Rel Proteins Research Articles

RelA binding of mRNAs modulates translation or sRNA-mRNA basepairing depending on the position of the GGAG site.

Previously, we reported that RelA protein facilitates Hfq-mediated mRNA-sRNA regulation by binding sRNAs carrying a Shine-Dalgarno-like GGAG sequence. In turn, sRNA-Hfq monomers are stabilized, enabling the attachment of more Hfq subunits to form a functional hexamer. Here, using CLIP-seq, we present a global analysis of RelA-bound RNAs showing that RelA interacts with sRNAs as well as with mRNAs carrying a GGAG motif. RelA binding of mRNAs carrying GGAG at position -7 relative to the initiation codon (AUG) inhibits translation by interfering with the binding of 30S ribosomes. The extent of inhibition depends on the distance of GGAG relative to the AUG, as shortening the spacing between GGAG and AUG abrogates RelA-mediated inhibition. Interestingly, RelA binding of target mRNAs carrying GGAG in the coding sequence or close to AUG facilitates target gene regulation by sRNA partners that lack GGAG. However, translation inhibition caused by RelA binding of mRNAs carrying GGAG at position -7 relative to the AUG renders sRNA-mRNA basepairing regulation ineffective. Our study indicates that by binding RNAs carrying GGAG the ribosome-associated RelA protein inhibits translation of specific newly synthesized incoming mRNAs or enables basepairing regulation by their respective sRNA partners, thereby introducing a new regulatory concept for the bacterial response.

Triptolide is a Promising Therapeutic Approach in Treating Thyroid Cancer Based on in silico and in vitro Experiment.

IntroductionThyroid cancer is a familiar kind of cancer. Natural products are promising therapeutic approaches in treating thyroid cancer. Triptolide is a diterpenoid epoxide extracted from Tripterygium wilfordii. The mechanism of triptolide in the treatment of thyroid cancer has not been investigated clearly.MethodsWe evaluated triptolide targets and thyroid cancer targets with related databases. The protein–protein interaction (PPI) networks of the triptolide targets and thyroid cancer targets were constructed with Cytoscape software. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the core PPI network were obtained. Molecular docking analysis was used to evaluated the binding of triptolide with core targets. Furthermore, apoptosis assays, real-time polymerase chain reaction (RT-PCR) and Western blotting were used to evaluate the anticancer functions of triptolide.ResultsTriptolide had 34 targets, and thyroid cancer had 210 targets. The core PPI network of merged PPI networks had 164 nodes and 4513 edges. GO and KEGG enrichment analyses showed that triptolide were related to the cell cycle, apoptosis, and inflammatory signaling pathways. Molecular docking analysis showed that triptolide directly reacted with four core targets: cyclin-dependent kinase inhibitor 1A (CDKN1A), c-JUN, RELA, and tumor protein p53 (TP53). CB-Dock analysis indicated that triptolide could stably bind to core targets. Triptolide inhibited the growth but induced apoptosis of thyroid cancer cells. Triptolide increased the mRNA expression of CDKN1A and TP53 but reduced the mRNA expression of c-JUN and RELA, as shown by RT-PCR. Triptolide increased the protein levels of CDKN1A and phospho-p53 but reduced those of phospho-c-JUN and phospho-NF-κB p65, as shown by Western blotting.DiscussionWe considered that triptolide could treat thyroid cancer by inhibiting cell proliferation, inducing apoptosis and inhibiting inflammatory pathways such as the NF-κB and MAPK signaling pathways. CDKN1A, c-JUN, RELA, and TP53 were involved in the antithyroid cancer mechanism of triptolide.

NF-κB Rel subunit exchange on a physiological timescale.

The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

A comparative study on chemical compositions and biological activities of four essential oils: Cymbopogon citratus (DC.) Stapf, Cinnamomum cassia (L.) Presl, Salvia japonica Thunb. and Rosa rugosa Thunb.

Ethnopharmacological relevanceEssential oils (EOs) are important active ingredients in Chinese herbal medicine. Cymbopogon citratus (DC.) Stapf, Cinnamomum cassia (L.) Presl, Salvia japonica Thunb., and Rosa rugosa Thunb. are used in local and traditional medicine to treat inflammation-related diseases. While the constituents and bioactivities of EOs of the above four Chinese herbals have been reported previously, but their anti-inflammatory and antioxidant activities have not been systematically evaluated. Aim of the studyThe purpose of this study was to research the chemical constituents, anti-inflammatory activities, and antioxidant activities of EOs of four Chinese herbal medicines. Materials and methodsFresh plant materials were collected both in August 2018. The compositions of EOs were determined by the gas chromatography-mass spectrometry (GC-MS) method. TPA (12-O-tetradecylphophenol-13-acetate)-induced mouse ear model was evaluated the anti-inflammatory activities of EOs. The reduction of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and RelA protein of the nuclear factor kappa-B (NF-κB) in the signal pathway (P65) were measured. The antioxidant activities of EOs from four plants were determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. ResultsA total of 217 compounds were separated and identified from four EOs, mainly including trans-cinnamylaldehyde (68.75%), citronellal (38.16%), linalool (1.02–33.73%), geraniol (19.39%) and citronellol (17.18%). All four EOs reduced the damage of the ear tissue and had certain anti-inflammatory effects, and the EOs of C. citratus (CyCEOs) and S. japonica (SJEOs) had the best anti-inflammatory activities among others, even better than ibuprofen. The four EOs exhibited different DPPH radical-scavenging activities (IC50, 0.101–1.017%), where most of them were much lower than that of EOs of C. citratus (CiCEOs) (IC50, 0.101%) and EOs of R. rugosa (RREOs) (IC50, 0.142%). ConclusionsThe main components determine diverse propertise of these four EOs, our results suggested that four EOs presented anti-inflammatory and antioxidant effect via reducing the expression of TNF-a, IL-6, COX-2, and NF-κB p65, which may provide a new approach for development of new anti-inflammatory drugs.

The RelA hydrolase domain acts as a molecular switch for (p)ppGpp synthesis

Bacteria synthesize guanosine tetra- and penta phosphate (commonly referred to as (p)ppGpp) in response to environmental stresses. (p)ppGpp reprograms cell physiology and is essential for stress survival, virulence and antibiotic tolerance. Proteins of the RSH superfamily (RelA/SpoT Homologues) are ubiquitously distributed and hydrolyze or synthesize (p)ppGpp. Structural studies have suggested that the shift between hydrolysis and synthesis is governed by conformational antagonism between the two active sites in RSHs. RelA proteins of γ-proteobacteria exclusively synthesize (p)ppGpp and encode an inactive pseudo-hydrolase domain. Escherichia coli RelA synthesizes (p)ppGpp in response to amino acid starvation with cognate uncharged tRNA at the ribosomal A-site, however, mechanistic details to the regulation of the enzymatic activity remain elusive. Here, we show a role of the enzymatically inactive hydrolase domain in modulating the activity of the synthetase domain of RelA. Using mutagenesis screening and functional studies, we identify a loop region (residues 114–130) in the hydrolase domain, which controls the synthetase activity. We show that a synthetase-inactive loop mutant of RelA is not affected for tRNA binding, but binds the ribosome less efficiently than wild type RelA. Our data support the model that the hydrolase domain acts as a molecular switch to regulate the synthetase activity.

Homology Model of a Catalytically Competent Bifunctional Rel Protein.

Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters’ temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis via pyrophosphate transfer from an ATP molecule to the 3’ position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on RelSeq (from S. equisimilis), still the only bifunctional Rel protein for which a GDP-bound “synthetase-ON” structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of RelSeq by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from S. aureus. Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.

A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-κB/RelA to Nucleolar Aggresomes.

Elevated NF-κB activity is a contributory factor in many hematologic and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here, we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intranucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to-nucleolar transport of the protein. Furthermore, using a dominant-negative mutant deleted for this nucleolar localization signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. IMPLICATIONS: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.

Curcumin ameliorates IL‐1β‐induced apoptosis by activating autophagy and inhibiting the NF‐κB signaling pathway in rat primary articular chondrocytes

Articular cartilage damage and chondrocyte apoptosis are common features of rheumatoid arthritis and osteoarthritis. Recently, curcumin has been reported to exhibit protective effects on degeneration in articular cartilage diseases. However, the effects and mechanisms of curcumin on articular chondrocyte injury remain to be elucidated. The aim of the present study is to investigate the chondroprotective mechanisms of curcumin on interleukin-1β (IL-1β)-induced chondrocyte apoptosis in vitro. The results revealed that IL-1β decreased cell viability and induced apoptosis in primary articular chondrocytes. Curcumin pretreatment reduced IL-1β-induced articular chondrocyte apoptosis. In addition, treatment with curcumin increased autophagy in articular chondrocytes and protected against IL-1β-induced apoptosis. The curcumin-mediated protection against IL-1β induced apoptosis was abolished when cells were treated with the autophagy inhibitor 3-methyladenine or transfected with Beclin-1 small interfering RNA. Furthermore, IL-1β stimulation significantly increased the phosphorylation levels of nuclear factor (NF)-κB p65 and glycogen synthase kinase-3β, and decreased the phosphorylation levels of β-catenin in articular chondrocytes, and these alterations to the phosphorylation levels were partly reversed by treatment with curcumin. Dual-luciferase and electrophoretic mobility shift assays demonstrated that IL-1β increased NF-κB p65 promoter activity in chondrocytes, and this was also reversed by curcumin. Pretreatment with the NF-κB inhibitor pyrrolidine dithiocarbamate enhanced the protective effects of curcumin on chondrocyte apoptosis, but Wnt/β-catenin inhibitor, XAV-939, did not exhibit this effect. Molecular docking and dynamic simulation studies results showed that curcumin could bound to RelA (p65) protein. These results indicate that curcumin may suppress IL-1β-induced chondrocyte apoptosis through activating autophagy and restraining NF-κB signaling pathway.

Induction of the Stringent Response Underlies the Antimicrobial Action of Aliphatic Isothiocyanates.

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain—enterohemorrhagic Escherichia coli (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress. The important source of potentially beneficial compounds are secondary plant metabolites, isothiocyanates (ITC), and phytoncides from the Brassicaceae family. We reported previously that sulforaphane and phenethyl isothiocyanate, already known for their chemopreventive and anticancer features, exhibit significant antibacterial effects against various pathogenic bacteria. The mechanism of their action is based on the induction of the stringent response and accumulation of its alarmones, the guanosine penta- and tetraphosphate. In this process, the amino acid starvation path is employed via the RelA protein, however, the precise mechanism of amino acid limitation in the presence of ITCs is yet unknown. In this work, we asked whether ITCs could act synergistically with each other to increase the antibacterial effect. A set of aliphatic ITCs, such as iberin, iberverin, alyssin, erucin, sulforaphen, erysolin, and cheirolin was tested in combination with sulforaphane against E. coli. Our experiments show that all tested ITCs exhibit strong antimicrobial effect individually, and this effect involves the stringent response caused by induction of the amino acid starvation. Interestingly, excess of specific amino acids reversed the antimicrobial effects of ITCs, where the common amino acid for all tested compounds was glycine. The synergistic action observed for iberin, iberverin, and alyssin also led to accumulation of (p)ppGpp, and the minimal inhibitory concentration necessary for the antibacterial effect was four- to eightfold lower than for individual ITCs. Moreover, the unique mode of ITC action is responsible for inhibition of prophage induction and toxin production, in addition to growth inhibition of EHEC strains. Thus, the antimicrobial effect of plant secondary metabolites by the stringent response induction could be employed in potential therapeutic strategies.

MicroRNA-520c-3p suppresses vascular endothelium dysfunction by targeting RELA and regulating the AKT and NF-κB signaling pathways.

Endothelial injury, which can cause endothelial inflammation and dysfunction, is an important mechanism for the development of atherosclerotic plaque. This study aims to investigate the functional role of miR-520c-3p in vascular endothelium during inflammatory diseases such as atherosclerosis. Quantitative real-time PCR was used to detect miR-520c-3p expression in in human umbilical vein endothelial cells (HUVECs) after treatment with platelet-derived growth factor (PDGF). Furthermore, the effects of miR-520c-3p overexpression and silencing on cell proliferation, adhesion, and apoptosis were assessed. Bioinformatics analysis and Biotin-labeled miRNA pull-down assay were used to confirm the targets of miR-520-3p. Then, the effects of miR-520c-3p on AKT and NF-κB signaling pathways were detected by western blot. Herein, we observed that the expression level of miR-520c-3p was downregulated in HUVECs under PDGF stimulation. Overexpression of miR-520c-3p not only decreased cell adhesion but also promoted proliferation and inhibited apoptosis to protect the viability of endothelial cells. It was confirmed that RELA is the target of miR-520c-3p. MiR-520c-3p inhibited the protein phosphorylation of AKT and RELA, and si-RELA reversed the promotion of AKT and RELA protein phosphorylation by anti-miR-520c-3p. In summary, our study suggested that miRNA-520c-3p targeting RELA through AKT and NF-κB signaling pathways regulated the proliferation, apoptosis, and adhesion of vascular endothelial cells. We conclude that miR-520c-3p may play an important role in the suppression of endothelial injury, which could serve as a biomarker and therapeutic target for atherosclerosis.

Computational Genomics of RELA gene of Pig and its protein prescription

RELA gene is a pleiotropic transcription that is present in almost all living cells where several signal transduction events end points were initiated by many arrays of stimuli that were related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis in the cells. The nucleotide and amino acid sequence (AAS) of RELA gene of pig and other mammalian species were downloaded from the National Center for Biotechnology information (NCBI) data base, United State of America and Universal protein resources (Uniprot) database, United Kingdom. Data generated were analyzed by bioinformatics tools. The results showed a high variation of AAS among the mammals where only four mammals have the same AAS length (551) with pig while others were not. The RELA gene of pig shared very high percent identity with all mammals ranging from 95.5% (warthog) to 83.5% (guinea pig) and similarity percentage ranged from 85.1 to 99.5% where the warthog had the highest percentage value while the least mammal was Guinea pig. The tree showed RELA gene with two clades; the first for mammals and the second for non-mammals. The RELA gene of pig was in the same cluster with the other Suidae family, warthog and babirusa. For the protein structure obtained, only 43% can be meaningfully predicted from the 61% residues modelled at more than 90%. The domain architecture of RELA protein structure contained mainly RNA Recognition motif and region of low complexity. The domainstarting position of all the organisms ranged from 191 to 210 while the termination of all the mammals ranged from 102 to 306 amino acids.The region of low complexity starting point ranged from position 173 to 462while termination positions ranged from 184 to 474. The evolution relationship of RELA gene of pig and other mammalian species were highly related and conserved among the mammalian species; they have high comparability and they evolved from common ancestors.

Survival Following Traumatic Brain Injury in Drosophila Is Increased by Heterozygosity for a Mutation of the NF-κB Innate Immune Response Transcription Factor Relish.

Traumatic brain injury (TBI) pathologies are caused by primary and secondary injuries. Primary injuries result from physical damage to the brain, and secondary injuries arise from cellular responses to primary injuries. A characteristic cellular response is sustained activation of inflammatory pathways commonly mediated by nuclear factor-κB (NF-κB) transcription factors. Using a Drosophila melanogaster TBI model, we previously found that the main proximal transcriptional response to primary injuries is triggered by activation of Toll and Imd innate immune response pathways that engage NF-κB factors Dif and Relish (Rel), respectively. Here, we found by mass spectrometry that Rel protein level increased in fly heads at 4-8 hr after TBI. To investigate the necessity of Rel for secondary injuries, we generated a null allele, Reldel , by CRISPR/Cas9 editing. When heterozygous but not homozygous, the Reldel mutation reduced mortality at 24 hr after TBI and increased the lifespan of injured flies. Additionally, the effect of heterozygosity for Reldel on mortality was modulated by genetic background and diet. To identify genes that facilitate effects of Reldel on TBI outcomes, we compared genome-wide mRNA expression profiles of uninjured and injured +/+, +/Reldel , and Reldel /Reldel flies at 4 hr following TBI. Only a few genes changed expression more than twofold in +/Reldel flies relative to +/+ and Reldel /Reldel flies, and they were not canonical innate immune response genes. Therefore, Rel is necessary for TBI-induced secondary injuries but in complex ways involving Rel gene dose, genetic background, diet, and possibly small changes in expression of innate immune response genes.

Escherichia coli RelA Regulation via Its C-Terminal Domain.

One of the most important stress responses in bacteria is the stringent response. The main player in this response is the signal molecule (p)ppGpp, which is synthesized by a Rel family protein. In Escherichia coli, RelA is the main synthetase of (p)ppGpp in response to amino acid starvation. Although the synthetic activity of RelA is well-understood, its regulation is not yet fully characterized. The C-terminus domain (CTD) of the E. coli RelA is responsible for the regulation of the protein and for its complete dependency on wild-type (WT) ribosome. The CTD contains three Cysteine residues, positioned in a very conserved order. Together with our previous results, we show in vitro the negative dominant effect of a part of the WT CTD (AA 564–744) named YG4 on RelA synthetic activity. This effect is abolished using mutated YG4 (YG4-638). In vitro and mass spectrometry (MS)-MS analysis of the native RelA and the mutated RelA in Cys-638 (Rel638) in the presence of the native and mutated YG4 (YG4-638) reveals that RelA forms a homodimer via its CTD by the formation of a disulfide bond between the two Cys-638 residues. This supports our previous data which showed, using a two-hybrid system, interactions between RelA proteins via the CTD. Finally, we show in vitro that excess of the native YG4 inhibited RelA synthetic activity but did not affect the amount of RelA bound to the ribosome. Our results suggest that the regulatory mechanism of RelA is by the dimerization of the protein via disulfide bonds in the CTD. Upon amino-acid starvation, the dimer changes its conformation, thus activating the stringent response in the cell.

Atomic structure of, and valine binding to the regulatory ACT domain of the Mycobacterium tuberculosis Rel protein.

The stringent response, regulated by the bifunctional (p)ppGpp synthetase/hydrolase Rel in mycobacteria, is critical for long-term survival of the drug-tolerant dormant state of Mycobacterium tuberculosis. During amino acid starvation, MtRel senses a drop in amino acid concentration and synthesizes the messengers pppGpp and ppGpp, collectively called (p)ppGpp. Here, we investigate the role of the regulatory 'Aspartokinase, Chorismate mutase and TyrA' (ACT) domain in MtRel. Using NMR spectroscopy approaches, we report the high-resolution structure of dimeric MtRel ACT which selectively binds to valine out of all other branched-chain amino acids tested. A set of MtRel ACT mutants were generated to identify the residues required for maintaining the head-to-tail dimer. Through NMR titrations, we determined the crucial residues for binding of valine and show structural rearrangement of the MtRel ACT dimer in the presence of valine. This study suggests the direct involvement of amino acids in (p)ppGpp accumulation mediated by MtRel independent to interactions with stalled ribosomes. Database Structural data are available in the PDB database under the accession number 6LXG.

Abstract PO-07: The FLI1 direct target ASB2 promotes NF-KB pathway activation in diffuse large B-cell lymphoma of the germinal center B-cell type

Abstract Introduction: Gains affecting chromosome 11 are recurrent events in lymphomas. The 11q24.3 gain occurs in 25% of diffuse large B-cell lymphoma (DLBCL), and it is associated with the overexpression of two ETS transcription factors, ETS1 and FLI1 (Blood 2013). Here, we have focused on the latter to identify the network of FLI1 regulated genes in GCB DLBCL. Methods: GCB and ABC cell lines. Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013, GSE10846, and EGAS00001002606. Anti-FLI1 antibody - ChIP Grade (ab15289). ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNA) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was analyzed using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. Results: The analysis of DLBCL cell lines showed that FLI1 protein levels were higher in GCB (n=12) than ABC (n=8) cell lines and was more commonly expressed at high levels in GCB (n= 414) than ABC (n= 518) DLBCL clinical specimens. Integration of identified binding sites from ChIP-Seq with RNA-Seq from GCB DLBCL cell lines (OCI-Ly1 and VAL) with genetically silenced FLI1 allowed the identification of putative FLI1 direct targets. The FLI1 negatively regulated genes included tumor-suppressor genes involved in negative regulation of cell cycle and p53 cascade. Among the FLI1 positively regulated targets we found genes annotated for immune response, MYC targets and B cell receptor, TNF-alpha and IL2 signaling pathways. Of note, direct targets of FLI1 overlapped with those genes regulated by ETS1, the other transcription factor co-gained in DLBCL, suggesting a functional convergence within the ETS family. ASB2 was downregulated after FLI1 silencing and had FLI1 binding sites in both promoter region and distal enhancer regions. Furthermore, ASB2 is known to promote NF-kB activation in T-cell acute lymphoblastic leukemia and might be an essential gene in DLBCL cells according to a genetic screening. Consistently, ASB2 gene silencing was toxic in GCB DLBCL lines. We observed inhibition of NF-kB pathway by a strong protein downregulation of RELB, along with increased IκBα upon ASB2 and FLI1 silencing, although with no differences in NF-kB2 levels. Only FLI1 silencing caused downregulation of NF-kB1 and RELA protein levels, but no effect on these two proteins was observed upon ASB2 silencing. These results indicate that FLI1 regulates either the classic NF-kB pathway at transcriptional level or the alternative pathway, via ASB2, in GCB DLBCL. Conclusions: FLI1 is expressed at higher levels in GCB than ABC DLBCL and directly regulates a network of biologically crucial genes and processes in DLBCL, contributing to the regulation of NF-kB pathway in GCB DLBCL. ASB2, a subunit of a multimeric E3 ubiquitin ligase complex, is a novel FLI1 direct target, and its inhibition might represent a therapeutic approach for GCB DLBCL. Citation Format: Giulio Sartori, Sara Napoli, Luciano Cascione, Elaine Y.L. Chung, Valdemar Priebe, Alberto J. Arribas, Andrea Rinaldi, Michela Dall'Angelo, Mattia Forcato, Silvio Bicciato, Margot Thome, Francesco Bertoni. The FLI1 direct target ASB2 promotes NF-KB pathway activation in diffuse large B-cell lymphoma of the germinal center B-cell type [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-07.

Purification and preliminary characterization of four Rel homologues from pathogenic bacteria: Implications for species-specific inhibitor design

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design.

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

Abstract 6042: TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer

Abstract Cytotoxic and genotoxic therapies are known to induce the prosurvival Inhibitor-kappaB kinase (IKK)-NF-κB/RELA pathway and DNA repair at the G2/M checkpoint, thereby promoting therapeutic resistance, but the coordination of these mechanisms is unclear. Our recent RNAi screen unexpectedly uncovered possible crosstalk between WEE1, a G2/M checkpoint kinase and the TNFα-IKK-NF-κB prosurvival pathway components. siRNAs targeting WEE1 and IKKs both inhibited TNFα-inducible NF-κB activity in a HNSCC reporter line, suggesting a mechanistic linkage. We investigated this potential connection further and studied the effect of WEE1 inhibition using AZD1775 in head and neck squamous cell carcinoma (HNSCC) cells and mouse xenograft models. Increased expression, phosphorylation and cellular localization of IKK-NF-κB and WEE1-CDC2 axis proteins co-occurred in a subset of HNSCC lines, and were inversely co-modulated by TNFα and WEE1 kinase inhibitor AZD1775. WEE1 co-immunoprecipitated with the IKKα/β and RELA protein complex, and AZD1775 or an IKK inhibitor, IKK16, inhibited phosphorylation and kinase activities, further suggesting that these two axes interact with each other. AZD1775 sensitized HNSCC cells to TNFα-induced cytotoxicity, in part by inhibiting IKK-dependent phosphorylation and nuclear localization of NF-κB activation, and expression of target prosurvival protein BCL-2. Combination of AZD1775 with radiation, a known inducer of TNFα- and DNA-mediated cytotoxicity, potently inhibited HPV+/- HNSCC tumor xenografts. Depleting TNFα abolished the anti-tumor activity of combined therapy with AZD1775 and radiation. The findings of our study reveal that TNFα co-modulates a novel interaction between the prosurvival IKK/NF-κB/RELA and WEE1-CDC2 G2/M checkpoint pathway and that components of both the pathways are inhibited by AZD1775. Our mouse xenograft experiments suggest that AZD1775, when combined with radiation, significantly delays the growth of xenograft HNSCC tumors differing in HPV status in a TNFα-dependent manner. The current findings reveal a novel mechanism whereby crosstalk and co-activation of WEE1- and NF-κB-signaling promotes resistance to TNFα-mediated cytotoxicity, which may be targeted using WEE1 inhibitors. Supported by NIDCD projects ZIA-DC-000016, -73 and 74. Citation Format: Ramya Viswanathan, Zhengbo Hu, Jianghong Chen, Xinping Yang, Angel Huynh, Paul Clavijo, Yi An, Yvette Robbins, Christopher Silvin, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. TNFa co-activates IKK/NF-kB/RELA prosurvival and WEE1-CDC2 G2/M checkpoint signaling and is targetable by WEE1 antagonist AZD1775 in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6042.

Protective Role of Optineurin Against Joint Destruction in Rheumatoid Arthritis Synovial Fibroblasts

Optineurin (OPTN) is an autophagy adaptor/receptor that acts as an intrinsic negative regulator of osteoclast differentiation. RANKL expressed by rheumatoid arthritis synovial fibroblasts (RASFs) is primarily responsible for the development of bone erosions in patients with RA. The aim of the present study was to explore the role of OPTN in the pathogenesis of joint destruction in RA. RASFs were left untreated or incubated with tumor necrosis factor (TNF) or interferon-γ (IFNγ), and expression of OPTN by RASFs was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Expression of RANKL and osteoprotegerin (OPG) was evaluated in cultures of OPTN-reduced RASFs with or without TNF or IFNγ treatment. OPTN-reduced RASFs were cocultured with monocytes and stained for tartrate-resistant acid phosphatase (TRAP). IκBα, NF-κB1, and RelA protein levels were measured to evaluate NF-κB signaling. Expression of messenger RNA (mRNA) for matrix metalloproteinase 3 (MMP3), interleukin-6 (IL6), GATA binding protein 3 (GATA3), carbohydrate sulfotransferase 15 (CHST15), hyaluronan synthase 1 (HAS1), and GATA1 was analyzed by RT-qPCR. In RASFs incubated with TNF or IFNγ, OPTN expression was up-regulated and RANKL expression was increased, and these effects were further pronounced in OPTN-reduced RASFs (all P < 0.05 versus controls). OPG mRNA levels remained unchanged. Monocytes cocultured with OPTN-reduced RASFs differentiated to a greater extent into TRAP+ multinucleated cells compared to monocytes cocultured with control RASFs (P < 0.05). IκBα degradation and nuclear NF-κB1 expression following TNF treatment were both prolonged in OPTN-reduced RASFs (each P < 0.05 versus controls). MMP3 mRNA levels were up-regulated, while GATA3, CHST15, and HAS1 mRNA levels were down-regulated in OPTN-reduced RASFs (each P < 0.05 versus controls). OPTN plays a protective role in RA when it is up-regulated in RASFs in the presence of proinflammatory cytokines. Absence of OPTN might worsen RA by generating a joint-destructive state, as indicated by evidence of increased RANKL expression on RASFs and subsequent osteoclast differentiation.

Synergistic role of abiotic factors driving viable but non-culturable Vibrio cholerae.

Vibrio cholerae O1, a natural inhabitant of estuarine environments, is found in a dormant, viable but non-culturable (VBNC) state during interepidemic periods. Although the individual roles of abiotic factors affecting VBNC formation have been extensively studied, their interplay in driving this phenomenon remains largely unaddressed. Here, we identified that major abiotic factors synergize with low nutrient conditions governing entry of cells into the VBNC state. Specifically, V. cholerae cells exposed to a combination of alkaline pH and high salinity under aeration at low temperatures (VBNC-inducing conditions) synergize to facilitate rapid entry into VBNC, whereas the opposite conditions prevented entry into the state. The major virulence regulator ToxR, and the stringent response protein RelA played opposing roles, repressing and facilitating VBNC entry respectively. Further, VBNC-inducing conditions negated the effects of ToxR and RelA, facilitating rapid formation of VBNC cells. In summary, this study highlights the synergy between critical abiotic factors and identified ToxR and RelA as two associated regulators, allowing for the persistence of V. cholerae in aquatic environments. Insights obtained in this study will help better understand environmental survival non-sporulating bacteria and transmission of facultative bacterial pathogens.