An intrinsically disordered transcription activation domain increases the DNA binding affinity and reduces the specificity of NFκB p50/RelA.

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with coactivators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. Whether these results can be generalized to more TADs is not clear. Here, we biophysically characterized the NFκB p50/RelA heterodimer including the RelA TAD and investigated the TAD's influence on NFκB-DNA interactions. In solution, we show the RelA TAD is disordered but compact, with helical tendency in two regions that interact with coactivators. We determined that the presence of the TAD increased the stoichiometry of NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. In addition, we measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it also increased the affinity for nonspecific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB DNA sequences. In contrast, previous studies have generally reported that TADs decrease DNA-binding affinity and increase sequence specificity. Our results reveal a novel function of the RelA TAD in promoting binding to nonconsensus DNA, which sheds light on previous observations of extensive nonconsensus DNA binding by NFκB in vivo in response to strong inflammatory signals.

Interactions of a Long Noncoding RNA with Domains of NF-κB and IκBα: Implications for the Inhibition of Non-Signal-Related Phosphorylation.

The transcription factor NF-κB is one of the central mediators of cellular signaling pathways. Under resting conditions, the canonical RelA-p50 (p65-p50) heterodimer NF-κB remains sequestered in the cytoplasm in complex with its inhibitor IκBα. Signal-mediated activation of NF-κB involves phosphorylation, ubiquitination and degradation of IκBα, and translocation of NF-κB to the nucleus. It was recently shown that a long noncoding RNA (termed NKILA) can modulate the NF-κB signaling circuit by interacting with the NF-κB-IκBα complex in the cytoplasm. In the current study, we investigated the interaction of RNA sequences derived from NKILA with domains of NF-κB and IκBα using NMR spectroscopy and native gel electrophoresis. Our results indicate that two RNA hairpin sequences interact with the DNA-binding domains of the Rel homology regions of RelA (p65) and p50 and that the same RNA sequences can affect the phosphorylation of the N-terminus of IκBα under low-salt conditions. We also observe that full-length RHR dimers (heterodimer of p65 and p50 and homodimer of p50) show a stronger interaction with the RNA hairpins than the individual domains of NF-κB. All of the interactions we observe between fragments of NKILA and domains of NF-κB are weak and nonspecific, consistent with the proposed function of the NKILA-NF-κB-IκBα interaction in protecting the NFκB-IκBα complex from aberrant activation of the NF-κB signaling pathway.

Voacanga globosa Spirobisindole Alkaloids Exert Antiviral Activity in HIV Latently Infected Cell Lines by Targeting the NF-kB Cascade: In Vitro and In Silico Investigations.

Since the efficiency in the transcription of the HIV genome contributes to the success of viral replication and infectivity, we investigated the downregulating effects of the spirobisindole alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) from the endemic Philippine medicinal plant, Voacanga globosa, during HIV gene transcription. Alkaloids 1-3 were explored for their inhibitory activity on TNF-α-induced viral replication in two latently HIV-infected cell lines, OM10.1 and J-Lat. The induction of HIV replication from OM10.1 and J-Lat cells elicited by TNF-α was blocked by globospiramine (1) within noncytotoxic concentrations. Furthermore, globospiramine (1) was found to target the NF-ĸB activation cascade in a dose-dependent manner when the transcriptional step at which inhibitory activity is exerted was examined in TNF-α-induced 293 human cells using transient reporter (luciferase) gene expression systems (HIV LTR-luc, ĸB-luc, and mutant ĸB-luc). Interrogation through molecular docking against the NF-ĸB p50/p65 heterodimer and target sites of the subunits comprising the IKK complex revealed high binding affinities of globospiramine (1) against the S281 pocket of the p65 subunit (BE = -9.2 kcal/mol) and the IKKα activation loop (BE = -9.1 kcal/mol). These findings suggest globospiramine (1) as a molecular inspiration to discover new alkaloid-based anti-HIV derivatives.

ZCL-082, a boron-containing compound, induces apoptosis of non-Hodgkin's lymphoma via targeting p90 ribosomal S6 kinase 1/NF-κB signaling pathway.

INTRODUCTION: Despite the rapid progress in the diagnosis and treatment, the prognosis of some types of non-Hodgkin's lymphoma (NHL), especially those with double-hit or double-expressor genotypes, remains poor. Novel targets and compounds are needed to improve the prognosis of NHL. METHODS: We investigated the effect of ZCL-082, a novel boron-containing compound with anti-proliferating activity against ovarian cancer cells, on NHL cells and human peripheral blood mononuclear cells by CCK-8 assay, Annexin V/PI double staining assay, RH123/PI double staining, Western blot, and immunohistochemistry. NF-κB pathway activity was analyzed using luciferase reporter gene assay and RT-PCR. The location of p65 was detected by immunofluorescence and nuclear/cytoplasmic fractionation assay. Immunoprecipitation and chromatin immunoprecipitation assays were used to detect the binding between p65 and p300. CETSA and molecular docking assay were carried out to test the interaction between ZCL-082 and p90 ribosomal S6 kinase 1 (RSK1). Kinase reaction was conducted to examine the inhibition of RSK1 kinase activity by ZCL-082. RESULTS: We found that ZCL-082 can induce the apoptosis of various NHL cell lines in vitro and in vivo. ZCL-082 significantly inhibits TNFα- or LPS-induced NF-κB activation without disturbing TNFα-induced IκBα degradation or the nuclear translocation and DNA-binding ability of p65. However, ZCL-082 markedly suppresses the phosphorylation of p65 on Ser536 and the interaction between p65 and p300. The overexpression of the phosphomimetic mutant of p65 at Ser536 partially abrogates ZCL-082-induced cell death. We further found that ZCL-082 directly binds to and inhibits the activity of RSK1. RSK1 can phosphorylate RelA/p65 on Ser536 and its overexpression is associated with the poor prognosis of lymphoma. The overexpression of RSK1 partially rescues ZCL-082-induced cell death. Molecular docking studies show that ZCL-082 fits well with the N-terminal kinase domain of RSK1. Furthermore, the combination of ZCL-082 and BCL-2 inhibitor ABT-199 has a synergistic apoptosis-inducing effect against double-hit lymphoma cell line OCI-Ly10. DISCUSSION: We found that ZCL-082 is a highly promising anti-lymphoma compound that targets RSK1 and interferes with the RSK1/NF-κB signaling pathway. The combination of ZCL-082 with BCL-2 inhibitor may represent a novel strategy to improve the outcome of double-hit or double-expressor lymphoma.

Single-molecule conformational dynamics of a transcription factor reveals a continuum of binding modes controlling association and dissociation.

Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.

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 10 min. 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.

PDTC ameliorates neuropathic pain by inhibiting microglial activation via blockage of the TNFα-CX3CR1 pathway.

Previous studies have suggested that pyrrolidine dithiocarbamate (PDTC), a nuclear factor κB (NF-κB) inhibitor, play a role in deterring nerve injury-induced neuropathic pain (NP) The activation of NF-κB pathway may contribute to spinal microglial activation, CX3CR1 and tumor necrosis factor-alpha (TNF-a) up-regulation. The aim of this study was to clarify whether PDTC could inhibit the development of neuropathic pain via decreasing TNF-a-induced CX3CR1 up-regulation. Sprague-Dawley rats were randomly divided into sham group and NP group. Rats in each group were treated with intrathecal infusion of PDTC (100 or 1000 pmol/d) or saline. The sciatic nerve chronic constriction injury (CCI) model was used to induce NP in rats. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. Spinal microglial marker OX42 and TNF-a were detected by immunohistochemistry. In vitro BV-2 microglia activation was induced by TNF-a incubation, and the levels of CX3CR1 were assessed by Western blot and reverse transcription-polymerase chain reaction. Pain behavior and immunohistochemistry results showed that intrathecal infusion of PDTC at 100 or 1000 pmol/d prevented the development of mechanical and thermal hyperalgesia, spinal microglial activation and TNF-a expression induced by sciatic nerve CCI in rats. In vitro experiment results showed that PDTC inhibited the TNF-a-induced CX3CR1 up-regulation in BV-2 microglial cells. In conclusion, intrathecal infusion of PDTC could attenuate the pain-related behaviors induced by sciatic nerve CCI through suppressing the spinal microglia activation and TNF-a up-regulation in rats. The NF-κB activation might be responsible for TNF-a-induced CX3CR1 up-regulation in microglia.

Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer.

The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA-p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA-RelA homodimer is critical as it facilitates the formation of the more stable RelA-p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA-RelA homodimer, the kinetics of the RelA-p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA-RelA specific target gene regulation in physiology.

1,7­Bis(4­hydroxy­3­methoxyphenyl)­1,4,6­heptatrien­3­one alleviates lipopolysaccharide­induced inflammation by targeting NF­κB translocation in murine macrophages and it interacts with MD2 in silico.

Trienones are curcuminoid analogues and are minor constituents in the rhizomes of numerous Curcuma plant species. Studies investigating the biological activities of trienones, particularly their anti­inflammatory activities, are limited. In the present study, the trienone 1,7­bis(4­hydroxy­3­methoxyphenyl)­1,4,6­heptatrien­3­one (HMPH) was structurally modified from curcumin using a novel and concise method. HMPH was shown to exhibit potential anti­inflammatory effects on lipopolysaccharide (LPS)­activated RAW264.7 macrophages. Furthermore, LPS­induced nitric oxide secretion in RAW264.7 cells was markedly and dose­dependently inhibited by HMPH; in addition, HMPH had a greater efficacy compared with curcumin. This inhibition was accompanied by the suppression of inducible nitric oxide synthase and cyclooxygenase­2 expression, as well as pro­inflammatory cytokine secretion. To elucidate the molecular mechanism underlying the anti­inflammatory effects of HMPH, the effects of this compound on nuclear factor­κB (NF­κB) translocation were assessed. HMPH significantly inhibited the translocation of p65 NF­κB into the nucleus to a greater extent than curcumin, thus indicating that HMPH has more potent anti­inflammatory activity than curcumin. In addition, an in silico modelling study revealed that HMPH possessed stronger binding energy to myeloid differentiation factor 2 (MD2) compared with that of curcumin, and indicated that the anti­inflammatory effects of HMPH may be through upstream inhibition of the inflammatory pathway. In conclusion, HMPH may be considered a promising compound for reducing inflammation via targeting p65 NF­κB translocation and interfering with MD2 binding.

Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods.

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA-DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Functional network analysis reveals potential repurposing of ß-blocker atenolol for pancreatic cancer therapy.

BACKGROUND: The survival rate of patients with pancreatic cancer is low; therefore, continuous discovery and development of novel pancreatic cancer drugs are required. Functional network analysis is an integrated bioinformatics approach based on gene, target, and disease networks interaction, and it is extensively used in drug discovery and development. OBJECTIVE: This study aimed to identify if atenolol, a selective adrenergic inhibitor, can be repurposed for the treatment of pancreatic cancer using functional network analysis. METHODS: Direct target proteins (DTPs) and indirect target proteins (ITPs) were obtained from STITCH and STRING databases, respectively. Atenolol-mediated proteins (AMPs) were collected from DTPs and ITPs and further analyzed for gene ontology, KEGG pathway enrichment, genetic alterations, overall survival, and molecular docking. RESULTS: We obtained 176 AMPs that consisted of 10 DTPs and 166 ITPs. Among the AMPs involved in the pancreatic cancer pathways, several AMPs such as MAPK1, RELA, MAPK8, STAT1, and STAT3 were identified. Genetic alterations in seven AMPs were identified in 0.9%-16% of patients. Patients with high mRNA levels of MAPK1, RELA, STAT3, GNB1, and MMP9 had significantly worse overall survival rates compared with patients with low expression. Molecular docking studies showed that RELA and MMP9 are potential target candidates of atenolol in the treatment of patients with pancreatic cancer. CONCLUSION: In conclusion, atenolol can potentially be repurposed to target pancreatic cancer cells by modulating MMP9 and NF-κB signaling. The results of this study need to be further validated in vitro and in vivo.

Fructose-1, 6-bisphosphatase 1 interacts with NF-κB p65 to regulate breast tumorigenesis via PIM2 induced phosphorylation.

Rationale: Fructose-1, 6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis, was recently shown to be a tumor suppressor and could mediate the activities of multiple transcriptional factors via its non-canonical functions. However, the underlying mechanism of posttranscriptional modification on the non-canonical functions of FBP1 remains elusive. Methods: We employed immunoaffinity purification to identify binding partner(s) and used co-immunoprecipitation to verify their interactions. Kinase reaction was used to confirm PIM2 could phosphorylate FBP1. Overexpression or knockdown proteins were used to assess the role in modulating p65 protein stability. Mechanistic analysis was involved in protein degradation and polyubiquitination assays. Nude mice and PIM2-knockout mice was used to study protein functions in vitro and in vivo. Results: Here, we identified Proviral Insertion in Murine Lymphomas 2 (PIM2) as a new binding partner of FBP1, which could phosphorylate FBP1 on Ser144. Surprisingly, phosphorylated FBP1 Ser144 abrogated its interaction with NF-κB p65, promoting its protein stability through the CHIP-mediated proteasome pathway. Furthermore, phosphorylation of FBP1 on Ser144 increased p65 regulated PD-L1 expression. As a result, phosphorylation of FBP1 on Ser144 promoted breast tumor growth in vitro and in vivo. Moreover, the levels of PIM2 and pSer144-FBP1 proteins were positively correlated with each other in human breast cancer and PIM2 knockout mice. Conclusions: Our findings revealed that phosphorylation noncanonical FBP1 by PIM2 was a novel regulator of NF-κB pathway, and highlights PIM2 inhibitors as breast cancer therapeutics.

Fragment-Based Stabilizers of Protein-Protein Interactions through Imine-Based Tethering.

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising concept in drug discovery, however the question how to identify or design chemical starting points in a "bottom-up" approach is largely unanswered. We report a novel concept for identifying initial chemical matter for PPI stabilization based on imine-forming fragments. The imine bond offers a covalent anchor for site-directed fragment targeting, whereas its transient nature enables efficient analysis of structure-activity relationships. This bond enables fragment identification and optimisation using protein crystallography. We report novel fragments that bind specifically to a lysine at the PPI interface of the p65-subunit-derived peptide of NF-κB with the adapter protein 14-3-3. Those fragments that subsequently establish contacts with the p65-derived peptide, rather than with 14-3-3, efficiently stabilize the 14-3-3/p65 complex and offer novel starting points for molecular glues.

Synthesis and Spectroscopic Analysis of Piperine- and Piperlongumine-Inspired Natural Product Scaffolds and Their Molecular Docking with IL-1ß and NF-κB Proteins.

Inspired by the remarkable bioactivities exhibited by the natural products, piperine and piperlongumine, we synthesised eight natural product-inspired analogues to further investigate their structures. For the first time, we confirmed the structure of the key cyclised dihydropyrazolecarbothioamide piperine analogues including the use of two-dimensional (2D) 15N-based spectroscopy nuclear magnetic resonance (NMR) spectroscopy. Prior investigations demonstrated promising results from these scaffolds for the inhibition of inflammatory response via downregulation of the IL-1ß and NF-κB pathway. However, the molecular interaction of these molecules with their protein targets remains unknown. Ab initio calculations revealed the electronic density function map of the molecules, showing the effects of structural modification in the electronic structure. Finally, molecular interactions between the synthesized molecules and the proteins IL-1ß and NF-κB were achieved. Docking results showed that all the analogues interact in the DNA binding site of NF-κB with higher affinity compared to the natural products and, with the exception of 9a and 9b, have higher affinity than the natural products for the binding site of IL-1ß. Specificity for the molecular recognition of 3a, 3c and 9b with IL-1ß through cation-π interactions was determined. These results revealed 3a, 3c, 4a, 4c and 10 as the most promising molecules to be evaluated as IL-1ß and NF-κB inhibitors.

Trichostatin A augments esophageal squamous cell carcinoma cells migration by inducing acetylation of RelA at K310 leading epithelia-mesenchymal transition.

Protein acetylation modification controlled by acetyltransferases (HATs) and histone deacetylases (HDACs) regulates multiple biologic processes including cell proliferation and migration. HDAC inhibitors (HDACi) are currently used as a promising epigenetic-based therapy for cancer treatment. Of the anticancer activity, accumulating evidence has shown that HDACi can enhance cell migration in subset of cancer cells. Thus, there is a critical need to identify such counter anticancer activity to HDACi in different cancer cell types and elucidate the rational in order to develop appropriate combination therapies in cancer treatment. In seeking to address the effect of HDACi on esophageal squamous cell carcinoma (ESCC) cells migration, trichostatin A (TSA), a canonical HDACi targeting class I and class II HDACs, was used. Here, we report the discovery that TSA augmented ESCC cells migration by increasing the acetylation of nuclear factor-κB/RelA at lysine 310 (K310). To elucidate the mechanism by which TSA promotes the migration of ESCC cells, plasmid of RelA K310R, a mutant precluding acetylation at K310, was transfected into ESCC cells. Blocking acetylation of RelA at K310 significantly arrogated TSA-induced cell migration. Mechanistic investigations revealed that TSA increased the level of acetylated RelA at K310 (RelA K310ac), thereby increasing the level of epithelia-mesenchymal transition (EMT) transcription factor slug mRNA, which in turn induced EMT. Overall, this study indicates that TSA promotes ESCC cells migration by RelA K310ac-slug-EMT pathway. Our findings provide a strategy to eradicate HDACi-induced ESCC cells migration by targeting RelA as a combination therapy with nonspecific HDACi in ESCC treatment.

Assaying Homodimers of NF-κB in Live Single Cells.

NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as "NF-κB" in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.

The NF-κB RelA Transcription Factor Is Critical for Regulatory T Cell Activation and Stability.

Regulatory T cells (Tregs) play a major role in immune homeostasis and in the prevention of autoimmune diseases. It has been shown that c-Rel is critical in Treg thymic differentiation, but little is known on the role of NF-κB on mature Treg biology. We thus generated mice with a specific knockout of RelA, a key member of NF-κB, in Tregs. These mice developed a severe autoimmune syndrome with multi-organ immune infiltration and high activation of lymphoid and myeloid cells. Phenotypic and transcriptomic analyses showed that RelA is critical in the acquisition of the effector Treg state independently of surrounding inflammatory environment. Unexpectedly, RelA-deficient Tregs also displayed reduced stability and cells that had lost Foxp3 produced inflammatory cytokines. Overall, we show that RelA is critical for Treg biology as it promotes both the generation of their effector phenotype and the maintenance of their identity.

Comparison of backbone dynamics of the p50 dimerization domain of NFκB in the homodimeric transcription factor NFκB1 and in its heterodimeric complex with RelA (p65).

The nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) transcription factors play a critical role in human immune response. The family includes homodimers and heterodimers of five component proteins, which mediate different transcriptional responses and bind preferentially to different DNA sequences. Crystal structures of DNA complexes show that the dimers of the Rel-homology regions are structurally very similar. Differing DNA sequence preference together with structural similarity suggests that the dimers may differ in their dynamics. In this study, we present the first near-complete 15 N, 13 Cα/ß , and HN backbone resonance assignments of two dimers of the dimerization domain (DD) of the NFκB1 (p50) protein (residues 241-351): the homodimer of two p50 domains and a heterodimer of the p50 DD with the p65 DD. As expected, the two dimers behave very similarly, with chemical shift differences between them largely concentrated in the dimer interface and attributable to specific differences in the amino acid sequences of p50 and p65. A comparison of the picosecond-nanosecond dynamics of the homo- and heterodimers also shows that the environment of p50 is similar, with an overall slightly reduced correlation time for the homodimer compared to the heterodimer, consistent with its slightly smaller molecular weight. These results demonstrate that NMR spectroscopy can be used to explore subtle changes in structure and dynamics that have the potential to give insights into differences in specificity that can be exploited in the design of new therapeutic agents.

Nerolidol attenuates cyclophosphamide-induced cardiac inflammation, apoptosis and fibrosis in Swiss Albino mice.

Incidence and prevalence of cancer is an alarming situation globally. For the treatment of cancer many anticancer drugs have been developed but, unfortunately, their potential cardiotoxic side effects raised serious concerns about their use among clinicians. Cyclophosphamide is a potent anticancer and immunosuppressant drug but its use is limited due to cardiotoxic side effect. Thus, there is a need for the development of certain drug which can reduce cardiotoxicity and can be used as an adjuvant therapy in cancer patients. In this direction we, therefore planned to evaluate nerolidol (NER) for its cardioprotective potential against cyclophosphamide-induced cardiotoxicity in Swiss Albino mice. Animals were divided into 6 groups. Vehicle control; Cyclophosphamide (CP 200); NER 400 per se; NER 200 + CP 200; NER 400 + CP 200; and fenofibrate (FF 80) + CP 200. Dosing was done for 14 days along with a single dose of CP 200 on the 7th day. On 15th day animals were sacrificed and various biochemical parameters pertaining to oxidative stress, nitrative stress, inflammation, apoptosis and fibrosis were estimated in the blood and heart tissues. Histopathological analysis (H & E and Masson's trichrome staining); ultrastructural analysis (transmission electron microscopy) and immunohistochemical analysis were also performed along with mRNA expression and molecular docking to establish the cardioprotective potential of nerolidol. Nerolidol acted as a potent cardioprotective molecule and attenuated CP-induced cardiotoxicity.

The E3 ubiquitin ligase RNF182 inhibits TLR-triggered cytokine production through promoting p65 ubiquitination and degradation.

The activation of Toll-like receptors (TLRs) leads to proinflammatory cytokine production, which is responsible for activating the innate immune system. Thus, TLR signaling is subject to multilayer regulatory control mechanisms that aim to prevent a protective response from causing injury. In the present study, we report that the E3 ubiquitin ligase RNF182 is highly expressed in macrophages and is specifically upregulated by TLR stimuli (TLR4, TLR3 and TLR9 agonists). Knockdown of RNF182 selectively amplifies TLR signaling by promoting the production of proinflammatory cytokines but not type I interferons in macrophages. Mechanistically, RNF182 promotes the degradation of p65 via K48-linked ubiquitination, resulting in the inhibition of TLR-triggered innate immune responses. Our findings highlight a feedback-negative mechanism for terminating TLR-induced inflammation and maintaining the immunological balance.