M2-type macrophage-targeted delivery of IKKß siRNA induces M2-to-M1 repolarization for CNV gene therapy.

Choroidal Neovascularization (CNV) is capable of inciting recurrent hemorrhage in the macular region, severely impairing patients' visual acuity. During the onset of CNV, infiltrating M2 macrophages play a crucial role in promoting angiogenesis. To control this disease, our study utilizes the RNA interference (RNAi)-based gene therapy to reprogram M2 macrophages to the M1 phenotype in CNV lesions. We synthesize the mannose-modified siRNA-loaded liposome specifically targeting M2 macrophages to inhibit the inhibitory kappa B kinase ß (IKKß) gene involved in the polarization of macrophages, consequently modulating macrophage polarization state. In vitro and in vivo, the mannose-modified IKKß siRNA-loaded liposome (siIKKß-ML) has been proven to effectively target M2 macrophages to repolarize them to M1 phenotype, and inhibit the progression of CNV. Collectively, our findings elucidate that siIKKß-ML holds the potential to control CNV by reprogramming the macrophage phenotype, indicating a promising therapeutic avenue for CNV management.

IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model.

Loss-of-function mutations have provided crucial insights into the immunoregulatory actions of Foxp3+ regulatory T cells (Tregs). By contrast, we know very little about the consequences of defects that amplify aspects of Treg function or differentiation. Here we show that mice heterozygous for an Ikbkb gain-of-function mutation develop psoriasis. Doubling the gene dose (IkbkbGoF/GoF) results in dactylitis, spondylitis, and characteristic nail changes, which are features of psoriatic arthritis. IkbkbGoF mice exhibit a selective expansion of Foxp3 + CD25+ Tregs of which a subset express IL-17. These modified Tregs are enriched in both inflamed tissues, blood and spleen, and their transfer is sufficient to induce disease without conventional T cells. Single-cell transcriptional and phenotyping analyses of isolated Tregs reveal expansion of non-lymphoid tissue (tissue-resident) Tregs expressing Th17-related genes, Helios, tissue-resident markers including CD103 and CD69, and a prominent NF-κB transcriptome. Thus, IKK2 regulates tissue-resident Treg differentiation, and overactivity drives dose-dependent skin and systemic inflammation.

The African swine fever virus MGF300-4L protein is associated with viral pathogenicity by promoting the autophagic degradation of IKKß and increasing the stability of IκBα.

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1ß and TNF-α, which are regulated by the NF-κB signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKKß and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and IκBα competitively inhibits the binding of the E3 ligase ß-TrCP to IκBα, thereby inhibiting the ubiquitination-dependent degradation of IκBα. Remarkably, although ASFV encodes other inhibitors of NF-κB, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1ß and TNF-α early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.

Dual regulation of NEMO by Nrf2 and miR-125a inhibits ferroptosis and protects liver from endoplasmic reticulum stress-induced injury.

Rationale: The surge of severe liver damage underscores the necessity for identifying new targets and therapeutic agents. Endoplasmic reticulum (ER) stress induces ferroptosis with Gα12 overexpression. NF-κB essential modulator (NEMO) is a regulator of inflammation and necroptosis. Nonetheless, the regulatory basis of NEMO de novo synthesis and its impact on hepatocyte ferroptosis need to be established. This study investigated whether Nrf2 transcriptionally induces IKBKG (the NEMO gene) for ferroptosis inhibition and, if so, how NEMO induction protects hepatocytes against ER stress-induced ferroptosis. Methods: Experiments were conducted using human liver tissues, hepatocytes, and injury models, incorporating NEMO overexpression and Gα12 gene modulations. RNA sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation analyses were done. Results: NEMO downregulation connects closely to ER and oxidative stress, worsening liver damage via hepatocyte ferroptosis. NEMO overexpression protects hepatocytes from ferroptosis by promoting glutathione peroxidase 4 (GPX4) expression. This protective role extends to oxidative and ER stress. Similar shifts occur in nuclear factor erythroid-2-related factor-2 (Nrf2) expression alongside NEMO changes. Nrf2 is newly identified as an IKBKG (NEMO gene) transactivator. Gα12 changes, apart from Nrf2, impact NEMO expression, pointing to post-transcriptional control. Gα12 reduction lowers miR-125a, an inhibitor of NEMO, while overexpression has the opposite effect. NEMO also counters ER stress, which triggers Gα12 overexpression. Gα12's significance in NEMO-dependent hepatocyte survival is confirmed via ROCK1 inhibition, a Gα12 downstream kinase, and miR-125a. The verified alterations or associations within the targeted entities are validated in human liver specimens and datasets originating from livers subjected to exposure to other injurious agents. Conclusions: Hepatic injury prompted by ER stress leads to the suppression of NEMO, thereby facilitating ferroptosis through the inhibition of GPX4. IKBKG is transactivated by Nrf2 against Gα12 overexpression responsible for the increase of miR-125a, an unprecedented NEMO inhibitor, resulting in GPX4 induction. Accordingly, the induction of NEMO mitigates ferroptotic liver injury.

IκB kinase phosphorylates cytoplasmic TDP-43 and promotes its proteasome degradation.

Cytoplasmic aggregation of TDP-43 in neurons is a pathological feature common to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We demonstrate that the IκB kinase (IKK) complex promotes the degradation of cytoplasmic TDP-43 through proteasomes. While IKKß is a major factor in TDP-43 degradation, IKKα acts as a cofactor, and NEMO functions as a scaffold for the recruitment of TDP-43 to the IKK complex. Furthermore, we identified IKKß-induced phosphorylation sites of TDP-43 and found that phosphorylation at Thr8 and Ser92 is important for the reduction of TDP-43 by IKK. TDP-43 phosphorylation at Ser92 was detected in a pattern different from that of C-terminal phosphorylation in the pathological inclusion of ALS. IKKß was also found to significantly reduce the expression level and toxicity of the disease-causing TDP-43 mutation. Finally, the favorable effect of IKKß on TDP-43 aggregation was confirmed in the hippocampus of mice. IKK and the N-terminal phosphorylation of TDP-43 are potential therapeutic targets for ALS and FTLD.

MEK-mediated CHPF2 phosphorylation promotes colorectal cancer cell proliferation and metastasis by activating NF-κB signaling.

The cytokine tumor necrosis factor (TNF) plays a crucial role in the proliferation and metastasis of colorectal cancer (CRC) cells, but the underlying mechanisms remain poorly understood. Here, we report that chondroitin polymerizing factor 2 (CHPF2) promotes CRC cell proliferation and metastasis mediated by TNF, independently of its enzymatic activity. CHPF2 is highly expressed in CRC, and its elevated expression is associated with poor prognosis of CRC patients. Mechanistically, upon TNF stimulation, CHPF2 is phosphorylated at the T588 residue by MEK, enabling CHPF2 to interact with both TAK1 and IKKα. This interaction enhances the binding of TAK1 and IKKα, leading to increased phosphorylation of the IKK complex and activation of NF-κB signaling. As a result, the expression of early growth factors (EGR1) is upregulated to promote CRC cell proliferation and metastasis. In contrast, introduction of a phospho-deficient T588A mutation in CHPF2 weakened the interaction between CHPF2 and TAK1, thus impairing NF-κB signaling. CHPF2 T588A mutation reduced the ability of CHPF2 to promote the proliferation and metastasis of CRC in vitro and in vivo. Furthermore, the NF-κB RELA subunit promotes CHPF2 expression, further amplifying TNF-induced NF-κB signaling activation. These findings identify a moonlighting function of CHPF2 in promoting tumor cell proliferation and metastasis and provide insights into the mechanism by which CHPF2 amplifies TNF-mediated NF-κB signaling activation. Our study provides a molecular basic for the development of therapeutic strategies for CRC treatment.

IKKß deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. METHODS: We used constitutive MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. RESULTS: Global depletion of IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Molecular cloning and functional characterization of pigeon IKKε.

Inhibitor of nuclear factor kappa-B kinase ε (IKKε), a member of the non-canonical IκB kinase family, plays a critical role in connecting various signaling pathways associated with the initiation of type I interferon (IFN) production. Although the importance of IKKε in innate immunity has been well established in mammals and fish, its characterization and function in pigeons have remained largely unexplored. In this study, we successfully cloned pigeon IKKε (piIKKε) from pigeon embryo fibroblasts (PEFs) for the first time. This gene encodes 722 amino acids and shares high amino acid similarity with its duck and goose counterparts. piIKKε showed a diffuse cytoplasmic distribution and broad expression in all tissues examined. Overexpression of piIKKε in PEFs significantly activated the IFN-ß promoter, with both the kinase and CC domains of piIKKε playing key roles in initiating IFN-ß expression. Knockdown of piIKKε using small interfering RNA significantly reduced the levels of IFN-ß induced by NDV, AIV, poly (I:C), or SeV. Furthermore, the presence of piIKKε resulted in a remarkable reduction in the replication of both avian influenza virus (AIV) H9N2 and Newcastle disease virus (NDV) in PEFs. Our results demonstrate that piIKKε plays a critical role in mediating antiviral innate immunity in pigeons.

A molluscan IRF interacts with IKKα/ß family protein and modulates NF-κB and MAPK activity.

Interferon regulatory factor (IRF) family proteins are key transcription factors involved in vital physiological processes such as immune defense. However, the function of IRF in invertebrates, especially in marine shellfish is not clear. In this study, a new IRF gene (CfIRF2) was identified in the Zhikong scallop, Chlamys farreri, and its immune function was analyzed. CfIRF2 has an open reading frame of 1107 bp encoding 368 amino acids. The N-terminus of CfIRF2 consists of a typical IRF domain, with conserved amino acid sequences. Phylogenetic analysis suggested close evolutionary relationship with shellfish IRF1 subfamily proteins. Expression pattern analysis showed that CfIRF2 mRNA was expressed in all tissues, with the highest expression in the hepatopancreas and gills. CfIRF2 gene expression was substantially enhanced by a pathogenic virus (such as acute viral necrosis virus) and poly(I:C) challenge. Co-immunoprecipitation assay identified CfIRF2 interaction with the IKKα/ß family protein CfIKK1 of C. farreri, demonstrating a unique signal transduction mechanism in marine mollusks. Moreover, CfIRF2 interacted with itself to form homologous dimers. Overexpression of CfIRF2 in HEK293T cells activated reporter genes containing interferon stimulated response elements and NF-κB genes in a dose-dependent manner and promoted the phosphorylation of protein kinases (JNK, Erk1/2, and P38). Our results provide insights into the functions of IRF in mollusks innate immunity and also provide valuable information for enriching comparative immunological theory for the prevention of diseases in scallop farming.

Terbuthylazine exposure induces innate immune response and inflammation through activating cGAS-STING/NF-κB pathway in myocardium of broiler chicken (Gallus gallus).

Terbuthylazine (TBA), a triazine herbicide, is extensively employed in agriculture for its wide range of effectiveness. However, prolonged utilization of TBA can pose a potential hazard to animals and human health. Here, a total of 180 broiler chickens (Gallus gallus) were stochastically assigned to three groups (control group, 0.4 mg/kg TBA group, and 4 mg/kg TBA group) for investigating the impact of TBA on cardiotoxicity. The results revealed that TBA exposure resulted in pathological alterations in the myocardium. Moreover, TBA exposure activated cGAS-STING pathway and markedly elevated the mRNA and protein expression levels of innate immune response (cGAS, STING, TBK1, and IRF3) in myocardium. Additionally, NF-κB signal was also activated under TBA exposure, which was characterized by the increasing mRNA expression levels of NF-κB, IKKα and the protein expression levels of p-NF-κB/NF-κB, IKKα, p-IκBα/IκBα in the TBA treatment groups. Meanwhile, the expression of pro-inflammatory cytokines (TNF-α and IL-1ß) were also significantly increased. In summary, our findings suggested that cGAS-STING/NF-κB pathway functionated in the innate immune response and inflammation in myocardium brought on by TBA exposure, which provided new insights into the TBA toxicology.

NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.

NEMO is a ubiquitin-binding protein which regulates canonical NF-κB pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identify an NF-κB-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO-encoding IKBKG gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including α-synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at α-synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.

Novel association between single-nucleotide polymorphisms of IKKß at rs17875746 and rs12676482 and periodontitis.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in the IKKß gene have been associated with susceptibility to various inflammatory illnesses, including periodontitis. OBJECTIVES: The aim of the present study was to investigate the association between IKKß SNPs (at rs17875746 and rs12676482) and periodontitis in an Iraqi Arab population. MATERIAL AND METHODS: In this case-control study, 94 Iraqi volunteers were split into 2 groups, with the case group including 62 periodontitis patients (37 men and 25 women) and the control group including 32 racially matched healthy people (19 men and 13 women). Periodontal parameters were recorded for each individual. Then, 2 mL of venous blood was taken from each participant to isolate their genomic DNA. In particular, the genotyping of rs17875746 and rs12676482 in IKKß was performed with the use of the polymerase chain reaction (PCR) sequencing methods. RESULTS: The effect of the distribution of IKKß SNPs on periodontitis was assessed by counting the odds ratio (OR), which was 5.264 for rs17875746 and 0.900 for rs12676482. Surprisingly, allele T revealed a significantly higher association with periodontitis for rs17875746 (OR = 6.750) than allele G (p = 0.038). Overall, the GT genotype in rs17875746 had a higher chance of developing the disease (OR = 3.321) as compared to other genotypes. Meanwhile, the GA genotype in rs12676482 had a higher chance of developing the disease (OR = 1.242) as compared to other genotypes. In addition, rs17875746 showed a significant positive association with tooth mobility, a family history, clinical attachment loss (CAL), and gingival recession (GR) in the study groups. CONCLUSIONS: The IKKß polymorphisms may increase genetic susceptibility to periodontitis in Iraqi Arab patients.

An IKBKE variant conferring functional cGAS/STING pathway deficiency and susceptibility to recurrent HSV-2 meningitis.

The mechanisms underlying susceptibility to recurrent herpes simplex virus type 2 (HSV-2) meningitis remain incompletely understood. In a patient experiencing multiple episodes of HSV-2 meningitis, we identified a monoallelic variant in the IKBKE gene, which encodes the IKKε kinase involved in induction of antiviral IFN genes. Patient cells displayed impaired induction of IFN-ß1 (IFNB1) expression upon infection with HSV-2 or stimulation with double-stranded DNA (dsDNA) and failed to induce phosphorylation of STING, an activation marker of the DNA-sensing cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway. The patient allele encoded a truncated IKKε protein with loss of kinase activity and also capable of exerting dominant-negative activity. In stem cell-derived microglia, HSV-2-induced expression of IFNB1 was dependent on cGAS, TANK binding kinase 1 (TBK1), and IKBKE, but not TLR3, and supernatants from HSV-2-treated microglia exerted IKBKE-dependent type I IFN-mediated antiviral activity upon neurons. Reintroducing wild-type IKBKE into patient cells rescued IFNB1 induction following treatment with HSV-2 or dsDNA and restored antiviral activity. Collectively, we identify IKKε to be important for protection against HSV-2 meningitis and suggest a nonredundant role for the cGAS/STING pathway in human antiviral immunity.

An Atypical Incontinentia Pigmenti Female with Persistent Mucocutaneous Hyperinflammation and Immunodeficiency Caused by a Novel Germline IKBKG Missense Mutation.

While most missense mutations of the IKBKG gene typically result in Ectodermal Dysplasia with Immunodeficiency, there have been rare reported instances of missense mutations of the IKBKG gene causing both Incontinentia Pigmenti (IP) and immunodeficiency in female patients. In this study, we described an atypical IP case in a 19-year-old girl, characterized by hyperpigmented and verrucous skin areas over the entire body. Remarkably, she experienced recurrent red papules whenever she had a feverish upper respiratory tract infection. Immunohistochemical staining unveiled a substantial accumulation of CD68+ macrophages alongside the TNF-α positive cells in the dermis tissue of new pustules, with increased apoptotic basal keratinocytes in the epidermis tissue of these lesions. Starting from the age of 8 years old, the patient suffered from severe and sustained chronic respiratory mucous membrane scar hyperplasia and occluded subglottic lumen. In addition to elevated erythrocyte sedimentation rate values, inflammatory cells were observed in the pathologic lesions of endobronchial biopsies and Bronchoalveolar Lavage Fluid (BALF) smear. Further histological analysis revealed a destructive bronchus epithelium integrity with extensive necrosis. Simultaneously, the patient experienced recurrent incomplete intestinal obstructions and lips contracture. The patient's BALF sample displayed an augmented profile of proinflammatory cytokines and chemokines, suggesting a potential link to systemic hyperinflammation, possibly underlying the pathogenic injuries affecting the subglottic, respiratory, and digestive systems. Furthermore, the patient presented with recurrent pneumonias and multiple warts accompanied by a T+BlowNKlow immunophenotype. Next generation sequencing showed that the patient carried a novel de novo germline heterozygous missense mutation in the IKBKG gene (c. 821T>C, p. L274P), located in the highly conserved CC2 domain. TA-cloning sequencing of patient's cDNA yielded 30 mutant transcripts out of 44 clones. In silico analysis indicated that the hydrogen bond present between Ala270 and Leu274 in the wild-type NEMO was disrupted by the Leu274Pro mutation. However, this mutation did not affect NEMO expression in peripheral blood mononuclear cells (PBMCs). Moreover, patient PBMCs exhibited significantly impaired TNF-α production following Lipopolysaccharide (LPS) stimulation. X-chromosome inactivation in T cells and neutrophils were not severely skewed. Reduced levels of IκBα phosphorylation and degradation in patient's PBMCs were observed. The NF-κB luciferase reporter assay conducted using IKBKG-deficient HEK293T cells revealed a significant reduction in NF-kB activity upon LPS stimulation. These findings adds to the ever-growing knowledge on female IP that might contribute to the better understanding of this challenging disorder.

SERPINB1 promotes Senecavirus A replication by degrading IKBKE and regulating the IFN pathway via autophagy.

IMPORTANCE: Senecavirus A (SVA) is an emerging picornavirus associated with vesicular disease, which wide spreads around the world. It has evolved multiple strategies to evade host immune surveillance. The mechanism and pathogenesis of the virus infection remain unclear. In this study, we show that SERPINB1, a member of the SERPINB family, promotes SVA replication, and regulates both innate immunity and the autophagy pathway. SERPINB1 catalyzes K48-linked polyubiquitination of IκB kinase epsilon (IKBKE) and degrades IKBKE through the proteasome pathway. Inhibition of IKBKE expression by SERPINB1 induces autophagy to decrease type I interferon signaling, and ultimately promotes SVA proliferation. These results provide importantly the theoretical basis of SVA replication and pathogenesis. SERPINB1 could be a potential therapeutic target for the control of viral infection.

TRIM25 negatively regulates IKKε-mediated interferon signaling in black carp.

IKKε plays an important role in the activation of IRF3/IRF7 and the production of interferon (IFN), however, its regulation remains obscure in human. E3 ligase TRIM25 has been reported to manipulate the K63-linked ubiquitination of RIG-I, leading to the activation of RIG-I/IFN signaling. To elucidate the role of TRIM25 in teleost, a TRIM25 homolog (bcTRIM25) was cloned and characterized from black carp (Mylopharyngodon piceus). bcTRIM25 contains 653 amino acids, possessing conservative RING, B-box and SPRY domain, which is highly expressed in muscle, spleen and skin. bcTRIM25 knock-down enhanced the antiviral ability of host cells. bcTRIM25 over-expression alone in EPC cells attenuated bcIFNa promoter transcription in the reporter assays and impeded PKR and MX1 expression in qRT-PCR. Interestingly, co-IP assays indicated that bcTRIM25 interacted with bcIKKε and the induced bcIFNa promoter transcription by bcIKKε was notably hindered by bcTRIM25. Furthermore, bcIKKε-induced expression of interferon stimulated genes (ISGs) and antiviral activity were dampened by bcTRIM25. Further exploration showed that bcTRIM25 visibly enhanced the ubiquitination of bcIKKε but significantly attenuated the phosphorylation of bcIKKε. Thus, our data demonstrate for the first time in vertebrate that TRIM25 negatively regulates IKKε through enhancing its ubiquitination, which sheds a light on the regulation of IKKε/IFN signaling.

Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling.

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades, although how these pathways intersect remains unclear. We discovered that nuclear factor kappa B (NF-κB) essential regulator NF-κB effector molecule (NEMO) is recruited to damaged mitochondria in a Parkin-dependent manner in a time course similar to recruitment of the structurally related mitophagy adaptor, optineurin (OPTN). Upon recruitment, NEMO partitions into phase-separated condensates distinct from OPTN but colocalizing with p62/SQSTM1. NEMO recruitment, in turn, recruits the active catalytic inhibitor of kappa B kinase (IKK) component phospho-IKKß, initiating NF-κB signaling and the upregulation of inflammatory cytokines. Consistent with a potential neuroinflammatory role, NEMO is recruited to mitochondria in primary astrocytes upon oxidative stress. These findings suggest that damaged, ubiquitinated mitochondria serve as an intracellular platform to initiate innate immune signaling, promoting the formation of activated IKK complexes sufficient to activate NF-κB signaling. We propose that mitophagy and NF-κB signaling are initiated as parallel pathways in response to mitochondrial stress.

IκB kinase ß (IKKß): Structure, transduction mechanism, biological function, and discovery of its inhibitors.

The effective approach to discover innovative drugs will ask natural products for answers because of their complex and changeable structures and multiple biological activities. Inhibitory kappa B kinase beta (IKKß), known as IKK2, is a key regulatory kinase responsible for the activation of NF-κB through its phosphorylation at Ser177 and Ser181 to promote the phosphorylation of inhibitors of kappa B (IκBs), triggering their ubiquitination and degradation to active the nuclear factor kappa-B (NF-κB) cascade. Chemical inhibition of IKKß or its genetic knockout has become an effective method to block NF-κB-mediated proliferation and migration of tumor cells and inflammatory response. In this review, we summarized the structural feature and transduction mechanism of IKKß and the discovery of inhibitors from natural resources (e.g. sesquiterpenoids, diterpenoids, triterpenoids, flavonoids, and alkaloids) and chemical synthesis (e.g. pyrimidines, pyridines, pyrazines, quinoxalines, thiophenes, and thiazolidines). In addition, the biosynthetic pathway of novel natural IKKß inhibitors and their biological potentials were discussed. This review will provide inspiration for the structural modification of IKKß inhibitors based on the skeleton of natural products or chemical synthesis and further phytochemistry investigations.

Classical swine fever virus NS5A protein antagonizes innate immune response by inhibiting the NF-κB signaling.

The NS5A non-structural protein of classical swine fever virus (CSFV) is a multifunctional protein involved in viral genomic replication, protein translation, assembly of infectious virus particles, and regulation of cellular signaling pathways. Previous report showed that NS5A inhibited nuclear factor kappa B (NF-κB) signaling induced by poly(I:C); however, the mechanism involved has not been elucidated. Here, we reported that NS5A directly interacted with NF-κB essential modulator (NEMO), a regulatory subunit of the IκB kinase (IKK) complex, to inhibit the NF-κB signaling pathway. Further investigations showed that the zinc finger domain of NEMO and the aa 126-250 segment of NS5A are essential for the interaction between NEMO and NS5A. Mechanistic analysis revealed that NS5A mediated the proteasomal degradation of NEMO. Ubiquitination assay showed that NS5A induced the K27-linked but not the K48-linked polyubiquitination of NEMO for proteasomal degradation. In addition, NS5A blocked the K63-linked polyubiquitination of NEMO, thus inhibiting IKK phosphorylation, IκBα degradation, and NF-κB activation. These findings revealed a novel mechanism by which CSFV inhibits host innate immunity, which might guide the drug design against CSFV in the future.