stability. The paracaspase MALT1 is the only CBM component having an enzymatic (proteolytic) activity and has therefore recently gained attention as a potential drug target. Here we review recent advances in the understanding of the molecular function of the protease MALT1 and summarize how MALT1 scaffold and protease function contribute to the transmission of CBM signals. Finally, we will highlight how dysregulation of MALT1 function can cause pathologies such as immunodeficiency, autoimmunity, psoriasis, and cancer.

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells.

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

Acetylation regulates the MKK4-JNK pathway in T cell receptor signaling.

T cell functions are regulated by multiple signaling cascades, including the MKK4-JNK (c-Jun NH2 terminal kinase) pathway. However, the mechanism regulating the MKK4-JNK axis in T cells remains unclear. Herein, we demonstrated that protein acetylation modulates JNK activity induced by T cell receptor (TCR) activation. The acetyltransferase, CREB-binding protein (CBP), is transported from the nucleus to the cytoplasm in response to TCR cross-linking. To investigate the role of CBP in TCR signaling, we overexpressed CBP in the cytoplasm of Jurkat cells, a human T lymphocyte line. Enforced expression of cytoplasmic CBP led to MKK4 acetylation and interfered with MKK4-mediated JNK phosphorylation. Insufficient JNK activity decreased the activity of the transcription factor, AP-1. In contrast, other transcription factors, NF-κB and NFAT, stimulated with anti-CD3 and anti-CD28 antibodies were activated normally in the presence of cytoplasmic-CBP. These results provide valuable insights into the role of acetylation in MKK4-JNK signaling in T cells.

Activation and inhibition of adaptive immune response mediated by mast cells.

Adaptive immune response plays an important role against bacteria and parasites, a reaction that also involves mast cell (MC) activation which participates in innate and adaptive immunity. In allergic reactions there is a TH2 immune response with generation of allergen-specific IgE antibodies. In MCs, IgE cross-link FcRI high affinity receptor and activate tyrosine kinase proteins, leading to stimulation of NF-κB and AP-1 resulting in the release of a number of cytokines/chemokines and other compounds. Through their proteolytic pathways, MCs may process the antigen for presentation to CD4+ cells which release TH2 cytokines and growth factors, which play an important role in asthma, allergy, anaphylaxis and inflammation. Thus, MCs can contribute to adaptive immunity. MCs may also be activated though the TLR-dependent pathway which is controlled by several proteins including myeloid differentiation factor 88 (MyD88) which can be inhibited by interleukin (IL)-37. Here, we describe the participation of MCs in adaptive immunity and inflammation, an effect that may be inhibited by IL-37.