Molecular mechanism of IKK catalytic dimer docking to NF-κB substrates.

The inhibitor of κB (IκB) kinase (IKK) is a central regulator of NF-κB signaling. All IKK complexes contain hetero- or homodimers of the catalytic IKKß and/or IKKα subunits. Here, we identify a YDDΦxΦ motif, which is conserved in substrates of canonical (IκBα, IκBß) and alternative (p100) NF-κB pathways, and which mediates docking to catalytic IKK dimers. We demonstrate a quantitative correlation between docking affinity and IKK activity related to IκBα phosphorylation/degradation. Furthermore, we show that phosphorylation of the motif's conserved tyrosine, an event previously reported to promote IκBα accumulation and inhibition of NF-κB gene expression, suppresses the docking interaction. Results from integrated structural analyzes indicate that the motif binds to a groove at the IKK dimer interface. Consistently, suppression of IKK dimerization also abolishes IκBα substrate binding. Finally, we show that an optimized bivalent motif peptide inhibits NF-κB signaling. This work unveils a function for IKKα/ß dimerization in substrate motif recognition.

Dehydroevodiamine targeting IKKß to alleviate acute gastric injury via inhibiting the p65/NLRP3 axis.

BACKGROUND: Acute gastric injury, a common and recurring global digestive disorder, significantly impairs patient quality of life and overall health. Dehydroevodiamine (DHE), a bioactive natural product derived from Tetradium ruticarpum (A. Juss.) Hartley, shows potential therapeutic effects on acute gastric injury. This study investigates the underlying mechanisms of DHE's alleviating effects on acute gastric injury. METHODS: The gastric mucosal protective effect of DHE was confirmed through in vivo and in vitro acute gastric injury models. Biotin pulldown MS and molecular dynamics simulations identified DHE's target. CETSA and SPR assays validated DHE's affinity for IKKß. Protein site mutation validation and MST pinpointed the direct binding sites of DHE on IKKß. Additionally, the potential mechanism by which DHE ameliorates acute gastric injury was elucidated using WB, IHC, and IF methods, and further confirmed by rescue experiments. RESULTS: DHE effectively ameliorated IDO-induced gastric injury in GES-1 cells and rat gastric mucosa, both in vitro and in vivo. Biotin pulldown MS identified IKKß as the target of DHE in alleviating gastric injury. CETSA and SPR assays confirmed DHE's direct binding to IKKß. Molecular dynamics simulations, protein mutation experiments, and MST results pinpointed GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket as the binding sites of DHE on IKKß. Notably, DHE was found to competitively bind to IKKß with ATP. Mechanistically, DHE attenuated IDO-induced gastric injury by inhibiting the IKKß-p65/NLRP3 signaling pathway. Importantly, exogenous activation of IKKß reversed the therapeutic effect of DHE, indicating that DHE's efficacy depends on IKKß. CONCLUSION: DHE attenuated IDO-induced gastric injury by inhibiting the IKKß-p65/NLRP3 signaling pathway. Notably, DHE is a novel ATP-competitive IKKß inhibitor that prevents phosphorylation by targeting GLU-149, GLU-49, and ASP-103 in the ATP-binding pocket. This study reveals new targets of action for DHE, providing a new molecular basis for using DHE in treating inflammation-related diseases.

IKKα-STAT3-S727 axis: a novel mechanism in DOX-induced cardiomyopathy.

Doxorubicin (DOX) is an effective chemotherapeutic drug, but its use can lead to cardiomyopathy, which is the leading cause of mortality among cancer patients. Macrophages play a role in DOX-induced cardiomyopathy (DCM), but the mechanisms undlerlying this relationship remain unclear. This study aimed to investigate how IKKα regulates macrophage activation and contributes to DCM in a mouse model. Specifically, the role of macrophage IKKα was evaluated in macrophage-specific IKKα knockout mice that received DOX injections. The findings revealed increased expression of IKKα in heart tissues after DOX administration. In mice lacking macrophage IKKα, myocardial injury, ventricular remodeling, inflammation, and proinflammatory macrophage activation worsened in response to DOX administration. Bone marrow transplant studies confirmed that IKKα deficiency exacerbated cardiac dysfunction. Macrophage IKKα knockout also led to mitochondrial damage and metabolic dysfunction in macrophages, thereby resulting in increased cardiomyocyte injury and oxidative stress. Single-cell sequencing analysis revealed that IKKα directly binds to STAT3, leading to the activation of STAT3 phosphorylation at S727. Interestingly, the inhibition of STAT3-S727 phosphorylation suppressed both DCM and cardiomyocyte injury. In conclusion, the IKKα-STAT3-S727 signaling pathway was found to play a crucial role in DOX-induced cardiomyopathy. Targeting this pathway could be a promising therapeutic strategy for treating DOX-related heart failure.

Non-Skewed X-inactivation Results in NF-κB Essential Modulator (NEMO) Δ-exon 5-autoinflammatory Syndrome (NEMO-NDAS) in a Female with Incontinentia Pigmenti.

PURPOSE: Genetic hypomorphic defects in X chromosomal IKBKG coding for the NF-κB essential modulator (NEMO) lead to ectodermal dysplasia and immunodeficiency in males and the skin disorder incontinentia pigmenti (IP) in females, respectively. NF-κB essential modulator (NEMO) Δ-exon 5-autoinflammatory syndrome (NEMO-NDAS) is a systemic autoinflammatory disease caused by alternative splicing and increased proportion of NEMO-Δex5. We investigated a female carrier presenting with IP and NEMO-NDAS due to non-skewed X-inactivation. METHODS: IKBKG transcripts were quantified in peripheral blood mononuclear cells isolated from the patient, her mother, and healthy controls using RT-PCR and nanopore sequencing. Corresponding proteins were analyzed by western blotting and flow cytometry. Besides toll-like receptor (TLR) and tumor necrosis factor (TNF) signaling, the interferon signature, cytokine production and X-inactivation status were investigated. RESULTS: IP and autoinflammation with recurrent fever, oral ulcers, hepatitis, and neutropenia, but no immunodeficiency was observed in a female patient. Besides moderately reduced NEMO signaling function, type I interferonopathy, and elevated IL-18 and CXCL10 were found. She and her mother both carried the heterozygous variant c.613 C > T p.(Gln205*) in exon 5 of IKBKG previously reported in NEMO-deficient patients. However, X-inactivation was skewed in the mother, but not in the patient. Alternative splicing led to increased ratios of NEMO-Dex5 over full-length protein in peripheral blood cell subsets causing autoinflammation. Clinical symptoms partially resolved under treatment with TNF inhibitors. CONCLUSION: Non-skewed X-inactivation can lead to NEMO-NDAS in females with IP carrying hypomorphic IKBKG variants due to alternative splicing and increased proportions of NEMO-∆ex5.

Design and Synthesis of Novel Aminoindazole-pyrrolo[2,3-b]pyridine Inhibitors of IKKα That Selectively Perturb Cellular Non-Canonical NF-κB Signalling.

The inhibitory-kappaB kinases (IKKs) IKKα and IKKß play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKß-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKß in vitro and explain the structure-activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKß Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKß Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKß in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents.

HDX-MS Analysis of Catalytic Activation of IKK2 in the IκB Kinase Complex.

The IκB Kinase (IKK) complex, containing catalytic IKK2 and noncatalytic NEMO subunits, plays essential roles in the induction of transcription factors of the NF-κB family. Catalytic activation of IKK2 via phosphorylation of its activation loop is promoted upon noncovalent association of linear or K63-linked polyubiquitin chains to NEMO within the IKK complex. The mechanisms of this activation remain speculative. To investigate interaction dynamics within the IKK complex during activation of IKK2, we conducted hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on NEMO and IKK2 proteins in their free and complex-bound states. Altered proton exchange profiles were observed in both IKK2 and NEMO upon complex formation, and changes were consistent with the involvement of distinct regions throughout the entire length of both proteins, including previously uncharacterized segments, in direct or allosteric interactions. Association with linear tetraubiquitin (Ub4) affected multiple regions of the IKK2:NEMO complex, in addition to previously identified interaction sites on NEMO. Intriguingly, observed enhanced solvent accessibility of the IKK2 activation loop within the IKK2:NEMO:Ub4 complex, coupled with contrasting protection of surrounding segments of the catalytic subunit, suggests an allosteric role for NEMO:Ub4 in priming IKK2 for phosphorylation-dependent catalytic activation.

Blockade of IKK signaling induces RIPK1-independent apoptosis in human macrophages.

Regulated cell death in response to microbial infection plays an important role in immune defense and is triggered by pathogen disruption of essential cellular pathways. Gram-negative bacterial pathogens in the Yersinia genus disrupt NF-κB signaling via translocated effectors injected by a type III secretion system, thereby preventing induction of cytokine production and antimicrobial defense. In murine models of infection, Yersinia blockade of NF-κB signaling triggers cell-extrinsic apoptosis through Receptor Interacting Serine-Threonine Protein Kinase 1 (RIPK1) and caspase-8, which is required for bacterial clearance and host survival. Unexpectedly, we find that human macrophages undergo apoptosis independently of RIPK1 in response to Yersinia or chemical blockade of IKKß. Instead, IKK blockade led to decreased cFLIP expression, and overexpression of cFLIP contributed to protection from IKK blockade-induced apoptosis in human macrophages. We found that IKK blockade also induces RIPK1 kinase activity-independent apoptosis in human T cells and human pancreatic cells. Altogether, our data indicate that, in contrast to murine cells, blockade of IKK activity in human cells triggers a distinct apoptosis pathway that is independent of RIPK1 kinase activity. These findings have implications for the contribution of RIPK1 to cell death in human cells and the efficacy of RIPK1 inhibition in human diseases.

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus.

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (encoded by IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKε. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a regulatory pathway involving rs2297550, Ikaros, and IKKε implicated by human genetics in risk for SLE.

Cyld restrains the hyperactivation of synovial fibroblasts in inflammatory arthritis by regulating the TAK1/IKK2 signaling axis.

TNF is a potent cytokine known for its involvement in physiology and pathology. In Rheumatoid Arthritis (RA), persistent TNF signals cause aberrant activation of synovial fibroblasts (SFs), the resident cells crucially involved in the inflammatory and destructive responses of the affected synovial membrane. However, the molecular switches that control the pathogenic activation of SFs remain poorly defined. Cyld is a major component of deubiquitination (DUB) machinery regulating the signaling responses towards survival/inflammation and programmed necrosis that induced by cytokines, growth factors and microbial products. Herein, we follow functional genetic approaches to understand how Cyld affects arthritogenic TNF signaling in SFs. We demonstrate that in spontaneous and induced RA models, SF-Cyld DUB deficiency deteriorates arthritic phenotypes due to increased levels of chemokines, adhesion receptors and bone-degrading enzymes generated by mutant SFs. Mechanistically, Cyld serves to restrict the TNF-induced hyperactivation of SFs by limiting Tak1-mediated signaling, and, therefore, leading to supervised NF-κB and JNK activity. However, Cyld is not critically involved in the regulation of TNF-induced death of SFs. Our results identify SF-Cyld as a regulator of TNF-mediated arthritis and inform the signaling landscape underpinning the SF responses.

Puerarin Attenuates Insulin Resistance by Inhibiting Endoplasmic Reticulum Stress and Suppresses Inflammation by Modulating the JNK and IKKß/NF-κB Pathways in Epididymal White Adipose Tissue of Mice on a High-Fat Diet.

SCOPE: Obesity is associated with insulin resistance (IR), which is characterized by endoplasmic reticulum (ER) stress in multiple organs. ER stress in adipose tissue causes metabolic disturbances and activates inflammatory signaling pathways. Puerarin, an isoflavone extracted from Pueraria lobata, exhibits antioxidant, anti-inflammatory, and antidiabetic effects. This study explores the potential mechanisms underlying puerarin's role in mitigating insulin resistance in high-fat diet (HFD)-induced obese mice. METHODS AND RESULTS: In this study, insulin resistant in mice is induced by a high-fat diet, followed by treatment with puerarin. The results demonstrate that puerarin effectively attenuates insulin resistance, including weight loss, improvement of glucose tolerance and insulin sensitivity, and activation of insulin signaling pathway. Additionally, puerarin administration suppresses ER stress by down-regulation of ATF6, ATF4, CHOP, GRP78 expressions in epididymal white adipose tissue (eWAT), along with decreased phosphorylation IRE1α, PERK, and eIF2α. Furthermore, puerarin exerts anti-inflammatory effects by inhibiting JNK and IKKß/NF-κB pathways, leading to reduction of TNF-α and IL-6. CONCLUSION: These findings suggest that puerarin mitigates insulin resistance by inhibiting ER stress and suppressing inflammation through the JNK and IKKß/NF-κB pathways. This highlights the promising clinical application of puerarin in the treatment of insulin resistance.

Thyroxine regulates pig Sertoli cell line proliferation and maturation through the IKK/NFκB and p38 MAPK signaling pathways.

The aim of this study was to investigate the signaling pathways involved in the proliferation and differentiation of pig Sertoli cells (SCs) mediated by thyroid hormone (T3) to provide a theoretical and practical basis for enhancing pig semen production. The effects of different concentrations of T3 on the proliferation of pig SCs were evaluated using the CCK8 assay. The impact of T3 on the proliferation and differentiation of pig SCs was further examined using RNA-seq, qPCR, and Western Blotting techniques. Additionally, the involvement of the p38 MAPK and NFκB pathways in mediating the effects of T3 on SCs proliferation and differentiation was investigated. Our findings revealed a strong correlation between the dosage of T3 and the inhibition of pig SCs proliferation and promotion of maturation. T3 regulated the activation state of the NFκB signaling pathway by upregulating IKKα, downregulating IKKß, and promoting IκB phosphorylation. Furthermore, T3 facilitated SCs maturation by upregulating AR and FSHR expression while downregulating KRT-18. In conclusion, T3 inhibits pig SCs proliferation and promote pig SCs maturation through the IKK/NFκB and p38 MAPK pathways. These findings provide valuable insights into the mechanisms by which T3 influences the proliferation and maturation of pig SCs.

LAIR1 promotes hepatocellular carcinoma cell metastasis and induces M2-macrophage infiltration through activating AKT-IKKß-p65 axis.

LAIR1, a receptor found on immune cells, is capable of binding to collagen and is involved in immune-related diseases. However, the precise contribution of LAIR1 expressed on hepatocellular carcinoma (HCC) cells to tumor microenvironment is still unclear. In our study, bioinformatics analysis and immunofluorescence were employed to study the correlation between LAIR1 levels and clinical indicators. Transwell and scratch tests were used to evaluate how LAIR1 affected the migration and invasion of HCC cells. The chemotactic capacity and alternative activation of macrophages were investigated using RT-qPCR, transwell, and immunofluorescence. To investigate the molecular mechanisms, transcriptome sequencing analysis, Western blot, nucleus/cytoplasm fractionation, ELISA, and cytokine microarray were employed. We revealed a significant correlation between the presence of LAIR1 and an unfavorable outcome in HCC. We indicated that LAIR1 promoted migration and invasion of HCC cells through the AKT-IKKß-p65 axis. Additionally, the alternative activation and infiltration of tumor-associated macrophages induced by LAIR1 were reliant on the upregulation of IL6 and CCL5 within this axis, respectively. In conclusion, blocking LAIR1 was found to be an effective approach in combating the cancerous advancement of HCC.

Secondary interactions in ubiquitin-binding domains achieve linkage or substrate specificity.

Small ubiquitin-binding domains (UBDs) recognize small surface patches on ubiquitin with weak affinity, and it remains a conundrum how specific cellular responses may be achieved. Npl4-type zinc-finger (NZF) domains are ∼30 amino acid, compact UBDs that can provide two ubiquitin-binding interfaces, imposing linkage specificity to explain signaling outcomes. We here comprehensively characterize the linkage preference of human NZF domains. TAB2 prefers Lys6 and Lys63 linkages phosphorylated on Ser65, explaining why TAB2 recognizes depolarized mitochondria. Surprisingly, most NZF domains do not display chain linkage preference, despite conserved, secondary interaction surfaces. This suggests that some NZF domains may specifically bind ubiquitinated substrates by simultaneously recognizing substrate and an attached ubiquitin. We show biochemically and structurally that the NZF1 domain of the E3 ligase HOIPbinds preferentially to site-specifically ubiquitinated forms of NEMO and optineurin. Thus, despite their small size, UBDs may impose signaling specificity via multivalent interactions with ubiquitinated substrates.

ATM-dependent Phosphorylation of Nemo SQ Motifs Is Dispensable for Nemo-mediated Gene Expression Changes in Response to DNA Double-Strand Breaks.

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

Cimicifugoside H-2 as an Inhibitor of IKK1/Alpha: A Molecular Docking and Dynamic Simulation Study.

One of the most challenging issues scientists face is finding a suitable non-invasive treatment for cancer, as it is widespread around the world. The efficacy of phytochemicals that target oncogenic pathways appears to be quite promising and has gained attention over the past few years. We investigated the effect of docking phytochemicals isolated from the rhizomes of the Cimicifuga foetida plant on different domains of the IκB kinase alpha (IKK1/alpha) protein. The Cimicifugoside H-2 phytochemical registered a high docking score on the activation loop of IKK1/alpha amongst the other phytochemicals compared to the positive control. The interaction of the protein with Cimicifugoside H-2 was mostly stabilized by hydrogen bonds and hydrophobic interactions. A dynamic simulation was then performed with the Cimicifugoside H-2 phytochemical on the activation loop of IKK1/alpha, revealing that Cimicifugoside H-2 is a possible inhibitor of this protein. The pharmacokinetic properties of the drug were also examined to assess the safety of administering the drug. Therefore, in this in silico study, we discovered that the Cimicifugoside H-2 phytochemical inhibits the actively mutated conformation of IKK1/alpha, potentially suppressing the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway.

Black carp A20 inhibits interferon signaling through de-ubiquitinating IKKß.

IkappaB kinase beta (IKKß) is a key member of IκB kinases and functions importantly in interferon (IFN) signaling. Phosphorylation and ubiquitination are involved in the activation of IKKß. A20 is a de-ubiquitin enzyme and functions as a suppressor in inflammation signaling, which has been reported to be phosphorylated and activated by IKKß. However, the role and relationship of IKKß and A20 in teleost remains unclear. In this study, IKKß (bcIKKß) and A20 (bcA20) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Overexpressed bcIKKß in EPC cells showed strong anti-viral ability by activating both NF-κB and IFN signaling. EPC cells stable expressing bcIKKß presented improved anti-viral activity as well. The interaction between bcA20 and bcIKKß was identified, and overexpression of bcA20 was able to suppress bcIKKß-mediated activation of NF-κB and IFN signaling. Meanwhile, knock-down of A20 increased host the antiviral ability of host cells. Importantly, it has been identified that bcA20 was able to remove K27-linked ubiquitination and decrease the phosphorylation of bcIKKß. Thus, our data conclude that bcA20 suppresses the anti-viral activity of bcIKKß and removes its K27-linked ubiquitination, which presents a new mechanism of IKKß regulation.

A mitochondrial checkpoint to NF-κB signaling.

Mitochondrial dysfunction can elicit multiple inflammatory pathways, especially when apoptotic caspases are inhibited. Such an inflammatory program is negatively regulated by the autophagic disposal of permeabilized mitochondria. Recent data demonstrate that the ubiquitination of mitochondrial proteins is essential for NEMO-driven NF-kB activation downstream of mitochondrial permeabilization.

Loss of Lymphatic IKKα Disrupts Lung Immune Homeostasis, Drives BALT Formation, and Protects against Influenza.

IκB kinase (IKK)α controls noncanonical NF-κB signaling required for lymphoid organ development. We showed previously that lymph node formation is ablated in IkkαLyve-1 mice constitutively lacking IKKα in lymphatic endothelial cells (LECs). We now reveal that loss of IKKα in LECs leads to the formation of BALT in the lung. Tertiary lymphoid structures appear only in the lungs of IkkαLyve-1 mice and are not present in any other tissues, and these highly organized BALT structures form after birth and in the absence of inflammation. Additionally, we show that IkkαLyve-1 mice challenged with influenza A virus (IAV) exhibit markedly improved survival and reduced weight loss compared with littermate controls. Importantly, we determine that the improved morbidity and mortality of IkkαLyve-1 mice is independent of viral load and rate of clearance because both mice control and clear IAV infection similarly. Instead, we show that IFN-γ levels are decreased, and infiltration of CD8 T cells and monocytes into IkkαLyve-1 lungs is reduced. We conclude that ablating IKKα in LECs promotes BALT formation and reduces the susceptibility of IkkαLyve-1 mice to IAV infection through a decrease in proinflammatory stimuli.

IκBα kinase inhibitor BAY 11-7082 promotes anti-tumor effect in RAS-driven cancers.

BACKGROUND: Oncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge. METHODS: In this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082. RESULTS: We demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition. CONCLUSION: Our study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers.

RNF31-mediated IKKα ubiquitination aggravates inflammation and intestinal injury through regulating NF-κB activation in human and mouse neonates.

AIMS: Neonatal necrotizing enterocolitis (NEC) is a leading cause of intestine inflammatory disease, and macrophage is significantly activated during NEC development. Posttranslational modifications (PTMs) of proteins, particularly ubiquitination, play critical roles in immune response. This study aimed to investigate the effects of ubiquitin-modified proteins on macrophage activation and NEC, and discover novel NEC-related inflammatory proteins. MATERIALS AND METHODS: Proteomic and ubiquitin proteomic analyses of intestinal macrophages in NEC/healthy mouse pups were carried out. In vitro macrophage inflammation model and in vivo NEC mouse model, as well as clinical human samples were used for further verification the inhibitor of nuclear factor-κB kinase α (IKKα) ubiquitination on NEC development through Western blot, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry. KEY FINDINGS: We report here that IKKα was a new ubiquitin-modified protein during NEC through ubiquitin proteomics, and RING finger protein 31 (RNF31) acted as an E3 ligase to be involved in IKKα degradation. Inhibition of IKKα ubiquitination and degradation with siRNF31 or proteasome inhibitor decreased nuclear factor-κB (NF-κB) activation, thereby decreasing the expression of pro-inflammatory factors and M1 macrophage polarization, resulting in reliving the severity of NEC. SIGNIFICANCE: Our study suggests the activation of RNF31-IKKα-NF-κB axis triggering NEC development and suppressing RNF31-mediated IKKα degradation may be therapeutic strategies to be developed for NEC treatment.