A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Association of REL Polymorphism with Cow's Milk Proteins Allergy in Pediatric Algerian Population.

INTRODUCTION: Cow's milk proteins allergy (CMPA) pathogenesis involves complex immunological mechanisms with the participation of several cells and molecules involved in food allergy. The association of polymorphisms in the interleukin 4, Forkhead box P3 and the avian reticuloendotheliosis genes was investigated in an infant population with CMPA of Western Algeria. MATERIALS AND METHODS: We obtained DNA and clinical data from milk allergic subjects during active phase and from a group of non-atopic control subjects. RESULTS: Our findings showed that the allele G of the cRel gene intronic polymorphism at +7883 positions was significantly higher among cow's milk proteins allergic patients compared to control subjects. CONCLUSION: The results of this study suggest a possible association of CMPA with cRel G+7883T polymorphism.

Treating psoriasis by targeting its susceptibility gene Rel.

Psoriasis is a chronic inflammatory disorder of the skin. Accumulating evidence indicates that the Rel gene, a member of the NF-κB family, is a risk factor for the disease. We sought to investigate whether psoriasis can be prevented by directly targeting the Rel gene transcript, i.e., the c-Rel mRNA. Using chemically-modified c-Rel specific siRNA (siRel) and poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) micelles, we successfully knocked down the expression of c-Rel, and showed that the expression of cytokine IL-23, a direct target of c-Rel that can drive the development of IL-17-producing T cells, was markedly inhibited. More importantly, treating mice with siRel not only prevented but also ameliorated imiquimod (IMQ)-induced psoriasis. Mechanistic studies showed that siRel treatment down-regulated the expression of multiple inflammatory cytokines. Taken together, these results indicate that the susceptibility gene Rel can be targeted to treat and prevent psoriasis.

Characterizations and expression analyses of NF-κB and Rel genes in the Yesso scallop (Patinopecten yessoensis) suggest specific response patterns against Gram-negative infection in bivalves.

Rel/NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) genes are evolutionarily conserved and play a pivotal role in several physiological events. They have been extensively studied from various species, including both vertebrates and invertebrates. However, the Rel/NF-κB genes have not been systematically characterized in bivalves. In this study, we identified and characterized PyNF-κB and PyRel in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine the identities and evolutionary relationships of Rel/NF-κB genes in Yesso scallop. Compared with the Rel/NF-κB genes from vertebrate species, the PyNF-κB and PyRel are relatively conserved in their structural features, but there were no paralogs found in P. yessoensis or other invertebrates. To gain insights into the roles of Rel/NF-κB genes during the innate immune response in scallop, quantitative real-time PCR was used to investigate the expression profiles of these genes at different developmental stages, in healthy adult tissues and in the hemolymph after bacterial infection with Micrococcus luteus and Vibrio anguillarum. The real-time PCR results indicated the abundance of PyNF-κB in the first four embryonic stages, including oocytes, fertilized eggs, morulae and blastulae. By contrast, PyRel was abundantly expressed in blastulae, trochophores and D-shaped larvae. In adult scallops, PyNF-κB and PyRel were ubiquitously expressed in most healthy tissues and highly expressed in most of the immune related tissues. Both genes were significantly up-regulated during the acute phase (3 h) after infection with Gram-positive (M. luteus) and negative (V. anguillarum) bacteria, while the much higher expression level of PyNF-κB suggested the involvement of the extra immune deficiency (IMD)-like pathway against the Gram-negative bacterial infection. The complex pattern of Rel/NF-κB induced expression suggested that PyNF-κB and PyRel both have specific and cooperative roles in the acute immune responses to bacterial infection.

Cleavage of roquin and regnase-1 by the paracaspase MALT1 releases their cooperatively repressed targets to promote T(H)17 differentiation.

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.

Identification and expression analysis of immune-related genes linked to Rel/NF-κB signaling pathway under stresses and bacterial challenge from the small abalone Haliotis diversicolor.

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB (SaIκB) and Akirin2 (SaAkirin2) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS56GIYS60) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB, SaAkirin2, and AbNF-κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues (P < 0.05) while the expression level of AbNF-κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB, SaAkirin2, and AbNF-κB were significantly up-regulated (P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus. These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress.

The velvet family of fungal regulators contains a DNA-binding domain structurally similar to NF-κB.

Morphological development of fungi and their combined production of secondary metabolites are both acting in defence and protection. These processes are mainly coordinated by velvet regulators, which contain a yet functionally and structurally uncharacterized velvet domain. Here we demonstrate that the velvet domain of VosA is a novel DNA-binding motif that specifically recognizes an 11-nucleotide consensus sequence consisting of two motifs in the promoters of key developmental regulatory genes. The crystal structure analysis of the VosA velvet domain revealed an unforeseen structural similarity with the Rel homology domain (RHD) of the mammalian transcription factor NF-κB. Based on this structural similarity several conserved amino acid residues present in all velvet domains have been identified and shown to be essential for the DNA binding ability of VosA. The velvet domain is also involved in dimer formation as seen in the solved crystal structures of the VosA homodimer and the VosA-VelB heterodimer. These findings suggest that defence mechanisms of both fungi and animals might be governed by structurally related DNA-binding transcription factors.

An investigation of rheumatoid arthritis loci in patients with early-onset psoriasis validates association of the REL gene.

BACKGROUND: Phenotypically diverse autoimmune conditions share common genetic susceptibility loci and underlying molecular pathways. OBJECTIVES: By systematically searching for single nucleotide polymorphisms (SNPs) associated with another autoimmune disease, rheumatoid arthritis (RA), we aimed to elucidate novel genetic markers of psoriasis. METHODS: We investigated 18 SNPs, previously confirmed as being associated with RA, in a U.K. cohort of 623 patients with early-onset psoriasis (presenting before age 40 years), comparing them with 2662 control subjects. RESULTS: Our findings confirm the association of early-onset psoriasis with REL (rs13031237, P=0·0027). The minor allele of REL had opposing effects upon susceptibility to disease in patients with psoriasis and RA. CONCLUSION: Similar exploration of additional autoimmune loci and fine mapping of such regions may provide further insight into the genetics and molecular pathophysiology of psoriasis.

Genome-wide meta-analysis of psoriatic arthritis identifies susceptibility locus at REL.

Psoriatic arthritis (PsA) is a chronic inflammatory musculoskeletal disease affecting up to 30% of psoriasis vulgaris (PsV) cases and approximately 0.25 to 1% of the general population. To identify common susceptibility loci, we performed a meta-analysis of three imputed genome-wide association studies (GWAS) on psoriasis, stratified for PsA. A total of 1,160,703 single-nucleotide polymorphisms (SNPs) were analyzed in the discovery set consisting of 535 PsA cases and 3,432 controls from Germany, the United States, and Canada. We followed up two SNPs in 1,931 PsA cases and 6,785 controls comprising six independent replication panels from Germany, Estonia, the United States, and Canada. In the combined analysis, a genome-wide significant association was detected at 2p16 near the REL locus encoding c-Rel (rs13017599, P=1.18 × 10(-8), odds ratio (OR)=1.27, 95% confidence interval (CI)=1.18-1.35). The rs13017599 polymorphism is known to associate with rheumatoid arthritis (RA), and another SNP near REL (rs702873) was recently implicated in PsV susceptibility. However, conditional analysis indicated that rs13017599, rather than rs702873, accounts for the PsA association at REL. We hypothesize that c-Rel, as a member of the Rel/NF-κB family, is associated with PsA in the context of disease pathways that involve other identified PsA and PsV susceptibility genes including TNIP1, TNFAIP3, and NFκBIA.

ERK and JNK activation is essential for oncogenic transformation by v-Rel.

v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

Apoptosis effects of Xrel3 c-Rel/Nuclear Factor-kappa B homolog in human cervical cancer cells.

Cervical cancer is considered a common yet preventable cause of death in women. In this report, we studied the role of the NF-kappaB gene family in HeLa human cervical cancer cells, using the Xrel3 c-Rel homologue of Xenopus laevis. The expression of Xrel3/c-Rel slowed cell growth 6-fold, consistent with an upregulated expression of the cell cycle inhibitor p21. The activated PARP apoptosis effector was significantly increased (P<0.01). Based on cell viability assays Xrel3 provided an anti-apoptotic effect in 1 microM cisplatin, and this was associated with significantly lower levels of the apoptotic proteins Bax and MDM-2 (P<0.05). Furthermore, there was a 3-fold drop in the level of the tumor suppressor protein p53. In 5 microM cisplatin, expression of HeLa Xrel3 enhanced apoptosis by significantly increasing the expression of the apoptotic proteins Bax and MDM-2 (P<0.05). However, the tumor suppressor protein p53 showed a significant decrease (P<0.05) relative to the control. Thus, c-Rel/NF-kappaB may potentially be of clinical significance, especially in tumors exhibiting resistance to high-level chemotherapy.

Epstein-Barr virus is integrated between REL and BCL-11A in American Burkitt lymphoma cell line (NAB-2).

Epstein-Barr virus (EBV) initially isolated from the cultured Burkitt lymphoma (BL) cells, is one of the well-known oncogenic virus. The NAB-2 line, which was established from a North American Burkitt's tumor, was indicated to contain one copy of EBV DNA as the integrated form into chromosome 2p13 of the host genome. To demonstrate the integration site of EBV directly, and to clarify the relation between the integration sites and the oncogenes, fragments containing the nucleotide sequence of NAB-2 integration sites were cloned. EBV was integrated via the terminal repeats (TR), and integration sites located in the clone RP11-440P5 on chromosome 2, between two oncogenes, REL and BCL11A, which is apart from approximately 350 kbp from each other. Expression level of REL in NAB-2 was increased. The flanking region of chromosome 2 at the bilateral junction sites showed no homology to the junction sites of EBV. The integration site 2p13 overlaps with common fragile site, FRA2E. NAB-2 cells expressed almost all latent genes but LMP-2A that flanks the TR, indicating the type III of latent infection of EBV. Integration event in NAB-2 might alter the regulation of the oncogenes and provide advantage for continuous cell proliferation.

Cg-Rel, the first Rel/NF-kappaB homolog characterized in a mollusk, the Pacific oyster Crassostrea gigas.

We report here the identification and functional characterization of Cg-Rel, a gene encoding the Crassostrea gigas homolog of Rel/NF-kappaB transcription factors found in insects and mammals. Sequence and phylogenetic analysis showed that Cg-Rel shares the structural organization of Rel/NF-kappaB transcription factors of class II. It includes a Rel homology domain as well as a C-terminal transactivation domain (TD). Overexpression of Cg-Rel in the Drosophila S2 cell line activated the expression of a NF-kappaB-dependent reporter gene, whereas transfection with a Cg-Rel construct containing a C-terminal deletion of the TD or using a reporter gene with mutated kappaB binding sites failed to activate expression. These results suggest that Cg-Rel is a functional member of the Rel family of transcription factors, making this the sixth structurally homologous component of the Rel/NF-kappaB pathway characterized in C. gigas. Based on homology to other invertebrates' Rel/NF-kappaB cascade, the function of the oyster pathway may serve to regulate genes involved in innate defense and/or development. These findings serve to highlight a potentially important regulatory pathway to the study of oyster immunology, hence allowing comparison of the immune system in vertebrates and invertebrates, an important key issue to understand its evolution.

NF-kappaB inducible genes BCL-X and cyclin E promote immature B-cell proliferation and survival.

B-cell receptor (BCR) ligation induces proliferation and survival in mature B-cells but conversely, can lead to apoptosis in immature B-cells. We have previously shown that c-Rel, a member of the NF-kappaB transcription factor family, is essential for mature B-cell survival and proliferation via regulation of the anti-apoptotic molecule Bcl-X and cell cycle genes E2F3a and cyclin E. Here, we report that c-Rel-deficient mature B-cells are rendered sensitive to BCR-induced growth arrest and apoptosis in a manner that strongly resembles the phenotypic response of immature B-cells to BCR signaling. We further demonstrate that BCR-stimulated immature B-cells are defective in NF-kappaB activation, but that introduction of two downstream c-Rel target genes, Bcl-X and cyclin E, can restore survival and proliferation to these cells. Our studies therefore suggest that specific blockade of NF-kappaB activation may be responsible for the growth arrest and apoptosis of BCR-activated immature B-cells.

Genes and genomics of autoimmune inflammation: from Rel to TRAIL.

Development of autoimmune diseases requires coordinated expression of a myriad of genes. To explore the spectrum and global patterns of gene expression during autoimmune inflammation, we have recently performed functional genomic studies of autoimmune inflammation in the central nervous system (CNS). Inflammation in the CNS not only induced the expression of many immune-related genes, but also significantly altered the gene expression profile of neural cells. A number of unique clusters of genes were identified, which represent putative immune and nervous responses in autoimmune inflammation. This review will focus on two clusters of genes that we have been studying during the past few years: the Rel/nuclear factor (NF)-kappaB family and the tumor necrosis factor (TNF) family.

Genomic organization and expression of the rearranged REL proto-oncogene in the human B-cell lymphoma cell line RC-K8.

The human large B-cell lymphoma cell line RC-K8 has a rearranged REL locus that is transcribed into a chimeric mRNA, termed REL-NRG (Non-Rel Gene). By analyzing the recently completed human genome sequence, we have found that the normal REL and NRG loci are separated by approximately 28 megabase pairs on chromosome 2, suggesting that a deletion created the REL-NRG locus in RC-K8 cells. Using computer-based and molecular approaches, we have determined the structure of the altered REL locus in RC-K8 cells. The REL-NRG transcript is encoded by 7 REL exons and 6 NRG-derived exons. Direct DNA sequencing has identified the site of the REL-NRG fusion in RC-K8 cells. We also show that both wild-type c-Rel and c-Rel-Nrg proteins are expressed and in a complex in RC-K8 cells. Furthermore, like c-Rel, c-Rel-Nrg is a cytoplasmic protein when overexpressed in fibroblasts in culture and can bind to a kappaB DNA site in vitro.

Classical Hodgkin lymphoma is characterized by recurrent copy number gains of the short arm of chromosome 2.

Hodgkin- and Reed-Sternberg (HRS) cells microdissected from 41 classical Hodgkin lymphomas (cHL) of 40 patients comprising 8 lymphocyte-rich (cHL-LR), 16 nodular sclerosis (cHL-NS), 15 mixed-cellularity (cHL-MC), and 2 lymphocyte-depletion (cHL-LD) subtypes were analyzed by comparative genomic hybridization for recurrently imbalanced chromosomal subregions. Chromosomal gains most frequently involved chromosome 2p (54%), 12q (37%), 17p (27%), 9p and 16p (24% each), and 17q and 20q (20% each), whereas losses primarily affected chromosome 13q (22%). Using fluorescence in situ hybridization, amplification of the REL oncogene was demonstrated within a distinct 2p15-p16 amplicon. The high frequency of 2p overrepresentations including REL, particularly in cHL-NS (88%), suggests that an alternative mechanism of constitutive activation of nuclear factor NF-kappaB is a hallmark of HRS cells. Hierarchical cluster analysis of chromosomal imbalances revealed a closer relationship among cHL-NS than other subtypes. Furthermore, there is a tendency for different subtypes of cHL-MC tumors characterized by different ages at the time of tumor onset and gain of chromosome 17p. The imbalance pattern of cHL subtypes suggests that different molecular pathways are activated, with REL or other genes on chromosomal band 2p15-p16 playing a fundamental role in the pathogenesis of classical Hodgkin lymphoma.