Search

Doxorubicin (Dox) use is limited by Dox-induced cardiotoxicity. TANK-blinding kinase 1 (TBK1) is an important kinase involved in the regulation of mitophagy, but the role of TBK1 in cardiomyocytes in chronic Dox-induced cardiomyopathy remains unclear. Cardiomyocyte-specific Tbk1 knockout (Tbk1 CKO) mice received Dox (6 mg/kg, injected intraperitoneally) once a week for 4 times, and cardiac assessment was performed 4 weeks after the final Dox injection. Adenoviruses encoding Tbk1 or containing shRNA targeting Tbk1 , or a TBK1 phosphorylation inhibitor were used for overexpression or knockdown of Tbk1 , or inhibit phosphorylation of TBK1 in isolated primary cardiomyocytes. Our results revealed that moderate Dox challenge decreased TBK1 phosphorylation (with no effect on TBK1 protein levels), resulting in compromised myocardial function, obvious mortality and overt interstitial fibrosis, and the effects were accentuated by Tbk1 deletion. Dox provoked mitochondrial membrane potential collapse and oxidative stress, the effects of which were exacerbated and mitigated by Tbk1 knockdown, specific inhibition of phosphorylation and overexpression, respectively. However, Tbk1 （Ser172A） overexpression did not alleviate these effects. Further scrutiny revealed that TBK1 exerted protective effects on mitochondria via SQSTM1/P62-mediated mitophagy. Tbk1 overexpression mediated cardioprotective effects on Dox-induced cardiotoxicity were cancelled off by Sqstm1/P62 knockdown. Moreover, TBK1-mitophagy-mitochondria cascade was confirmed in heart tissues from dilated cardiomyopathy patients. Taken together, our findings denoted a pivotal role of TBK1 in Dox-induced mitochondrial injury and cardiotoxicity possibly through its phosphorylation and SQSTM1/P62-mediated mitophagy. [Display omitted] • Cardiomyocyte-specific Tbk1 deletion aggravated chronic Doxorubicin-induced cardiotoxicity. • Specific inhibition of TBK1（Ser172） phosphorylation exacerbated the cardiomyocyte toxicity of Doxorubicin. • Overexpression of Tbk1 could mitigate the cardiotoxicity of Doxorubicin, while overexpression of Tbk1 (Ser172A) could not. • The TBK1-P62-mitophagy cascade may play a crucial role in the pathogenesis of Doxorubicin-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

Tank-binding kinase 1 (TBK1) is a critical signal transducer in the nuclear factor κB (NF-κB) and interferon regulatory factor (IRF) pathways, essential for innate immunity. However, its negative regulation mechanisms remain unclear. This study demonstrates that TBK1 succinylation, regulated by desuccinylase SIRT5, inhibits lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4)-mediated NF-κB and IRF signaling activation. We identified three key succinylation sites on TBK1: K38, K154, and K692. In endotoxemia and sepsis models, reduced SIRT5 levels in macrophages increased TBK1 succinylation, inhibiting its binding to IRF3 and TRAF2 and suppressing the inflammatory response. In vivo , adoptive transfer of macrophages expressing the succinylation-resistant TBK1-2KR (K154/692R) mutant reversed the inflammatory cytokine suppression caused by SIRT5 deficiency, exacerbating sepsis-induced lung injury. These findings reveal a novel mechanism by which SIRT5 modulates TBK1 activity and macrophage-mediated inflammation during sepsis. [Display omitted] • TBK1 is succinylated at K38, K154, and K692 in macrophages • Succinylation inhibits TBK1 binding to IRF3 and TRAF2 • SIRT5 directly desuccinylates TBK1 • Succinylation is a key negative regulator of TBK1 in inflammatory responses Zhang et al. reveal that TBK1 is succinylated at K38, K154, and K692, inhibiting the activation of NF-κB and IRF signaling pathways in macrophages during sepsis. This process is regulated by the desuccinylase SIRT5, highlighting a potential strategy for negatively regulating macrophage-related inflammatory responses. [ABSTRACT FROM AUTHOR]

Macroautophagy (autophagy) involves the formation of phagophores that mature into autophagosomes. The impact of inhibiting autophagosome closure remains unclear. Here, we report the generation and analysis of mice with impaired autophagosome closure by targeting the ubiquitin E2 variant-like (UEVL) β strands of the endosomal sorting complex required for transport (ESCRT) I subunit VPS37A. The VPS37A UEVL mutation (Δ43–139) impairs bulk autophagic flux without disrupting ESCRT-I complex assembly and endosomal function. Homozygous mutant mice exhibit signs of autophagy impairment, including p62/SQSTM1 and ubiquitinated protein accumulation, neuronal dysfunction, growth retardation, antioxidant gene upregulation, and tissue abnormalities. However, about half of the mutant neonates survive to adulthood without severe liver injury. LC3 proximity proteomics reveals that the VPS37A UEVL mutation leads to active TANK-binding kinase 1 (TBK1) accumulation on phagophores, resulting in increased p62 phosphorylation and inclusion formation. These findings reveal a previously unappreciated role of LC3-conjugated phagophores in facilitating protein aggregation and sequestration, potentially alleviating proteotoxicity. [Display omitted] • Autophagosome closure-defective mice were generated by targeting VPS37A UEVL • Homozygous mutant mice showed signs of autophagy impairment • About half of the mutant neonates survived to adulthoodwithout hepatomegaly • LC3-conjugated phagophores facilitate TBK1-dependent p62 phosphorylation and aggregation Hamamoto et al. report that disrupting the VPS37A ubiquitin E2 variant-like domain impairs autophagosome closure, resulting in milder autophagy-defective phenotypes in mice compared to those lacking upstream autophagy-related genes. They found that LC3-conjugated phagophores facilitate TBK1-dependent p62 phosphorylation, potentially alleviating proteotoxicity by sequestering protein aggregates. [ABSTRACT FROM AUTHOR]

E26-transforming specific (ETS) variant 6 (ETV6) is a transcription factor regulating the expression of interferon stimulating genes (ISGs) and involved in the embryonic development and hematopoietic regulation, but the role of ETV6 in host response to virus infection is not clear. In this study, we show that ETV6 was upregulated in DF-1 cells with poly(I:C) stimulation or IBDV, AIV and ARV infection via engagement of dsRNA by MDA5. Overexpression of ETV6 in DF-1 cells markedly inhibited IBDV-induced type I interferon (IFN-I) and ISGs expressions. In contrast, knockdown, or knockout of ETV6 remarkably inhibited IBDV replication via promoting IFN-I response. Furthermore, our data show that ETV6 negatively regulated host antiviral response to IBDV infection by interaction with TANK binding kinase 1 (TBK1) and subsequently inhibited its phosphorylation. These results uncovered a novel role of ETV6 as a pro-viral factor in host response by inhibiting TBK1 phosphorylation, furthering our understandings of RNA virus immunosuppression and providing a valuable clue to the development of antiviral reagents for the control of avian RNA virus infection. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Non-canonical NF-κB pathways are activated in the liver, spinal cord, and intestine in animal models of ALS.2. • The administration of the NIK inhibitor B022 significantly prolonged lifespan and grip strength and improved neuroinflammation in an animal model of ALS. • SOD1G93A in conjunction with TBK1-UKO produces intestinal inflammation possibly due to activation of non-canonical NF-κB pathway. Amyotrophic lateral sclerosis (ALS) is a typical neurodegenerative disorder typically characterized by inflammation activation. However, the relationship between non-canonical NF-κB (ncNF-κB) pathway activation and ALS progression is not clear. We tested the ncNF-κB pathway in the ALS animal model including hSOD1-G93A transgenic mice and TBK1 deletion mice.We treated age-matched SOD1-G93A mice with B022 (a NIK inhibitor) to investigate the role of NIK in the ALS animal model. We also established a new mice model by crossing SOD1-G93A mice with NIK+/- mice to further evaluate the interrelationship between the NIK and the disease progression in ALS animal model. In this study, we found the ncNF-κB pathway was activated in SOD1-G93A animal model and TBK1 deletion model. Inhibition of NIK activity by small molecule B022 significantly improved the motor performance of the ALS animal model. However, NIK deletion enhanced the mutant SOD1 toxicity by inflammatory infiltration. TBK1 deletion and mutant SOD1 shared the common pathological feature possibly via effects on NIK activation and inhibitor of NIK could be a novel strategy for treating ALS. [ABSTRACT FROM AUTHOR]

Postoperative cognitive decline (POCD) is a common complication after surgery and anesthesia among the elderly. Yet the potential mechanism of POCD remains ambiguous, with limited therapeutic measures currently available. Ketamine has been reported to attenuate POCD after cardiac surgery. Herein, we tried to determine the effect of esketamine (the S-enantiomer of ketamine) on POCD and the possible molecular mechanisms. We investigated the effects of esketamine (10 mg/kg) on POCD using an exploratory laparotomy model in aged SD rats (24 months). Open field, novel object recognition, and morris water maze tests were performed on day 30 post-surgery. 24 h or 30 d post-surgery, brain tissue from the hippocampus and ventromedial prefrontal cortex (vmPFC) was harvested and subjected to histopathology and molecular biology analysis. During the in vitro experiment, primary astrocytes from the hippocampus and vmPFC were exposed to lipopolysaccharide (LPS) to investigate the pathological changes in astrocytes during the process of POCD. Our results indicated that exploratory laparotomy could induce significant cognitive and memory decline, accompanied by A2-type astrocytes phenotype loss and increased expression of neuron Aβ-42, astrocytes GABA, stimulator of interferon genes (STING) and TANK‐binding kinase 1 (TBK1). In addition, LPS exposure significantly decreased the mitochondrial membrane potential and upregulated the level of pyroptosis-associated proteins, including cleaved caspase-1 and IL-18. Notably, treatment with esketamine reversed these abnormalities in vivo and vitro. However, ADU-S100, a special STING activator, suppressed the protective effects of esketamine to a certain extent. Finally, C-176, an antagonist of STING, further enhanced the protective effects of esketamine against POCD. Findings of our study suggest that esketamine can alleviate surgery-induced POCD in rats via inhibition of the STING/TBK1 signaling pathway. • Esketamine alleviates postoperative cognitive decline in aged rats. • Esketamine can upregulate A2-type astrocytes in the hippocampus and vmPFC. • Esketamine can downregulate Aβ-42 in neurons and GABA in astrocytes. • STING/TBK1 may be closely related to esketamine-induced neurofunctional protection. • Esketamine can alleviate the pyroptosis of astrocytes after LPS exposure. [ABSTRACT FROM AUTHOR]

Members of the inhibitor of nuclear factor kappa-B kinase (IKK) family are pivotal components of the innate immune signaling pathway responsible for regulating immune and inflammatory responses. Nevertheless, the various roles that IKKs play in the physiology of invertebrates, particularly marine mollusks, remain largely unexplored. In this study, an IKK gene was cloned from the Zhikong scallop, Chlamys farreri , and denoted as CfIKK2. CfIKK2 belongs to the TANK-binding kinase 1 (TBK1)/IKKε family. Real-time quantitative reverse transcription-polymerase chain reaction analysis showed that CfIKK2 was expressed in various scallop tissues and that its transcript levels substantially increased under different pathogenic stress conditions (including lipopolysaccharide, peptidoglycan, poly(I:C), acute viral necrosis virus, and Vibrio challenge). Co-immunoprecipitation experiments revealed that CfIKK2 interacts with MyD88 (a crucial adaptor in the Toll-like receptor signaling pathway) proteins through its C-terminal TBK1 coiled-coil domain 1. Moreover, CfIKK2 has been shown to interact with itself. In HEK293T cells, CfIKK2 overexpression resulted in an augmentation of mitogen-activated protein kinase phosphorylation. Furthermore, overexpression of CfIKK2 resulted in the activation of interferon reporter genes. In summary, our findings offer valuable insights into the mechanisms through which CfIKK2 responds to stress generated by pathogen-associated molecular patterns, participates in Toll-like receptor signaling, and ultimately activates some of the key cytokines involved in innate immunity in C. farreri. As a critical signaling molecule and immunoregulatory factor, CfIKK2 plays a vital role in the innate immune system of the Zhikong scallop. • CfIKK2 was cloned and characterized in the Zhikong scallop Chlamys farreri. • CfIKK2 gene was expressed across various tissues and exhibited responsive behavior to multiple pathogen-induced stresses. • CfIKK2 interacts with the MyD88 proteins and facilitated the formation of homodimers. • Overexpression of CfIKK2 amplified the phosphorylation of MAPKs and activated interferon reporter genes. [ABSTRACT FROM AUTHOR]

Growing evidence highlights that tripartite motif (TRIM) family members exert crucial functions in host defense upon infection with microbial pathogens. Of note, the roles of a subfamily of fish-specific TRIM (FTR, also named as finTRIM) which have no true orthologues in mammals were largely unexplored. In this study, a novel FTR gene from grouper (EcFTR14) was identified, and its effects on fish virus infection and host interferon response were investigated. EcFTR14 contained RING, B-Box, and PRY-SPRY domains, and shared 54.81% identity to zebrafish FTR14. The transcription levels of EcFTR14 were markedly induced upon fish virus infection. EcFTR14 was mainly localized in the cytoplasm as small spots or bright aggregates. The overexpression of EcFTR14 could markedly enhance the replication of RGNNV or SGIV. Moreover, overexpression of EcFTR14 reduced the mRNA level of IFN-associated immune signaling molecules, suggesting that the antiviral action of EcFTR14 was due to the negative regulation on IFN response. EcFTR14 was found to interact with EcTBK1, EcIRF3 and EcIRF7, but not with EcSTING. Overexpression of EcFTR14 markedly suppressed the transcription levels of IFN-signaling related molecules evoked by EcSTING and EcTBK1. In addition, overexpression of EcFTR14 could degrade EcTBK1, EcIRF3 and EcIRF7 in a dose-dependent manner, that resulted in the suppression of antiviral effects induced by EcIRF3 and EcTBK1. Collectively, our findings illuminated that EcFTR14, a novel member of FTR family, acted as a pro-viral factor through negatively regulating targeting TBK1-IRF3 activated antviral action. The present study will for the first time shed light on the regulatory roles of FTR14 in innate immune response upon fish virus infection. • EcFTR14 was a novel FTR protein which shared 54.81% identity to zebrafish FTR14. • EcFTR14 were significantly up-regulated upon SGIV and RGNNV infection. • EcFTR14 acted as a pro-viral factor during SGIV and RGNNV infection. • EcFTR14 interacted with EcTBK1, EcIRF3, EcIRF7, and negatively regulated IFN response. • EcFTR14 weakened the antiviral effects induced by EcIRF3 and EcTBK1. [ABSTRACT FROM AUTHOR]

TRAF-associated NF-κB activator (TANK)-binding kinase 1 (TBK1), a nonclassical IκB kinase (IKK), and its effect on inflammation have not been entirely clarified. Here, we identified that TBK1 participates in the catabolism of glutamine by mediating the phosphorylation of receptor-interacting protein kinase 3 (RIPK3) and promoting macrophage endotoxin tolerance (ET). We found that the TBK1 protein directly interacts with the RIPK3 protein and mediates the phosphorylation of RIPK3 in macrophages. Activated RIPK3 can directly bind to glutamate dehydrogenase 1 (GLUD1), which is known to be a critical enzyme for catalyzing glutamine decomposition, to improve its catalytic activity and increase the production of α-ketoglutarate (α-KG) in macrophages. α-KG generated from glutaminolysis can promote M2 activation and restrict M1 polarization, which plays a crucial role in promoting lipopolysaccharide (LPS)-induced ET. As a result of TBK1 regulating the phosphorylation level of RIPK3, overexpressed TBK1 could enhance the tolerance of macrophages to endotoxin through glutaminolysis. Overall, these findings reveal a novel mechanism for the metabolic control of inflammation and for the induction of ET by modulating glutamine metabolism. • TBK1 mediates inflammatory regulation in endotoxin tolerance through protein phosphorylation. • Activated RIPK3 promotes the enzyme activity of GLUD to strengthen the effect of α-KG resulting in inducing ET. • TBK1 can mediate the phosphorylation of RIPK3, thereby inducing GLUD1 to decompose glutamine to produce α-KG. [ABSTRACT FROM AUTHOR]

• The expression of RNF19a is increased in macrophages upon virus infection. • RNF19a selectively inhibits type I interferon production upon RNA virus infection. • RNF19a inhibits the RIG-I signaling upon RNA virus infection. • RNF19a promotes the degradation of TBK1 through K48-linked ubiquitination. TANK-binding kinase 1 (TBK1) plays a pivotal role in antiviral innate immunity. TBK1 mediates the activation of interferon regulatory factor (IRF) 3, leading to the induction of type I IFNs (IFN-α/β) and of NF-κB signal transduction following viral infections. TBK1 must be tightly regulated to effectively control viral infections and maintain immune homeostasis. Here, we found that E3 ubiquitin ligase RNF19a mediated K48-linked ubiquitination and proteasomal degradation of TBK1. Specifically, the silence of RNF19a enhanced the production of type I interferons and suppressed RNA viral replication. Our results uncover that RNF19a acts as a negative mediator in the RIG-I signaling pathway to attenuate antiviral immune responses and suggest RNF19a as a potential therapy target in clinical infectious and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs. The authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro. Blocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFP expression of 40–50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality. The authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors' protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific peptidase 46 (USP46) functions as a deubiquitinating enzyme, facilitating the removal of ubiquitin molecules attached to substrate proteins and playing a critical role in cancer and neurodegenerative diseases. However, its function in innate antiviral immunity is unknown. In this study we cloned and identified bcUSP46, a homolog of USP46 from black carp. We discovered that overexpression of bcUSP46 enhanced the transcription of interferon (IFN) promoters and increased the expression of IFN , PKR , and Mx1. In addition, bcUSP46 knockdown significantly inhibited the expression of ISG genes, as well as the antiviral activity of the host cells. Interestingly, when bcUSP46 was co-expressed with the RLR factors, it significantly enhanced the activity of the IFN promoter mediated by these factors, especially TANK-binding kinase 1 (TBK1). The subsequent co-immunoprecipitation (co-IP) and immunofluorescence (IF) assay confirmed the association between bcUSP46 and bcTBK1. Noteworthily, co-expression of bcUSP46 with bcTBK1 led to an elevation of bcTBK1 protein level. Further analysis revealed that bcUSP46 stabilized bcTBK1 by eliminating the K48-linked ubiquitination of bcTBK1. Overall, our findings highlight the unique role of USP46 in modulating TBK1/IFN signaling and enrich our knowledge of the function of deubiquitination in regulating innate immunity in vertebrates. • Black carp USP46 positively regulates the black carp TBK1-mediated antiviral innate immune responses. • Black carp USP46 promotes the protein stabilization of black carp TBK1. • Black carp USP46 removes the K48-linked ubiquitination of black carp TBK1. [ABSTRACT FROM AUTHOR]

Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery. [Display omitted] • TRIM5α ubiquitylates TBK1 in response to mitochondrial damage • TRIM5α facilitates the activation of TBK1 on damaged mitochondria • TBK1 ubiquitylation enables its interactions with autophagy adaptors • Ubiquitination is a key positive regulator of TBK1 action in mitophagy Saha et al. demonstrate the coordination between two antiviral proteins, TRIM5α and TBK1, in maintaining mitochondrial quality control. TRIM5α mediates K63-linked ubiquitination of TBK1. These ubiquitin chains scaffold the formation of a multi-protein complex that favors TBK1 activation on damaged mitochondria and subsequent activation of the mitophagy machinery. [ABSTRACT FROM AUTHOR]

• TBK1 is a kinase at the crossroads of immunity and autophagy signaling. • Selective autophagy pathways mitophagy and xenophagy are regulated by TBK1. • Aberrant TBK1 signaling contributes to oncogenesis and neurodegenerative diseases. TANK-binding kinase 1 (TBK1) is a druggable multifunctional kinase that exerts a broad spectrum of functions in cells. These range from innate immunity, inflammation and interferon (IFN) signaling, through selective autophagy, specifically mitophagy and xenophagy, to energy homeostasis. Thus, it is not surprising that TBK1 is involved in many cellular signaling pathways that contribute to diverse pathologies. In this review the role of TBK1 in autophagic signaling, especially mitophagy and xenophagy, and its contribution to oncogenesis or neurodegenerative diseases will be discussed. [ABSTRACT FROM AUTHOR]

[Display omitted] While its innate immune function has been known, recent works of literature have focused on the role of Tank binding kinase 1 (TBK1) in regulating autophagy and it is unknown whether TBK1 protects against intervertebral disc degeneration (IVDD) through affecting autophagy. Here, we aim to explore whether TBK1 is implicated in the pathogenesis of IVDD, and investigated the potential mechanism. Western blotting and immunohistochemistry were used to detect the TBK1 expression in human and rat NP tissue. After TBK1 overexpression in NP cells with lentivirus transfection, autophagic flux, apoptosis and senescence percentage were assessed. Si-RNA , a utophagy inhibitors and protein phosphatase inhibitors were applied to study the mechanism of autophagy regulation. In vivo study, we further evaluated the therapeutic action of lentivirus-TBK1(Lv-TBK1)injection in a rodent IVDD model. The TBK1 level was reduced in rat and human NP tissue. TBK1 overexpression protected against apoptosis and premature senescence. These functions of TBK1 were abolished by chloroquine-medicated autophagy inhibition.P-TBK1, an activation form of TBK, is involved in selective autophagy through directly phosphorylating P62 at Ser 403, and the activation of TBK1 is also dependent on Parkin manner. TBK1 also activated NPCs autophagy to relieve puncture injury in vivo. We demonstrated that TBK1 overexpression attenuated senescence and apoptosis and promoted NPCs survival via upregulating autophagy. TBK1 represents a promising avenue for IVDD treatment. [ABSTRACT FROM AUTHOR]

The inhibitor of kappa B kinase (IKK) family consists of IKKα, IKKβ, and the IKK-related kinases TBK1 and IKKε. These kinases are considered master regulators of inflammation and innate immunity via their control of the transcription factors NF-κB, IRF3, and IRF7. Novel phosphorylated substrates have been attributed to these kinases, a subset of which is not directly related to either inflammation or innate immunity. These findings have greatly expanded the perspectives on the biological activities of these kinases. In this review we highlight some of the novel substrates for this kinase family and discuss the biological implications of these phosphorylation events. The IKK family is most well studied in the context of control of inflammatory gene expression, particularly in the regulation of NF-κB and IRF transcription factors. While the major targets of IKK in regulation of NF-κB and IRF signaling have been extensively studied, IKK family members are now known to phosphorylate proteins not related to NF-κB- or IRF-dependent transcription mechanisms. Recently, a variety of novel IKK family-dependent phosphorylation events have been described. This aspect of IKK function, in our opinion, is currently underappreciated and is critical for understanding the biology of these kinases and understanding effects of IKK family-directed inhibitors. The nuances of these regulatory mechanisms are especially important since aberrant regulation of IKK family kinases has been implicated in a wide range of human diseases, including cancer, obesity, diabetes, heart disease, and pathogen infection. [ABSTRACT FROM AUTHOR]

Multiple cancer immunotherapies including chimeric antigen receptor T cell and immune checkpoint inhibitors (ICIs) have been successfully developed to treat various cancers by motivating the adaptive anti-tumor immunity. Particularly, the checkpoint blockade approach has achieved great clinic success as evidenced by several U.S. Food and Drug Administration (FDA)-approved anti-programmed death receptor 1/ligand 1 or anti-cytotoxic T lymphocyte associated protein 4 antibodies. However, the majority of cancers have low clinical response rates to these ICIs due to poor tumor immunogenicity. Indeed, the cyclic guanosine monophosphate-adenosine monophosphate synthase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS‒STING‒TBK1) axis is now appreciated as the major signaling pathway in innate immune response across different species. Aberrant signaling of this pathway has been closely linked to multiple diseases, including auto-inflammation, virus infection and cancers. In this perspective, we provide an updated review on the latest progress on the development of small molecule modulators targeting the cGAS‒STING‒TBK1 signaling pathway and their preclinical and clinical use as a new immune stimulatory therapy. Meanwhile, highlights on the clinical candidates, limitations and challenges, as well as future directions in this field are also discussed. Further, small molecule inhibitors targeting this signaling axis and their potential therapeutic use for various indications are discussed as well. The cGAS‒STING‒TBK1 axis is appreciated as the major signaling pathway in innate immune response, which is closely linked to multiple diseases. This review summarizes the latest progress on the development of small molecule modulators targeting the cGAS‒STING‒TBK1 signaling pathway and their clinical use as a new immune stimulatory therapy. Image 1 [ABSTRACT FROM AUTHOR]

Singapore grouper iridovirus (SGIV), a highly pathogenic member of iridoviruses, has become a global threat in the aquaculture industry due to its economic losses and damage to ecological diversity worldwide. Recent reports have shown that SGIV inhibits host interferon (IFN) immune response induced by poly(I:C), however, the potential mechanisms for immune evasion remain largely uncertain. Here, VP149 protein encoded by SGIV ORF149R was identified to be a pro-viral factor, evidenced by the increase of SGIV gene expression and viral production in VP149-overexpressing cells. SGIV VP149 overexpression suppressed IFN-1 and ISRE promoter activation, and the transcription of IFN-stimulated genes triggered by Epinephelus coioides stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), melanoma differentiation-associated gene 5 (EcMDA5), or mitochondrial antiviral signaling protein (EcMAVS). Mutational analyses suggested that VP149 interacted with EcSTING and EcTBK1, and their interactions were not dependent on the domains of adaptor protein. In addition, VP149 impaired EcSTING-EcTBK1 or EcTBK1-EcIRF3 (interferon regulatory factor 3) complex formations, and degraded EcSTING or EcTBK1 through the lysosome pathway to promote red-spotted grouper nervous necrosis virus (RGNNV) infection. Collectively, a novel mechanism through which SGIV evaded fish immune via its protein VP149 by targeting STING and TBK1 to suppress type I IFN expression was revealed, and the findings will provide insights into pathogenesis and prevention of SGIV. • SGIV-encoded protein VP149 was identified to be a pro-viral factor. • VP149 interacted with EcSTING and EcTBK1 to suppress type I interferon expression triggered by EcSTING, and EcTBK1. • VP149 impaired EcSTING-EcTBK1 or EcTBK1-EcIRF3 association. • VP149 degraded EcSTING or EcTBK1 in a dose manner through the lysosome pathway. [ABSTRACT FROM AUTHOR]

Neuroinflammation plays a vital role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). The hypothesis of this study was that activation of melanocortin 1 receptor (MC1R) with BMS-470539 attenuates EBI by suppression of neuroinflammation after SAH. We utilized BMS-470539, MSG-606, and MRT-68601 to verify the neuroprotective effects of MC1R. We evaluated brain water content, short-term and long-term neurobehavior after SAH. Western blotting and immunofluorescence staining were utilized to assess the changes of protein levels. The results of western blotting suggested that the expressions of MC1R, phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK), and phosphorylated-TANK binding kinase 1 (p-TBK1) were increased and reached their peak points at 24 h following SAH. Moreover, BMS-470539 treatment notably attenuated neurological deficits caused by SAH, and also notably improved long-term spatial learning and memory abilities after SAH. The underlying mechanisms of the neuroprotection of BMS-470539 involved the suppression of microglia activation, promotion of CD206+ microglia transformation and reduction of neutrophil infiltration by increasing the levels of p-AMPK and p-TBK1 while decreasing the levels of NF-κB, IL-1β, and TNFα. The neuroprotective effects of BMS-470539 were significantly abolished by MSG-606 and MRT-68601. The activation of MC1R with BMS-470539 notably attenuates EBI after SAH by suppression of microglial activation and neutrophil infiltration via the AMPK/TBK1/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

There is currently no specific drug for the treatment of sepsis and antibiotic administration is considered the best option, despite numerous issues. Therefore, the development of drugs to control the pathogen-induced inflammatory responses associated with sepsis is essential. To address this, our study examined the transcriptomes of lipopolysaccharide (LPS)-induced dendritic cells (DCs), identifying TANK-binding kinase1 (Tbk1) as a key factor involved in the inflammatory response. These data suggested drug repositioning of the Tbk1 inhibitor CYT387, currently used for the treatment of myelofibrosis and some cancers, as a candidate for regulating the LPS-induced inflammatory response. CYT387 also inhibited pro-inflammatory cytokine and surface molecule expression by mature DCs after LPS exposure. These effects correlated with both Akt phosphorylation and IκBα degradation. Finally, CYT387 demonstrated therapeutic effects in LPS-induced endotoxemia and Escherichia coli K1-induced mouse models of sepsis and decreased the expression of pro-inflammatory cytokines. In conclusion, our study suggests that drug repositioning of CYT387 may serve as a potential therapeutic for sepsis. • Tbk1 is the most relevant kinase in the "dendritic cell maturation" pathway induced by LPS. • CYT387 inhibits pro-inflammatory gene expression by interfering with the Akt and NF-κB signaling cascades. • CYT387 prevents organ dysfunction in E. coli K1-induced sepsis and LPS-induced endotoxemia. [ABSTRACT FROM AUTHOR]

Highlights • Many ALS-causing genetic factors share a common link to autophagy. • Diverse mechanisms in ALS impair or overactivate autophagy. • Autophagic activation or repression has variable effects in ALS models. • The nodes of autophagy amenable to therapeutic intervention remain uncertain. Abstract Age-dependent neurodegenerative diseases are associated with a decline in protein quality control systems including autophagy. Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disease of complex etiology with increasing connections to other neurodegenerative conditions such as frontotemporal dementia. Among the diverse genetic causes for ALS, a striking feature is the common connection to autophagy and its associated pathways. There is a recurring theme of protein misfolding as in other neurodegenerative diseases, but importantly there is a distinct common thread among ALS genes that connects them to the cascade of autophagy. However, the roles of autophagy in ALS remain enigmatic and it is still unclear whether activation or inhibition of autophagy would be a reliable avenue to ameliorate the disease. The main evidence that links autophagy to different genetic forms of ALS is discussed. [ABSTRACT FROM AUTHOR]

Activation of type I interferon (IFN-1) signaling is essential to protect host cells from viral infection. The full spectrum of IFN-I induction requires the activation of a number of cellular factors, including I κ B kinase epsilon (IKKϵ). However, the regulation of IKKϵ activation in response to viral infection remains largely unknown. Here, we show that factor inhibiting hypoxia-inducible factor (HIF) (FIH), an asparaginyl hydroxylase, interacts with IKKϵ and catalyzes asparagine hydroxylation of IKKϵ at Asn-254, Asn-700, and Asn-701, resulting in the suppression of IKKϵ activation. FIH-mediated hydroxylation of IKKϵ prevents IKKϵ binding to TBK1 and TRAF3 and attenuates the cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex-catalyzed K63-linked polyubiquitination of IKKϵ at Lys-416. In addition, Fih -deficient mice and zebrafish are more resistant to viral infection. This work uncovers a previously unrecognized role of FIH in suppressing IKKϵ activation for IFN signaling and antiviral immune responses. [Display omitted] • FIH catalyzes the asparagine hydroxylation of IKKϵ at Asn-254, Asn-700, and Asn-701 • FIH suppresses IKKϵ activation • FIH-mediated hydroxylation of IKKϵ prevents IKKϵ binding to TBK1 and TRAF3 • FIH attenuates K63-linked polyubiquitination of IKKϵ at Lys-416 Cai et al. demonstrate that FIH catalyzes asparagine hydroxylation of IKKϵ at Asn-254, Asn-700, and Asn-701, resulting in the prevention of IKKϵ binding to TBK1 and TRAF3. As a result, FIH attenuates K63-linked polyubiquitination of IKKϵ at Lys-416, thereby suppressing IKKϵ activation and negatively regulating antiviral immunity. [ABSTRACT FROM AUTHOR]

During viral infections, nucleic acid sensing by intracellular receptors can trigger type I interferon (IFN-I) production, key mediators in antiviral innate immunity. However, many flaviviruses use non-structural proteins to evade immune sensing favoring their survival. These mechanisms remain poorly characterized. Here, we studied the role of Zika virus (ZIKV) NS4B protein in the inhibition of IFN-I induction pathway and its biophysical interaction with host proteins. Using different cell-based assays, we studied the effect of ZIKV NS4B in the activation of interferon regulatory factors (IRFs), NF-κB, cytokines secretion and the expression of interferon-stimulating genes (ISG). We also analyzed the in vitro interaction between recombinant ZIKV NS4B and TANK-binding kinase 1 (TBK1) using surface plasmon resonance (SPR). Transfection assays showed that ZIKV NS4B inhibits IRFs activation involved in different nucleic acid sensing cascades. Cells expressing NS4B secreted lower levels of IFN-β and IL-6. Furthermore, early induction of ISGs was also restricted by ZIKV NS4B. For the first time, we demonstrate by SPR assays that TBK1, a critical component in IFN-I production pathway, binds directly to ZIKV NS4B (K D of 3.7 × 10−6 M). In addition, we show that the N-terminal region of NS4B is directly involved in this interaction. Altogether, our results strongly support that ZIKV NS4B affects nucleic acid sensing cascades and disrupts the TBK1/IRF3 axis, leading to an impairment of IFN-β production. This study provides the first biophysical data of the interaction between ZIKV NS4B and TBK1, and highlights the role of ZIKV NS4B in evading the early innate immune response. [Display omitted] • ZIKV NS4B protein disrupts nucleic acid sensing cascades. • ZIKV NS4B reduces the production of IFN-β, IL-6 and ISGs upon poly(I:C) stimulation. • Surface plasmon resonance assays demonstrate that ZIKV NS4B interacts with TBK1 by direct binding. • N-terminal domain of ZIKV NS4B protein is involved in TBK1 binding. [ABSTRACT FROM AUTHOR]

Surveys indicated that stroke classified among the leading cause of death as well as combined death and disability worldwide resulting in a great loss for the global economy. The present study aims to evaluate the neuroprotective potential of the biflavonoid amentoflavone (AMNT) in alleviating cerebral ischemia/reperfusion (IR) injury in rats, and to elucidate the possible underlying mechanism of an experimental condition with similar circumstances to stroke. Cerebral ischemia was achieved through left common carotid artery occlusion for 60 min, followed by blood flow restoration. Sham-operated control rats subjected to the same surgical process except for brain IR. Rats were orally administered AMNT/ or vehicle for three days' prior surgical operation, and for another three days after left brain IR. Rats of all groups were assessed for neurological deficits 24 h following brain IR. Each group was divided into two subgroups one for the rotarod testing and biochemical assessment while the other subgroup to perform the activity cage testing, histopathological study, immunohistochemistry, and gene expression analysis. AMNT enhanced brain levels of GSH and CAT activities, suppressed neuroinflammation via reducing the inflammatory cytokines in the serum, and enhanced brain contents of TBK1 and IFNβ. AMNT downregulated TLR4-/NF-κB signaling pathway as a result of the HMGB1 suppression. Moreover, AMNT blocked apoptotic cell death by suppressing the NF-κB signaling pathway and reducing the activation of caspase-3. These findings revealed that AMNT attenuates I/R–induced cerebral injury possibly by regulating the HMGB1-mediated TLR4/NF-kB pathway. Thus, AMNT could provide potential preventive and therapeutic option for cerebral stroke. [ABSTRACT FROM AUTHOR]

Abstract RIG-I like receptor (RLR) signaling functions importantly in host innate immune response against RNA virus, which is tightly regulated by a number of mechanisms to prevent aberrant interferon production. The s uppressor of IK K ε (SIKE) has been identified as a suppressor of IKKε and TBK1, which are key components of RLR signaling. In this study, SIKE homologue (bcSIKE) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcSIKE varied in host cells in response to the stimulation of LPS, poly (I:C) and viruses. bcSIKE migrated around 27 KDa in immunoblot assay and distributed in both cytoplasm and nucleus of host cell in immunofluorescent (IF) staining test. bcSIKE showed no IFN-inducing ability in reporter assay and EPC cells expressing bcSIKE showed no enhanced antiviral ability against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV). However, bcSIKE obviously dampened the IFN-inducing ability of RLR signaling members in reporter assay when bcSIKE was co-expressed with these molecules in EPC cells. The association between bcSIKE and bcTBK1 has been identified through IF and co-immunoprecipitation (co-IP) assay. The plaque assay demonstrated clearly that bcTBK1-mediated antiviral activity in EPC cells against both GCRV and SVCV was down regulated by bcSIKE. All the data generated in this paper support the conclusion that bcSIKE interacts with bcTBK1 and inhibits bcTBK1-mediated antiviral signaling during host innate immune activation, which is reported in teleost for the first time. Highlights • bcSIKE obviously dampens the IFN-inducing ability of RLR signaling components. • bcSIKE associates with bcTBK1 directly. • bcTBK1-mediated IFN production and antiviral activity are down-regulated by bcSIKE. [ABSTRACT FROM AUTHOR]

Abstract The Toll-like receptor 4 (TLR4) plays a crucial role in innate inflammatory responses, as it recognizes gram-negative bacteria (or their products) and contributes greatly to host defense against invading pathogens. Though TLR4 overexpressing transgenic sheep, resistant to certain diseases related with gram-negative bacteria, had been bred in our previous research, the effects of overexpression of TLR4 on innate immune response remained unclear. In this study, TLR4 overexpressing ovine macrophages were obtained from peripheral blood, and it was found that the overexpression of TLR4 initially promoted the production of proinflammatory cytokines TNFα and IL-6 by activating TLR4-mediated IRAK4-dependent NF-κB and MAPK (JNK and ERK1/2) signaling following LPS stimulation. However, this effect was later impaired due to increased internalization of TLR4 into endosomal compartment of the macrophages. Then the overexpression of TLR4 triggered TBK1-dependent interferon-regulatory factor-3 (IRF-3) expression, which in turn led to the induction of IFN-β and IFN-inducible genes (i.e. IP10 , IRG1 and GARG16). Understandably, an increased IFN-β level facilitated phosphorylation of STAT1 to induce expression of innate antiviral genes Mx1 and ISG15 , suggesting that TLR4 overexpressing macrophages were equipped better against viral infection. Correspondingly, the bacterial burden in these macrophages, after infection with live S. Typhimurium , was decreased significantly. In summary, the results indicated that overexpression of TLR4 could enhance innate inflammatory responses, initiate the innate antiviral immunity, and control effectively S. Typhimurium growth in ovine macrophages. [ABSTRACT FROM AUTHOR]

Abstract Optineurin (Optn) is an autophagy receptor that performs various functions in cargo-selective and non-selective autophagy. Here, we have identified and characterized a splice variant of mouse optineurin mRNA, which produces a truncated protein lacking N-terminal 157 amino acids (d157mOptn). This mRNA and protein are expressed in several tissues and cells. d157mOptn has an intact LC3-interacting region and a serine (S187) in it. However, unlike normal optineurin, the d157mOptn was not phosphorylated at this site when expressed in mammalian cells, and showed reduced interaction with TBK1 (tank binding kinase) that mediates phosphorylation at S187 (S177 in human OPTN). This phosphorylation of Optn required intact N-terminal sequence as well as functional C-terminal ubiquitin-binding domain. Unlike normal optineurin, d157mOptn was unable to promote autophagosome and autolysosome formation upon expression in Optn-deficient cells. d157mOptn was recruited to mutant huntingtin aggregates, but unlike wild type optineurin, it was unable to clear these aggregates by autophagy in neuronal NSC-34 cells. Phospho-TBK1 was seen around mutant Huntingtin aggregates in Optn overexpressing cells but it was reduced in cells overexpressing d157mOptn. Thus, we have identified an isoform of mouse optineurin which is defective in cargo-selective and non-selective autophagy possibly due to loss of phosphorylation and impaired interaction with TBK1. This isoform, which inhibits autophagosome formation in neuronal cells, might be involved in selectively modulating some of the functions of Optn, such as autophagy. Our results provide an insight into the role of N-terminal domain of Optn in various autophagic functions. Highlights • A splice variant of Optn lacking 157 amino acids is expressed in mouse tissues. • This protein is defective in selective as well as non-selective autophagy. • This protein, d157mOptn is defective in phosphorylation by TBK1. • Results reveal role of N-terminal domain of Optn in various autophagic functions. • This isoform might be involved in selectively modulating some functions of Optn. [ABSTRACT FROM AUTHOR]

Abstract Objective TANK Binding Kinase 1 (TBK1) has been implicated in the regulation of metabolism through studies with the drug amlexanox, an inhibitor of the IκB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since amlexanox is a dual IKKε/TBK1 inhibitor, the specific metabolic contribution of TBK1 is not clear. Methods To distinguish metabolic functions unique to TBK1, we examined the metabolic profile of global Tbk1 mutant mice challenged with an obesogenic diet and investigated potential mechanisms for the improved metabolic phenotype. Results and conclusion We report that systemic loss of TBK1 kinase function has an overall protective effect on metabolic readouts in mice on an obesogenic diet, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor. Highlights • Obesogenic diet induces TBK1 activity and promotes insulin receptor interaction. • Functional TBK1 impedes insulin receptor signaling in response to metabolic challenge, resulting in insulin resistance. • Tbk1 mutant mice expend more energy than Tbk1 wild-type mice and are protected from diet-induced weight gain. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) play a crucial role in danger recognition and induction of innate immune response against bacterial and viral infections. The TLR adaptor molecule, toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF), facilitates TLR3 and TLR4 signaling, leading to the activation of the transcription factor, NF-κB and interferon regulatory factor 3 (IRF3). Andrographolide, the active component of Andrographis paniculata , exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the role of andrographolide in TLR signaling pathways. Andrographolide suppressed NF-κB activation as well as COX-2 expression induced by TLR3 or TLR4 agonists. Andrographolide also suppressed the activation of IRF3 and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. Andrographolide attenuated ligand-independent activation of IRF3 following overexpression of TRIF, TBK1, or IRF3. Furthermore, andrographolide inhibited TBK1 kinase activity in vitro . These results indicate that andrographolide modulates the TRIF-dependent pathway of TLRs by targeting TBK1 and represents a potential new anti-inflammatory candidate. [ABSTRACT FROM AUTHOR]

Nontypeable Haemophilus influenzae (NTHI) is one of the leading causes of acute exacerbations of COPD (AECOPD). Although the immunoregulation function of NTHI outer member protein and endotoxin were confirmed, the role of NTHI DNA in activating immune responses remains to be elucidated. In this study, we found expression of IFN-β and IFN stimulated gene CXCL10 in host cells was forcefully elevated after treating with NTHI and NTHI DNA. Interestingly, we tested increased level of STING in NTHI infected mice lung. Meanwhile, STING expression in lung of mimic COPD murine model was higher than healthy mice after NTHI infection. Importantly, knockout of STING or overexpression of STING, TBK1 and IRF3 respectively impaired or enhanced IFN-β and CXCL10 expression during treating with NTHI and NTHI DNA. NTHI and NTHI DNA-induced I-IFN response appeared to be mediated by cGAS. Collectively, we suggested that NTHI DNA as a PAMP triggered I-IFN response, which was STING/TBK1/IRF3 dependent. Summary NTHI is the leading cause of acute exacerbations of COPD (AECOPD). Since AECOPD is an immune event, it is meaningful to elucidate the mechanism under NTHI induced immune response. It has been revealed that lipooligosaccharides and protein of NTHI could induce host immune response, but the function of NTHI nuclide acid during infection is unclear. In this research, we demonstrate NTHI DNA is a trigger for I-IFN expression, and the STING/TBK1/IRF3 pathway plays an integral role in sensing NTHI DNA to induce I-IFN expression. Moreover, by long-term intrabronchial infection of LPS, we constructed a mimic COPD murine model, in which the STING expression in lung tissues were higher than healthy mice after NTHI infection, which led us to surmise that NTHI cause AECOPD by inducing I-IFN production via STING signal pathway. [ABSTRACT FROM AUTHOR]

In recent years, there has been a notable increase in cancer incidence and mortality, and immune abnormalities have been closely linked to malignancy development. TANK-binding kinase 1 (TBK1) is a non-classical IκB kinase that regulates interferon and NF-κB signaling pathways and plays a crucial role in innate immunity. Recent studies have shown high expression levels of TBK1 and increased activity in various tumor cells, suggesting its involvement in the development and progression of multiple cancers. Targeting TBK1 for tumor therapy may be a possibility. However, little is known about the abnormal activation and dynamic regulation of TBK1 in cancer. First, we utilized the BioID biotinylation technique combined with TMT-based quantitative proteomics to analyze the TBK1 interacting proteins. Our results revealed that TXLNA interacts with TBK1 and binds to the α-helical scaffold of TBK1. The expression of TXLNA could affect the S172 phosphorylation of TBK1. PPM1B is a phosphatase that can dephosphorylate TBK1 S172, so we used the APEX2 proximity labeling technique combined with TMT-based quantitative proteomics to explore the interacting proteins of PPM1B and search for the regulatory pathway of TXLNA on TBK1 phosphorylation. We found that PPM1B interacts with TXLNA. Based on these results, we further found that TXLNA impairs the binding of PPM1B to TBK1, inhibiting the dephosphorylation of TBK1 and contributing to the abnormal enhancement of TBK1 activity in cancer cells. This study sheds light on the potential mechanism of aberrant activation and dynamic regulation of TBK1 in tumors and provides a potential target for tumor therapy. • TXLNA interacts with TBK1 and binding to the α-helical scaffold of TBK1. • The expression of TXLNA affects the phosphorylation of TBK1(S172). • TXLNA interacts with PPM1B and enhances TBK1 activity by inhibiting PPM1B recruitment. [ABSTRACT FROM AUTHOR]

Summary Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies. [ABSTRACT FROM AUTHOR]

Obesity mediates immune inflammatory response and induces IL-17 expression. Adipgenesis can be regulated by IL-17 and it causes TBK1 activation. The inhibition of TBK1 and the inhibition of I IKBKE reduces inflammatory response and improves obesity. It is hypothesized that IL-17 deficiency inhibits obesity progression and inflammation. 3T3-L1 preadipocytes were differentiated in vitro and treated with IL-17. RAW264.7 cells and differentiated 3T3-L1 were pretreated with TBK1 inhibitor and then stimulated with IL-17. Wild-type and IL-17 knock out mice were fed with high-fat diet. IL-17 inhibits adipocyte differentiation from mouse-derived 3T3-L1 preadipocytes and reduces mRNA expression of proadipogenic transcription factors and adipokines in adipocyte cells. IL-17 also showed up-regulation of mRNA levels of inflammatory cytokines in RAW cells. The inhibitor of TBK1 and IKBKE attenuates the effect of IL-17. Loss of IL-17 deficiency improves diet-induced obesity, fatty liver, glucose and lipid metabolism in mice. The expression of TBK1 and IKBKE decreased in the spleen and liver of IL-17 deficiency mice. Moreover, the inflammatory response within the visceral adipose tissue and Th1 cells were inhibited, however, M2 macrophage and Th2 cells increased in IL-17 deficiency mice. IL-17 inhibits adipogenesis where a lack of IL-17 ameliorates glucose metabolism. As well, the inhibition of TBK1 reduces inflammation induced by IL-17. Therefore, IL-17 may be involved in the development of obesity and metabolic dysfunction in a TBK1-dependent manner. [ABSTRACT FROM AUTHOR]

The aquatic spring viremia of carp virus (SVCV) causes significant mortality in common carp ( Cyprinus carpio ), and TBK1 plays a crucial role in the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system by phosphorylating its substrates to induce type I interferons (IFNs) and cellular antiviral responses. In this study, we report that zebrafish STAT6 is induced during SVCV infection and reduces IFNφ1 expression by suppressing TBK1 phosphorylation. A typical IFN stimulatory response element (ISRE) motif was found in the promoter region of zebrafish STAT6, and zebrafish STAT6 transcription was significantly upregulated in the early stages of virus infection. Overexpression of STAT6 interfered with IFNφ1 promoter activity in response to SVCV infection. Additionally, TBK1-, but not MITA-mediated activation of the IFNφ1 promoter was impaired by STAT6. Co-immunoprecipitation and Western blot experiments indicated that MITA and IRF3 were significantly phosphorylated by TBK1, and that the N-terminal kinase domain of TBK1 was critical in this process. In the final step, STAT6 interacted with the N-terminal kinase domain of TBK1 causing dephosphorylation, which resulted in reductions in the phosphorylation of IRF3 and the production of IFNφ1. These results indicate that fish STAT6 can attenuate the kinase activity of TBK1, leading to suppression of IFNφ1 expression which may in turn facilitate virus replication. [ABSTRACT FROM AUTHOR]

Membrane-associated RING-CH-type finger (MARCH) proteins have been reported to regulate type I IFN production during host antiviral innate immunity. The present study reported the zebrafish MARCH family member, MARCH7, as a negative regulator in virus-triggered type I IFN induction via targeting TANK-binding kinase 1 (TBK1) for degradation. As an IFN-stimulated gene (ISG), we discovered that MARCH7 was significantly induced by spring viremia of carp virus (SVCV) or poly(I:C) stimulation. Ectopic expression of MARCH7 reduced the activity of IFN promoter and dampened the cellular antiviral responses triggered by SVCV and grass carp reovirus (GCRV), which concomitantly accelerated the viral replication. Accordingly, the knockdown of MARCH7 by siRNA transfection significantly promoted the transcription of ISG genes and inhibited SVCV replication. Mechanistically, we found that MARCH7 interacted with TBK1 and degraded it via K48-linked ubiquitination. Further characterization of truncated mutants of MARCH7 and TBK1 confirmed that the C-terminal RING of MARCH7 is essential in the MARCH7-mediated degradation of TBK1 and the negative regulation of IFN antiviral response. This study reveals a molecular mechanism by which zebrafish MARCH7 negatively regulates the IFN response by targeting TBK1 for protein degradation, providing new insights into the essential role of MARCH7 in antiviral innate immunity. [ABSTRACT FROM AUTHOR]

In mammals, right open reading frame kinases (RIOKs) are initially reported to participate in cancer cell proliferation, apoptosis, migration and invasion, and recently they have been related to host immune response. Little is known about the homologs of RIOKs in fish. In the current study, we cloned three homologous genes of RIOK family in yellow catfish (Pelteobagrus fulvidraco), termed Pfriok1, Pfriok2 and Pfriok3. Pfriok1, Pfriok2 and Pfriok3 were constitutively expressed at relatively high levels in yellow catfish tissues, and their mRNA levels were not changed under viral infection. Individual overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 attenuated fish interferon (IFN) response, thereby promoting viral replication in fish cells. Mechanistically, yellow catfish RIOK proteins downregulated fish IFN response through attenuating TBK1 protein levels in cytoplasm. Our findings suggest that yellow catfish RIOK1, RIOK2 and RIOK3 are involved in downregulating fish IFN antiviral response. • Pfriok1 , Pfriok2 and Pfriok3 are constitutively expressed in yellow catfish. • Overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 promotes viral replication by downregulating IFN production. • PfRIOK1, PfRIOK2 and PfRIOK3 downregulates IFN-mediated antiviral response by attenuating TBK1 protein level in cytoplasm. [ABSTRACT FROM AUTHOR]

Type I interferon (IFN-I) is essential for the regulation of host–virus interactions, and viruses have evolved strategies to escape the host immune response. Duck hepatitis A virus type 1 (DHAV-1) causes severe liver necrosis and hemorrhage, neurological symptoms, and high mortality in ducklings. However, how DHAV-1 interacts with the duck innate immune system remains unclear. In this study, DHAV-1-encoded proteins were cloned, and DHAV-1 2A2 was shown to strongly suppress IFN-β–luciferase activity, triggered by Sendai virus and polyriboinosinic polyribocytidylic acid [poly(I:C)], along with the transcription of IFN-β and downstream antiviral genes, including OASL, PKR, and TNF-a. In addition, 2A2 interacts with the central adaptor proteins mitochondrial antiviral signaling (MAVS) and TANK-binding kinase 1 (TBK1) by its N-terminal 1–100 amino acids (aa), thus leading to the inhibition of IFN-β production. Importantly, the deletion of the N-terminal 1–100 aa region of 2A2 abolished inhibition of IFN-I production. Moreover, the transmembrane domain of the MAVS protein and the ubiquitin domain of TBK1 were demonstrated to be required for interaction with DHAV-1 2A2. These findings revealed a novel strategy by which DHAV-1 hijacks cellular immunosurveillance and provided new insights into controlling the disease. • DHAV-1 2A2 inhibited the expression of IFN-β and downstream antiviral genes. • The 1–100 aa of 2A2 protein was critical for interaction with MAVS and TBK1. • The TMD of MAVS and UBD of TBK1 were required for interaction with DHAV-1 2A2. • 2A2 had no significant effect on DHAV-1 replication in DEFs. [ABSTRACT FROM AUTHOR]

The clinical response to immune checkpoint blockade (ICB) correlates with tumor-infiltrating cytolytic T lymphocytes (CTLs) prior to treatment. However, many of these inflamed tumors resist ICB through unknown mechanisms. We show that tumors with transcription elongation deficiencies (TEdef+), which we previously reported as being resistant to ICB in mouse models and the clinic, have high baseline CTLs. We show that high baseline CTLs in TEdef+ tumors result from aberrant activation of the nucleic acid sensing-TBK1-CCL5/CXCL9 signaling cascade, which results in an immunosuppressive microenvironment with elevated regulatory T cells and exhausted CTLs. ICB therapy of TEdef+ tumors fail to increase CTL infiltration and suppress tumor growth in both experimental and clinical settings, suggesting that TEdef+, along with surrogate markers of tumor immunogenicity such as tumor mutational burden and CTLs, should be considered in the decision process for patient immunotherapy indication. [Display omitted] • Aberrant NAS in TEdef+ tumors triggers TBK1-mediated release of pro-inflammatory chemokine • CD8+ T cell infiltration is increased in ICB-resistant TEdef+ tumors • TEdef+ tumors attract regulatory T cells and promote CD8+ T cell exhaustion • Inert inflammation in TEdef+ human tumors is associated with ICB therapy resistance Modur et al. show that tumors with transcription elongation deficiencies (TEdef+) resist ICB despite having a high baseline of tumor-infiltrating cytolytic T lymphocytes (CTLs), resulting from aberrant activation of TBK1-induced inert inflammation with elevated regulatory T cells and exhausted CTLs, making TEdef+ important in patient immunotherapy indication. [ABSTRACT FROM AUTHOR]

• CIAV VP1 protein inhibits IFN-I expression induced by cGAS-STING signaling. • VP1 inhibits TBK1 phosphorylation. • The 121–150 aa in VP1 is essential for VP1 to interact with TBK1 and inhibit cGAS-STING signaling. Chicken infectious anemia virus (CIAV) infection induces immunosuppression or subclinical immunosuppression in chickens. CIAV infection has been reported to repress type I interferon (IFN-I) expression, but the underlying mechanisms are not yet understood. Here we reported that VP1, the capsid protein of CIAV, the major immunogenic protein that triggers the production of neutralizing antibodies in chickens, inhibited type I interferon (IFN-I) expression induced by cGAS-STING signaling. We showed that VP1 inhibited TBK1 phosphorylation and down stream signal transduction, leading to the inhibition of IFN-I expression. Subsequently, we demonstrated that VP1 interacted with TBK1. Finally, we clarified that aa 120–150 in VP1 was essential for VP1 to interact with TBK1 and inhibit cGAS-STING signaling. These findings will help us further understand the pathogenesis of CIAV in chickens. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is one of the most aggressive tumors in the adult central nervous system. We previously revealed that circadian regulation of glioma stem cells (GSCs) affects GBM hallmarks of immunosuppression and GSC maintenance in a paracrine and autocrine manner. Here, we expand the mechanism involved in angiogenesis, another critical GBM hallmark, as a potential basis underlying CLOCK's pro-tumor effect in GBM. Mechanistically, CLOCK-directed olfactomedin like 3 (OLFML3) expression results in hypoxia-inducible factor 1-alpha (HIF1α)-mediated transcriptional upregulation of periostin (POSTN). As a result, secreted POSTN promotes tumor angiogenesis via activation of the TANK-binding kinase 1 (TBK1) signaling in endothelial cells. In GBM mouse and patient-derived xenograft models, blockade of the CLOCK-directed POSTN-TBK1 axis inhibits tumor progression and angiogenesis. Thus, the CLOCK-POSTN-TBK1 circuit coordinates a key tumor-endothelial cell interaction and represents an actionable therapeutic target for GBM. [Display omitted] • The CLOCK-BMAL1 complex promotes tumor angiogenesis in GBM • CLOCK-directed OLFML3-HIF1α axis upregulates pro-angiogenic factor POSTN • POSTN promotes angiogenesis via activation of the TBK1 signaling in endothelial cells • Inhibition of the CLOCK-directed POSTN-TBK1 axis impairs GBM progression and angiogenesis Pang et al. show that circadian regulator CLOCK promotes tumor angiogenesis in glioblastoma (GBM) through regulation of the OLFML3-HIF1α-POSTN-TBK1 circuit. Disrupting the tumor-endothelial cell interaction by depletion of POSTN and TBK1 inhibits angiogenesis and tumor growth in GBM mouse and PDX models. [ABSTRACT FROM AUTHOR]

The importance of TANK binding kinase-1 (TBK1), a multimeric kinase that modulates inflammation and autophagy, in human health has been highlighted for the first time by the recent discoveries of mutations in TBK1 that underlie amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), normal tension glaucoma (NTG) or childhood herpes simplex encephalitis (HSE). Gain-of-function of TBK1 are associated with NTG, whereas loss-of-function mutations result in ALS/FTD or in HSE. In light of these new findings, we review the role of TBK1 in these seemingly unrelated, yet allelic diseases, and discuss the role of TBK1 in neuroinflammatory diseases. This discovery has the potential to significantly increase our understanding of the molecular basis of these poorly understood diseases. [ABSTRACT FROM AUTHOR]

• IRF5 was mainly located in the nucleus of TDP-25 cells. • IRF5 deficiency promotes motor neuron survival. • IRF5 deficiency alleviates oxidative stress and protects Mitochondria in TDP-25 cells. • TBK1 blockade disrupts protective effect of IRF5 deficiency on TDP-25 cells. Interferon-regulatory factor 5 (IRF5) participates in the regulation of apoptosis, affects the phenotype of inflammatory macrophages and plays an essential role in the inflammatory response. However, the role of IRF5 in the progression of amyotrophic lateral sclerosis (ALS) remains largely unknown. Here, we show that IRF5 mainly accumulated in the nucleus in cells expressing the truncated 25 k D C-terminal fragments of TDP-43 (TDP-25, named TDP-25 cells hereafter). IRF5 knockdown using a lentivirus carrying an shRNA in TDP-25 cells exerted a protective effect and reduced the level of the apoptosis-related protein cleaved caspase-9 and the cell cycle arrest protein p21, while increasing the expression of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its target molecule glutamate-cysteine ligase modulatory subunit (GCLM). Furthermore, IRF5-knockdown cells showed improved mitochondrial swelling and cristae dilation. In addition, we found that IRF5 mediated neuronal injury partly through the negative regulation of TANK-binding kinase 1 (TBK1). These data indicate that the loss of IRF5 in TDP-25 cells exerts a protective effect mainly by inhibiting apoptosis, regulating cell cycle arrest and alleviating oxidative stress. [ABSTRACT FROM AUTHOR]

• ASFV MGF505-7R could inhibit cGAS-STING-mediated activation of IFN-β and ISRE promoters. • ASFV MGF505-7R specifically associated with TBK1, IRF7. • ASFV MGF505-7R interacted with IRF7, TBK1, degrading IRF7 expression by autophagy, cysteine, proteasome pathways and TBK1 expression by proteasome. African swine fever virus (ASFV) employs diverse strategies to confront or evade host type I interferon (IFN-I)-induced antiviral responses. Moreover, the mechanisms of this process are largely unknown. Here, we assessed 27 ASFV proteins to determine whether any of them suppressed the cGAS-STING pathway to facilitate immune evasion. Using dual-luciferase assays, we found that ASFV MGF505-7R suppressed the activity of the IFN-β and ISRE promoters and the expression of IFN-I and ISGs. MGF505-7R interacted with IRF7 and TBK1, degrading IRF7 by autophagy, cysteine, and proteasome pathways and TBK1 by the proteasome pathway. Moreover, TBK1 and IRF3 were phosphorylated by cGAS-STING stimulation. Finally, small interfering RNA (siRNA)-based silencing of MGF505-7R enhanced IFN-I antiviral activity. Taken together, these results preliminarily clarified the immune escape mechanism of ASFV MGF505-7R, which provides a potential target for developing antiviral agents. [ABSTRACT FROM AUTHOR]

TANK-binding kinase 1 (TBK1), a member of IκB Kinase (IKK)-related kinases, plays a role in regulating innate immunity, inflammation and oncogenic signaling. This study aims to investigate the role of BX795, an inhibitor of TBK1, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antitumor effects and mechanisms of BX795 were assessed by MTT assays, flow cytometry, Western blotting, and confocal microscopy. BX795 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound caused apoptosis as evidenced by PARP cleavage, the presence of pyknotic nuclei in the TUNEL assay, and fragmented DNA tails in the Comet assay. BX795 inhibits Akt and NF-κB signaling, arrests cells in the mitotic phase, and increases generation of autophagy in oral cancer cells. Interestingly, the antiproliferative activity of BX795 does not correlate with TBK1 protein expression level in OSCC cells. We propose that the TBK1-independet effect is related to mitotic phase arrest. Pleiotropic anticancer activity with relative sparing of normal oral keratinocytes underscores the potential value of BX795 and warrants its further study in oral squamous cell carcinoma therapy. [ABSTRACT FROM AUTHOR]

Background: Tumor-associated macrophages (TAMs) with the M2-like phenotype are regulated by mainly NF-kB pathway including TBK1, which can influence tumor progression by secretion of proangiogenic factors such as vascular endothelial growth factor. The CCL2/CCR2 axis, histidine-rich glycoprotein (HRG), and placenta growth factor (PIGF) play a critical role in the polarization of M1/M2 phenotypes and the recruitment of TAMs to tumor microenvironment. We therefore hypothesized that variations in genes involved in regulating TAMs may predict clinical outcomes of bevacizumab treatment in patients with metastatic colorectal cancer (mCRC). Patients and methods: We analyzed genomic DNA extracted from samples of patients receiving bevacizumab plus FOLFIRI as a first-line treatment using PCR-based direct sequencing. Twelve functional single-nucleotide polymorphisms in eight genes (CCL2, CCR2, HRG, PIGF, NFKB1, TBK1, CCL18, and IRF3) were tested for associations with clinical outcomes in a discovery cohort of 228 participants in TRIBE trial (NCT00719797), then validated in 248 KRAS exon2 (KRAS) wild-type participants in FIRE3 trial (NCT00433927). FIRE3-cetuximab cohort served as a negative control. Results: TBK1 rs7486100 was significantly associated with overall survival in 95 KRAS wild-type patients of TRIBE cohort in univariate analysis and had a strong trend in multivariable analysis; furthermore, the association of the T allele was observed for progression-free survival (PFS) in both univariate and multivariable analyses in FIRE3-bevacizumab but not cetuximab cohort. CCL2 rs4586, CCL18 rs14304, and IRF3 rs2304205 had univariate and multivariable correlations with PFS in KRAS mutant patients of the TRIBE cohort, whereas they had no correlations in KRAS wild-type patients of the TRIBE cohort. No association was seen in control cohort. Conclusions: Our study demonstrates for the first time that variations in genes regulating TAMs-related functions are significantly associated with clinical outcomes in mCRC patients treated with bevacizumab-containing chemotherapy. These results also suggest that some TAM-related gene variations may predict outcomes of bevacizumab treatment in KRAS status-dependent manner. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance As documented in the Chinese Materia Medica Grand Dictionary (中藥大辭典), a herbal formula (RL) consisting of Rosae Multiflorae Fructus (multiflora rose hips) and Lonicerae Japonicae Flos (Japanese honeysuckle flowers) has traditionally been used in treating inflammatory disorders. RL was previously reported to inhibit the expression of various inflammatory mediators regulated by NF-κB and MAPKs that are components of the TLR4 signalling pathways. Aim of the study This study aims to provide further justification for clinical application of RL in treating inflammatory disorders by further delineating the involvement of the TLR4 signalling cascades in the effects of RL on inflammatory mediators. Materials and methods RL consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos (in 5:3 ratio) was extracted using absolute ethanol. We investigated the effect of RL on the production of cytokines and chemokines that are regulated by three key transcription factors of the TLR4 signalling pathways AP-1, NF-κB and IRF3 in LPS-stimulated RAW264.7 cells using the multiplex biometric immunoassay. Phosphorylation of AP-1, NF-κB, IRF3, IκB-α, IKKα/β, Akt, TAK1, TBK1, IRAK-1 and IRAK-4 were examined in LPS-stimulated RAW264.7 cells and THP-1 cells using Western blotting. Nuclear localizations of AP-1, NF-κB and IRF3 were also examined using Western blotting. Results RL reduced the secretion of various pro-inflammatory cytokines and chemokines regulated by transcription factors AP-1, NF-κB and IRF3. Phosphorylation and nuclear protein levels of these transcription factors were decreased by RL treatment. Moreover, RL inhibited the activation/phosphorylation of IκB-α, IKKα/β, TAK1, TBK1 and IRAK-1. Conclusions Suppression of the IRAK-1/TAK1 and TBK1/IRF3 signalling pathways was associated with the effect of RL on inflammatory mediators in LPS-stimulated RAW264.7 and THP-1 cells. This provides further pharmacological basis for the clinical application of RL in the treatment of inflammatory disorders. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) are essential innate immune proteins that maintain tissue homeostasis through tonic expression and can be upregulated to drive antiviral resistance and inflammation upon stimulation. However, the mechanisms that inhibit aberrant IFN upregulation in homeostasis and the impacts of tonic IFN production on health and disease remain enigmatic. Here, we report that caspase-8 negatively regulates type I IFN production by inhibiting the RIPK1-TBK1 axis during homeostasis across multiple cell types and tissues. When caspase-8 is deleted or inhibited, RIPK1 interacts with TBK1 to drive elevated IFN production, leading to heightened resistance to norovirus infection in macrophages but also early onset lymphadenopathy in mice. Combined deletion of caspase-8 and RIPK1 reduces the type I IFN signaling and lymphadenopathy, highlighting the critical role of RIPK1 in this process. Overall, our study identifies a mechanism to constrain tonic type I IFN during homeostasis which could be targeted for infectious and inflammatory diseases. [Display omitted] • Caspase-8 regulates tonic type I IFN in homeostasis through the RIPK1-TBK1 axis • Caspase-8 inhibits the interaction between RIPK1 and TBK1 to regulate type I IFN • When caspase-8 is absent, RIPK1 drives pathogenic IFN production and lymphadenopathy • RIPK1-mediated IFN leads to natural viral resistance when caspase-8 is absent Wang et al. report the mechanistic regulation of homeostatic type I IFN production by caspase-8 through the RIPK1-TBK1 axis. Hyper-activation of this pathway due to loss of caspase-8 has profound physiological impacts on natural resistance to viral infection and the pathogenesis of lymphadenopathy. [ABSTRACT FROM AUTHOR]

• Screening of 12 ASFV ORFs that modulate the cGAS-STING pathway. • M1249L inhibits the IFN-β promoter activity and transcriptional activity. • M1249L suppresses the phosphorylation of TBK1 mediated by cGAS and STING. • M1249L degrades endogenous and exogenous IRF3 by lysosomal pathways. Cyclic GMP-AMP synthase (cGAS) is a major DNA sensor. The recognition of cytosolic DNA by cGAS triggers a robust innate immune response that restricts the replication of diverse viral pathogens through the type I interferon (IFN) and nuclear factor-κB (NF-κB) pathways. African swine fever virus (ASFV) is a large and complex DNA virus reported to strongly inhibit the cGAS-STING signaling pathway. Herein, 12 ASFV structural proteins were screened to determine their effects on the cGAS-STING pathway. Ectopic expression of the ASFV caspid protein M1249L significantly inhibited the IFN-β promoter activity induced by the cGAS-STING pathway in a dose-dependent manner. And it could also downregulate the levels of IFN-β and several interferon-stimulating genes (ISGs) induced by cGAS-STING and 2'3'-cGAMP. Moreover, ASFV M1249L also suppressed phosphorylation of TBK1 by cGAS and STING overexpression. Further study showed that M1249L co-localized and interacted with interferon regulatory factor 3 (IRF3), which led to induce IRF3 degradation by lysosomal pathway. Taken together, our study revealed a novel strategy utilized by ASFV for cGAS-STING-related immune evasion. [ABSTRACT FROM AUTHOR]