Rel Homology Domain Research Articles

Functional analysis of a NF-κB transcription factor in the immune defense of Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae).

Although some novel antimicrobial peptides (AMP) have been successfully isolated from Bactrocera dorsalis Hendel, the mechanisms underlying the induction of these peptides are still elusive. The homolog of NF-κB transcription factor Relish, designated as BdRelish, was cloned from B. dorsalis. The full length cDNA of BdRelish is 3954 bp with an open reading frame that encodes 1013 amino acids. Similar to Drosophila Relish and the mammalian p100, it is a compound protein containing a conserved Rel homology domain, an IPT (Ig-like, plexins, transcription factors) domain and an IκB-like domain (four ankyrin repeats), the nuclear localization signal RKRRR is also detected at the residues 449-453, suggesting that it has homology to Relish and it is a member of the Rel family of transcription activator proteins. Reverse transcription quantitative polymerase chain reaction analysis reveals that BdRelish mRNAs are detected in different quantities from various tissues and the highest transcription level of BdRelish is determined in fat body. The injection challenge of Escherichia coli and Staphylococcus aureas significantly upregulated the expression of BdRelish. The injection of BdRelish dsRNA markedly reduced the expression of BdRelish and decreased the transcription magnitude of antimicrobial peptides. Individuals injected BdRelish dsRNA died at a significantly faster rate compared with the control groups. Therefore, BdRelish is vital for the transcription of AMPs to attack the invading bacteria.

The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription.

RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481bp along with 9bp 5' UTR, 639bp 3' UTR and the largest open reading frame (1833bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C.idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Invitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. Invivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.

Asymmetric arginine dimethylation of RelA provides a repressive mark to modulate TNFα/NF-κB response

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.

Co-expression of Dorsal and Rel2 Negatively Regulates Antimicrobial Peptide Expression in the Tobacco Hornworm Manduca sexta.

Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. Here we report the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. We for the first time confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggesting that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. Our results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides.

Identification of nuclear factor kappaB (NF-κB) binding motifs in Biomphalaria glabrata

Biomphalaria glabrata acts as the intermediate host to the parasite, Schistosoma mansoni, and for this reason, the immune system of B. glabrata has been researched extensively. Several studies have demonstrated that the transcriptome profile of B. glabrata changes following exposure to a variety of pathogens, yet very little is known regarding the regulation of gene expression in this species. Nuclear factor kappaB (NF-κB) homologues have recently been identified in B. glabrata but few functional studies have been carried out on this family of transcription factors. The aims of this study therefore were to identify NF-κB binding sites (κB motifs) in B. glabrata and examine them via functional assays. Two different κB motifs were predicted. Furthermore, the Rel homology domain (RHD) of a B. glabrata NF-κB was able to bind these κB motifs in EMSAs, as well as a vertebrate κB motif.

Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector.

The Werner Protein Acts as a Coactivator of Nuclear Factor κB (NF-κB) on HIV-1 and Interleukin-8 (IL-8) Promoters

The Werner syndrome helicase (WRN) plays a role in maintaining genomic stability. The lack of WRN results in Werner syndrome, a rare autosomal recessive genetic disorder, which causes premature aging accompanied by many complications such as rare forms of cancer and type 2 diabetes. However, the underlying mechanisms of these complications, arising due to the loss of WRN, are poorly understood. In this study, we demonstrated the function of WRN in transcriptional regulation of NF-κB targets. WRN physically interacts via its RecQ C-terminal (RQC) domain with the Rel homology domain of both the RelA (p65) and the p50 subunits of NF-κB. In the steady state, WRN is recruited to HIV-1 long terminal repeat (LTR), a typical NF-κB-responsive promoter, as well as the p50/p50 homodimer, in an NF-κB site-dependent manner. The amount of WRN on LTR increased along with the transactivating RelA/p50 heterodimer in response to TNF-α stimulation. Further, a knockdown of WRN reduced the transactivation of LTR in exogenous RelA/p50-introduced or TNF-α-stimulated cells. Additionally, knockdown of WRN reduced TNF-α stimulation-induced activation of the endogenous promoter of IL-8, an NF-κB-responsive gene, and WRN increased its association with the IL-8 promoter region together with RelA/p50 after TNF-α stimulation. In conjunction with studies that have shown NF-κB to be a key regulator of aging and inflammation, our results indicate a novel role of WRN in transcriptional regulation. Along with NF-κB, the loss of WRN is expected to result in incorrect regulation of downstream targets and leads to immune abnormalities and homeostatic disruption.

Molecular cloning and expression analysis of Relish gene from the ridgetail white prawn Exopalaemon carinicauda

Rel/NF-κB transcription factors play critical roles in induction and regulation of innate immune response in organisms. In this study, the full length of a Relish homolog cDNA from Exopalaemon carinicauda named EcRelish was 2141 bp encoding a 660 amino-acid polypeptide. EcRelish cDNA contained a conserved Rel homology domain and two nucleus localization signals. Sequence analysis indicated that the deduced amino acid sequence of the EcRelish showed high similarities to that of other crustaceans. Real time RT-PCR analysis showed that EcRelish mRNA expressed with different levels in tested tissues, and the highest expression was observed in the hemocytes. With longer infection time, the cumulative mortality rates increased gradually followed by the proliferation of Vibrio anguillarum and WSSV. The expression profiles of EcRelish gene were analyzed after V. a nguillarum, white spot syndrome virus (WSSV) challenge, and ammonia-N stress. The results showed that the expression levels of EcRelish mRNA in the hemocytes were up-regulated at 1–24 h after V. a nguillarum challenge. Meanwhile, the expression levels of EcRelish mRNA were up-regulated at 3 h after WSSV challenge. The expression of EcRelish in hemocytes was down-regulated significantly under ammonia-N stress during the experimental time. The results indicated that EcRelish might be involved in immune defense against pathogens and ammonia-N stress in E. c arinicauda.

Involvement of Relish gene from Macrobrachium rosenbergii in the expression of anti-microbial peptides

Relish is an NF-kB transcription factor involved in immune-deficiency (IMD) signal pathway. In this study, a Relish gene (MrRelish) was identified from Macrobrachium rosenbergii. The full length of MrRelish comprises 5072 bp, including a 3510 bp open reading frame encoding a 1169 bp amino acid protein. MrRelish contains a Rel homology domain (RHD), a nucleus localization signal, an IκB-like domain (6 ankyrin repeats), and a death domain. Phylogenetic analysis showed that MrRelish and other Relish from crustaceans belong to one group. MrRelish was expressed in all detected tissues, with the highest expression level in hemocytes and intestines. MrRelish was also upregulated in hepatopancreas at 6 h after Vibrio anguillarum challenge. The over-expression of MrRelish could induce the expression of antimicrobial peptides (AMPs), such as Drosophila Metchnikowin (Mtk), Attacin (Atta), Drosomycin (Drs), and Cecropin (CecA) and shrimp Penaeidin (Pen4). The RNAi of MrRelish in gills showed that the expression of crustin (cru) 2, Cru5, Cru8, lysozyme (Lyso) 1, and Lyso2 was inhibited. However, the expression of anti-lipopolysaccharide factor (ALF) 1 and ALF3 did not change when MrRelish was knocked down. These results indicate that MrRelish may play an important role in innate immune defense against V. anguillarum in M. rosenbergii.

MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

DNA element downstream of the κB site in the Lcn2 promoter is required for transcriptional activation by IκBζ and NF-κB p50.

The nuclear protein IκBζ activates transcription of a subset of NF-κB-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. IκBζ functions as a coactivator via its interaction with NF-κB p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in IκBζ function have remained unknown. Here, we show that, although IκBζ by itself is unable to associate with the Lcn2 promoter, IκBζ interacts with the promoter via p50 binding to the NF-κB-binding site (κB site) and the interaction also requires the pyrimidine-rich site (CCCCTC) that localizes seven bases downstream of the κB site. The pyrimidine-rich site is also essential for IκBζ-mediated activation of the Lcn2 gene. Introduction of both sites into an IκBζ-independent gene culminates in IκBζ-p50-DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both IκBζ-DNA interaction and IκBζ-mediated gene activation. Thus, the pyrimidine-rich IκBζ-responsive site plays an essential role in productive interaction of IκBζ with the p50-DNA complex.

Strategies in diabetic nephropathy: apelin is making its way

Diabetic nephropathy, one of the major microvascular complications of diabetes, is a leading cause of end-stage renal disease. This pathological entity is morphologically characterized by glomerular hypertrophy and thickening of tubular basal membranes, in association with progressive mesangial expansion. As the disease progresses, clinical manifestations include albuminuria, hypertension and declining glomerular filtration rate. Experimental and clinical studies have demonstrated the significant role of various inflammatory molecules in the setting of diabetic nephropathy, including acute phase reactants, inflammatory cytokines, adhesion molecules and chemokines (for review see Navarro-Gonzalez & Mora-Fernandez, 2008). Current treatment approaches include renin–angiotensin–aldosterone system inhibition, dietary sodium restriction and diuretic therapy. Nevertheless, new strategies are needed to slow down progression of diabetic nephropathy. In this issue of The Journal of Physiology, Chen and colleagues (2014) examined a possible mechanism involved in the beneficial effects of apelin-13 on experimental diabetic nephropathy. The adipokine apelin is a peptide known as the endogenous ligand of the G-protein-coupled receptor APJ. Apelin-13, the most active member of the apelin group, has emerged as a beneficial peptide with anti-obesity and antidiabetic properties, and thus, as a promising therapeutic target in metabolic disorders (for review see Castan-Laurell et al. 2011). Recently, Day et al. (2013) showed, for the first time, that apelin-13 exerts renoprotective effects in diabetic FVB/Ove26 mice. These authors observed that the whole-kidney and glomerular hypertrophy, as well as renal inflammation, was inhibited after treatment with apelin-13. These effects correlated with upregulation of the antioxidant catalase (Day et al. 2013). Chen et al. (2014) have added an important contribution by investigating the role of histone acetylation in apelin-induced effects on the diabetic kidney. Experiments carried out by these investigators demonstrated that apelin-13 inhibits histone hyperacetylation induced by either diabetes in the Akita mouse or high glucose concentration in cultured mesangial cells. This effect is mediated by selective upregulation of the expression of histone deacetylase HDAC1 (Chen et al. 2014). It is known that histone acetylation levels are regulated through the opposing activities of histone acetyltransferases and deacetylases, and that histone acetylation–deacetylation equilibrium plays a critical role in the control of gene expression. The participation of histone acetylation in the pathophysiology of diabetic nephropathy is still not well understood. As Chen et al. (2014) explain in their article (see their Discussion), previous studies demonstrated that histone hyperacetylation induces overexpression of inflammation-associated genes; and on the contrary, evidence has been accumulating that HDAC inhibitors exert renoprotective effects on the diabetic kidney. These controversies remain to be resolved. Probably, the identification of the individual transacetylase/deacetylase enzyme isoform involved in each case could contribute to elucidation of this issue. In the studies performed by Chen et al. (2014), the levels of two HDACs (HDAC1 and HDAC2) and two histone acetyltransferases (PCAF and GCN5) were determined. The expression level of only HDAC1 was affected by apelin-13 treatment in Akita mouse kidneys. In parallel with the diminution in histone acetylation levels, apelin-13 significantly reduced the diabetes-induced increase in renal monocyte chemotactic protein 1, intercellular adhesion molecule 1, inducible nitric oxide synthase and p65 [the active form of the transcriptional subunit of nuclear factor-κB (NF-κB)] phosphorylation levels of Akita mice (Chen et al. 2014). Interestingly, Ashburner et al. (2001) demonstrated that HDAC1 expression represses tumour necrosis factor-induced NF-κB-dependent gene expression. Consistent with this, the authors showed a direct association of HDAC1 with the Rel homology domain of p65. In diabetic nephropathy, an increase in the NF-κB p65 subunit and NF-κB–DNA binding depends on Toll-like receptor protein 2 (TLR2) upregulation (Mudaliar et al. 2013). In this connection, trichostatin A, a histone deacetylase inhibitor, upregulates the expression of both TLR2 mRNA and protein in the human THP-1 cell line (Li et al. 2013). Taking this information into account, it could be interesting to investigate whether the apelin-induced anti-inflammatory effects in diabetic nephropathy are mediated by direct association of the increased HDAC with p65 and/or TLR2 downregulation. In summary, the recently described effects of apelin on whole-body metabolism (for review see Castan-Laurell et al. 2011) and, especially, on the diabetic kidney (Day et al. 2013; Chen et al. 2014) lead to the proposal that apelin may be a novel therapeutic tool for treatment of diabetic nephropathy. The investigation by Chen et al. (2014) showed that apelin-13 alters the histone acetylation–deacetylation balance, exposing a wide spectrum of potential targets for the beneficial effects of this adipokine on diabetes-induced renal injury. Further investigations focused on elucidation of these mechanisms could contribute to evaluation of the risk–benefit ratio of apelin-13 treatment.

Release of High Mobility Protein Box-1 is Greatly Regulated by Nuclear Factor of Activated T Cell-2 in Human Monocytes

Close talk between inflammatory mediators and immunological cytokines has been discovered and reported. In this study, the role of nuclear factor of activated T cell-2 (NFAT2) in regulation of high mobility group box-1 (HMGB1) release was investigated. THP-1 cell and HEK293T cell were incubated and stimulated by lipopolysaccharide (LPS). Firstly, binding site between HMGB1 and NFAT2 was identified by co-immunoprecipitation (IP). Box A, Box B and CT domain of HMGB1 were constructed, as well as Rel-homology-domain (RHD), pre-RHD and pro-RHD of NFAT2. THP-1 cell was harvested, cell lysate and culture medium were collected at appointed times. Binding between HMGB1 and NFAT2 was measured, HMGB1 protein level in culture medium was analyzed at the same time. Secondly, the role of NFAT2 in regulating HMGB1 release was investigated. When THP-1 cell was cultured for 24 h, HMGB1 protein level was measured at appointed times with or without siRNA to inhibit NFAT2 expression. Our data show that HMGB1 bound to NFAT2 in THP-1 cell cytoplasm. Further experiments showed that box B domain of HMGB1 could bind to pre-RHD of NFAT2. After stimulation by LPS, interaction between HMGB1 and NFAT2 was discovered decreasing gradually. However, HMGB1 protein level increased in culture medium at the same time. Furthermore, HMGB1 release could be enhanced by NFAT2 inhibition. Taken together, release of HMGB1 could be regulated by NFAT2 in human monocytes.

The Velvet Family of Fungal Regulators Contains a DNA-Binding Domain Structurally Similar to NF-κB

Author SummaryIn many fungi, developmental processes and the synthesis of nonessential chemicals (secondary metabolites) are regulated by various external stimuli, such as light. Although fungi employ them for defensive purposes, secondary metabolites range from useful antibiotics to powerful toxins, so understanding the molecular processes that regulate their synthesis is of particular interest to us. In the mold Aspergillus nidulans the main regulators of these processes are the so-called “velvet” proteins VeA, VelB, and VosA, which share a 150-amino acid region known as the velvet domain. Velvet proteins interact with each other, alone (“homodimers”), in various combinations (“heterodimers”), and also with other proteins, but the molecular mechanism by which these proteins exert their regulatory function has been unclear. In this work we show that velvet proteins form a family of fungus-specific transcription factors that directly bind to target DNA, even though analysis of their amino acid sequence does not reveal any known DNA-binding domains or motifs. We determined the three-dimensional structure of the VosA-VosA homodimer and the VosA-VelB heterodimer and found that the structure of the velvet domain is strongly reminiscent of the N-terminal immunoglobulin-like domain found in the mammalian transcription factor NFκB-p50, despite the very low sequence similarity. We propose that, like NFκB, various homo- or heterodimers of velvet proteins modulate gene expression to drive development and defensive pathways in fungi.

The Velvet Underground Emerges

The Velvet Underground Emerges

Herpes Simplex Virus 1 E3 Ubiquitin Ligase ICP0 Protein Inhibits Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Interacting with p65/RelA and p50/NF-κB1

NF-κB plays central roles in regulation of diverse biological processes, including innate and adaptive immunity and inflammation. HSV-1 is the archetypal member of the alphaherpesviruses, with a large genome encoding over 80 viral proteins, many of which are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrated that the HSV-1 ICP0 protein, a viral E3 ubiquitin ligase, was shown to significantly suppress tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. ICP0 was demonstrated to bind to the NF-κB subunits p65 and p50 by coimmunoprecipitation analysis. ICP0 bound to the Rel homology domain (RHD) of p65. Fluorescence microscopy demonstrated that ICP0 abolished nuclear translocation of p65 upon TNF-α stimulation. Also, ICP0 degraded p50 via its E3 ubiquitin ligase activity. The RING finger (RF) domain mutant ICP0 (ICP0-RF) lost its ability to inhibit TNF-α-mediated NF-κB activation and p65 nuclear translocation and degrade p50. Notably, the RF domain of ICP0 was sufficient to interact with p50 and abolish NF-κB reporter gene activity. Here, it is for the first time shown that HSV-1 ICP0 interacts with p65 and p50, degrades p50 through the ubiquitin-proteasome pathway, and prevents NF-κB-dependent gene expression, which may contribute to immune evasion and pathogenesis of HSV-1.

Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis

Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was 2493bp with a 2022-bp open reading frame (ORF) encoding a 673-amino acid protein. This protein contained a 171-residue conserved Rel homology domain (RHD) and a 102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that (a) EsDorsal had higher expression level in immune organs; (b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.

A novel mechanism of crosstalk between the p53 and NFκB pathways

The inflammation regulating transcription factor NFκB and the tumor-suppressing transcription factor p53 can act as functional antagonists. Chronic inflammation (NFκB activity) may contribute to the development of cancer through the inhibition of p53 function, while, conversely, p53 activity may dampen inflammation. Here we report that the E3 ubiquitin ligase MDM2, whose gene is transcriptionally activated by both NFκB and p53, can bind and inhibit the p65RelA subunit of NFκB. The interaction is mediated through the N-terminal and the acidic/zinc finger domains of MDM2 on the one hand and through the N-terminal Rel homology domain of p65RelA on the other hand. Co-expression of MDM2 and p65RelA caused ubiquitination of the latter in the nucleus, and this modification was dependent of a functional MDM2 RING domain. Conversely, inhibition of endogenous MDM2 by small-molecule inhibitors or siRNA significantly reduced the ubiquitination of ectopic and endogenous p65RelA. MDM2 was able to equip p65RelA with mutated ubiquitin moieties capable of multiple monoubiquitination but incapable of polyubiquitination; moreover, MDM2 failed to destabilize p65RelA detectably, suggesting that the ubiquitin modification of p65RelA by MDM2 was mostly regulatory rather than stability-determining. MDM2 inhibited the NFκB-mediated transactivation of a reporter gene and the binding of NFκB to its DNA binding motif in vitro. Finally, knockdown of endogenous MDM2 increased the activity of endogenous NFκB as a transactivator. Thus, MDM2 can act as a direct negative regulator of NFκB by binding and inhibiting p65RelA.

Role of lysine methylation of NF-κB in differential gene regulation

Lysine methylation of the p65 subunit of nuclear factor κB (NF-κB) on K218 and K221 together or K37 alone strongly enhances gene expression in response to cytokines. We analyzed the effects of K-to-Q mutations in the REL homology domain of p65 on the response to IL-1β in 293 cells with low levels of p65. The K218/221Q mutation greatly reduced the expression of 39 of 82 genes, whereas the K37Q mutation reduced the expression of 23 different genes. Enhanced expression of the lysine demethylase FBXL11, which catalyzes the demethylation of K218 and K221 specifically, inhibited the expression of most of the genes that were inhibited by the DKQ mutation. CHIP-Seq analysis showed that the K218/221Q mutation greatly reduces the affinity of p65 for many promoters and that the K37Q mutation does not. Structural modeling showed that the newly introduced methyl groups of K218 and K221 interact directly with DNA to increase the affinity of p65 for specific κB sites. Thus, the K218/221Q and K37Q mutations have dramatically different effects because methylations of these residues affect different genes by distinct mechanisms.