TANK-binding Kinase 1 Activation Research Articles

TBC1D9 regulates TBK1 activation through Ca2+ signaling in selective autophagy

Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca2+ levels to activate TBK1 for xenophagy via the Ca2+-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca2+-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca2+ chelator impairs TBC1D9–ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca2+-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca2+-dependent ubiquitin-recognition.

TBK1 Limits mTORC1 by Promoting Phosphorylation of Raptor Ser877

While best known for its role in the innate immune system, the TANK-binding kinase 1 (TBK1) is now known to play a role in modulating cellular growth and autophagy. One of the major ways that TBK1 accomplishes this task is by modulating the mechanistic Target of Rapamycin (mTOR), a master regulator that when activated promotes cell growth and inhibits autophagy. However, whether TBK1 promotes or inhibits mTOR activity is highly cell type and context dependent. To further understand the mechanism whereby TBK1 regulates mTOR, we tested the hypothesis that TBK1 phosphorylates a key component of the mTOR complex 1 (mTORC1), Raptor. Using kinase assays coupled with mass spectrometry, we mapped the position of the TBK1 dependent phosphorylation sites on Raptor in vitro. Among the sites identified in vitro, we found that TBK1 promotes Raptor Ser877 phosphorylation in cells both basally and in response to pathogen-associated molecules known to induce TBK1 activity. The levels of Raptor Ser877 phosphorylation were inversely correlated with the levels of mTOR activity. Expression of a mutant Raptor that could not be phosphorylated at Ser877 led to an increase in mTORC1 activity. We conclude that TBK1 limits mTORC1 activity by promoting Raptor Ser877 phosphorylation.

Sting is a critical regulator of spinal cord injury by regulating microglial inflammation via interacting with TBK1 in mice

Spinal cord injury (SCI) is a devastating neurological condition that results in progressive tissue loss, secondary to vascular dysfunction and inflammation. Lack of effective pharmacotherapies for SCI is mainly attributable to an incomplete understanding of its pathogenesis. Stimulator of interferon gene (Sting), also known as Transmembrane protein 173 (TMEM173), activates the type I interferon-regulated innate immune response, playing crucial role in modulating inflammation. However, the mechanism underlying Sting activation in SCI is still unclear. Here, we reported that Sting functioned as a positive regulator of SCI. Sting expression was increased in the injured spinal cord samples of SCI mice, along with significantly up-regulated levels of pro-inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6. Suppressing Sting expression in lipopolysaccharide-incubated mouse microglia markedly reduced the activation of nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPKs) signaling pathways, as illustrated by the decreased phosphorylation of IKKβ, IκBα, NF-κB/p65, p38, ERK1/2 and JNK. Furthermore, LPS-stimulated release of pro-inflammatory cytokines in microglial cells was also reversed by Sting knockdown. In contrast, LPS-induced inflammation was further accelerated in microglial cells with Sting over-expression through potentiating NF-κB and MAPKs signaling. Mechanistically, Sting directly interacted with the TANK-binding kinase 1 (TBK1), thus promoting its phosphorylation and the activation of down-streaming NF-κB and MAPKs signaling pathways. Notably, the effects of Sting on SCI progression were verified in mice. Consistently, Sting knockout alleviated inflammatory response and facilitated recovery after SPI in mice through blocking TBK1 activation and subsequent NF-κB and MAPKs phosphorylation. In summary, our findings may provide a novel strategy for prevention and treatment of SCI by targeting Sting.

GCRV hijacks TBK1 to evade IRF7-mediated antiviral immune responses in grass carp Ctenopharyngodon idella

TANK-binding kinase 1 (TBK1) is an important kinase that regulates the activation of interferon regulatory factor 3/7 (IRF3/7) to induce type I interferon (IFN–I) production in antiviral immune responses. However, in long-term virus-host crosstalk, viruses have evolved elaborate strategies to evade host immune defense mechanisms. In the present study, we found that grass carp (Ctenopharyngodon idella) reovirus (GCRV) hijacks TBK1 to escape IRF7-IFN-Is signaling activation. In brief, GCRV inhibited TBK1 activation by restaining K63-linked ubiquitination of TBK1 and promoting its K48-linked ubiquitination. This regulation resulted in that under low titer of GCRV infection, TBK1 overexpression specifically supressed promoter activity and phosphorylation of IRF7 and induction of downstream IFN1and IFN3. qRT-PCR data uncovered that TBK1 negatively regulated IRF7, IFN1 and IFN3 transcription levels under low viral titer infection. Along with enhancement of GCRV titers, TBK1 swiched its function to up-regulate IRF7, IFN1 and IFN3 mRNA levels. Accordingly, TBK1 promoted GCRV replication at low infected titer, but inhibited GCRV replication at high infected titer. All these results revealed a viral evasion strategy that GCRV utilizes TBK1 to block cellular IFN responses at low titers or early stages in fish species, which will lay a foundation for further researching on host-virus interactions and developing novel antiviral strategies in lower vertebrates.

Abstract 1033: Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that higher levels of TBK1 mRNA are associated with poorer overall survival in human PDA patients and that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB-dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl-TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease. Citation Format: Emily N. Arner, Victoria H. Cruz, Wenting Du, Rolf Brekken. Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1033.

Herpes Simplex Virus 1 Tegument Protein UL46 Inhibits TANK-Binding Kinase 1-Mediated Signaling.

TANK-binding kinase 1 (TBK1) is a key component of the antiviral immunity signaling pathway. It activates downstream interferon regulatory factor 3 (IRF3) and subsequent type I interferon (IFN-I) production. Herpes simplex virus type 1 (HSV-1) can antagonize host antiviral immune responses and lead to latent infection. Here, HSV-1 tegument protein UL46 was demonstrated to downregulate TBK1-dependent antiviral innate immunity. UL46 interacted with TBK1 and reduced TBK1 activation and its downstream signaling. Our results showed that UL46 impaired the interaction of TBK1 and IRF3 and downregulated the activation of IRF3 by inhibiting the dimerization of TBK1 to reduce the IFN-I production induced by TBK1 and immunostimulatory DNA. The IFN-I and its downstream antiviral genes induced by UL46-deficient HSV-1 (ΔUL46 HSV-1) were higher than those of wild-type HSV-1 (WT HSV-1). In addition, the stable knockdown of TBK1 facilitated the replication of ΔUL46 HSV-1, but not WT HSV-1. Together, these findings reveal a novel mechanism of immune evasion by HSV-1.IMPORTANCE HSV-1 has evolved multiple strategies to evade host antiviral responses and establish a lifelong latent infection, but the molecular mechanisms by which HSV-1 interrupts antiviral innate immunity are not completely understood. As TBK1 is very critical for antiviral innate immunity, it is of great interest to reveal the immune evasion mechanism of HSV-1 by targeting TBK1. In the present study, HSV-1 UL46 was found to inhibit the activation of IFN-I by targeting TBK1, suggesting that the evasion of TBK1 mediated antiviral innate immunity by HSV-1 UL46. Findings in this study will provide new insights into the host-virus interaction and help develop new approaches against HSV-1 infection.

Axl-mediated activation of TBK1 drives epithelial plasticity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an activating mutation in KRAS. Direct inhibition of KRAS through pharmacological means remains a challenge; however, targeting key KRAS effectors has therapeutic potential. We investigated the contribution of TANK-binding kinase 1 (TBK1), a critical downstream effector of mutant active KRAS, to PDA progression. We report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. Further, we identify that the receptor tyrosine kinase Axl induces TBK1 activity in a Ras-RalB-dependent manner. These findings demonstrate that TBK1 is central to an Axl-driven epithelial-mesenchymal transition in KRAS-mutant PDA and suggest that interruption of the Axl-TBK1 signaling cascade above or below KRAS has potential therapeutic efficacy in this recalcitrant disease.

A conserved PLPLRT/SD motif of STING mediates the recruitment and activation of TBK1.

Nucleic acids from bacteria or viruses induce potent immune responses in infected cells1–4. The detection of pathogen-derived nucleic acids is a central strategy by which the host senses infection and initiates protective immune responses5,6. Cyclic GMP-AMP synthase (cGAS) is a double-stranded DNA sensor7,8. It catalyzes the synthesis of cyclic GMP-AMP (cGAMP)9–12, which stimulates the induction of type I interferons (IFN-Is) through the STING-TBK1-IRF-3 signaling axis13–15. Stimulator of interferon genes (STING) oligomerizes upon cGAMP binding, leading to the recruitment and activation of tank-binding kinase 1 (TBK1)8,16. Interferon regulatory factor 3 (IRF-3) is then recruited to the signaling complex and activated by TBK18,17–20. Phosphorylated IRF-3 translocates to the nucleus and initiates the expression of IFN-Is21. However, the precise mechanisms governing STING activation by cGAMP and subsequent TBK1 activation by STING remained poorly understood. Here we show that a conserved PLPLRT/SD motif within the C-terminal tail of STING mediates the recruitment and activation of TBK1. Crystal structures of TBK1 bound to STING reveal that the PLPLRT/SD motif binds to the dimer interface of TBK1. Cell-based studies confirm that the direct interaction between TBK1 and STING is essential for IFN-β induction upon cGAMP stimulation. Moreover, we show that full-length STING oligomerizes upon cGAMP binding and highlight this as an essential step in the activation of STING-mediated signaling.

TANK-binding kinase 1 as a novel therapeutic target for viral diseases

ABSTRACTIntroduction: TANK-binding kinase 1 (TBK1) is vital for the induction of antiviral innate immune responses. Both RNA and DNA viral infection induces TBK1 activation, triggers phosphorylation of interferon regulatory factor (IRF) 3 and subsequent expression of type I interferons (IFNs; IFN-α/β). Type I IFNs can induce the expression of numerous antiviral genes called interferon-stimulated genes (ISGs) to build a remarkable antiviral state and limit viral replication. Thus, optimal TBK1 activity is crucial for IRF3-induced type I IFNs expression and ISGs-mediated viral elimination.Areas covered: This review provides an overview of the diverse roles of TBK1 in antiviral innate immune responses, the regulatory mechanisms of TBK1 activity and the implication in antiviral development.Expert opinion: TBK1 is a key kinase against antiviral infection via inducing type I IFNs expression. Multiple types of post-translational modifications of TBK1 tightly regulate TBK1 activity and subsequent TBK1-dependent antiviral responses. The identified regulators of TBK1 unveil regulatory mechanisms of host antiviral innate immunity and immuno-escape mechanism of virus provide strategies to control viral diseases by modulating TBK1 activity.

Loss of HAX-1 May Contribute to the Neurodegeneration Caused by a Kv3.3 Mutation

Spinocerebellar Ataxia type 13 (SCA13) is caused by mutations in the KCNC3 gene, which encodes the voltage-dependent potassium channel Kv3.3. These channels are expressed at particularly high levels in cerebellum Purkinje cells. SCA13 is an autosomal dominant disease characterized primarily by degeneration of the cerebellum. We have found the Kv3.3 channel differs from other potassium channels in that it binds Hax-1, an anti-apoptotic protein that is absolutely required for the survival of cerebellar neurons. We have also found that depolarization of Kv3.3 channels directly activates Tank Binding Kinase 1 (TBK1), an enzyme that plays a key role in the formation of multivesicular bodies, autophagy and mitophagy. A disease-causing mutation, G592R Kv3.3, produces enhanced TBK1 activation both in cell lines and in the cerebellum of knock-in mice bearing this mutation. By electron microscopy, we find the enhanced activation of TBK1 in G592R Kv3.3 knock-in mice is associated with increased numbers of intracellular multivesicular bodies containing Hax-1, and increased levels of CD63, a molecular marker for these structures. We also found that the expression level of Hax-1 is reduced in G592R Kv3.3 knock-in mice by Western blotting. Mitochondrial function may also be impacted by the mutation because levels of mitofusin-1 and mitofusin-2, proteins that are required for fusion and maintenance of mitochondrial structure, are altered in the mutant mice. Using cell lines expressing the mutant channel, we have shown that the G592R Kv3.3-induced multivesicular bodies contain Hax-1. Inhibition of TBK1 in the cell lines prevents the increase in CD63 levels produced by the Kv3.3 mutation. Our findings suggest that Kv3.3 channels are directly coupled to pathways that regulate the trafficking of proteins into multivesicular bodies and that loss of the Hax-1 may contribute to the neurodegeneration caused by SCA13.

Zebrafish MVP Recruits and Degrades TBK1 To Suppress IFN Production.

IFN production is crucial for hosts to defend against viral infection, yet it must be tightly controlled to maintain immune homeostasis. TANK-binding kinase 1 (TBK1) is a pivotal kinase in the IFN induction signaling pathway, but it is negatively regulated by multiple molecules to avoid the excessive expression of IFN in mammals. However, the identified TBK1 suppressors and the mechanisms are rare in fish. In this study, we show that zebrafish major vault protein (MVP) recruits and degrades TBK1 in a lysosome-dependent manner to inhibit IFN production. Through viral infection, polyinosinic:polycytidylic acid and RIG-I-like receptor factor stimulation upregulated IFN expression, but overexpression of MVP significantly subverted these inductions. On the protein level, MVP interacted with TBK1, and interestingly, MVP recruited TBK1 from a uniformly distributed state in the cytoplasm to an aggregated state. Finally, MVP mediated the lysosome-dependent degradation of TBK1 and decreased the IFN response and IFN-stimulated genes expression. Our findings reveal that zebrafish MVP is a negative regulator of IFN production by restricting the activation of TBK1, supplying evidence of the balanced mechanisms of IFN expression in lower vertebrates.

Zika virus NS5 protein antagonizes type I interferon production via blocking TBK1 activation

Zika virus (ZIKV) is a mosquito-borne positive-sense single-stranded RNA virus in the family of Flaviviridae. Unlike other flaviviruses, ZIKV infection of pregnant women may result in birth defects in their newborns, such as microcephaly or vision problem. ZIKV is known to antagonize the interferon (IFN) production in infected cells. However, the exact mechanism of this interference is not fully understood. Here, we demonstrate that NS5 protein of ZIKV MR766 strain antagonizes IFN production through inhibiting the activation of TANK-binding kinase 1 (TBK1), which phosphorylates the transcription activator IFN regulatory factor 3 (IRF3). Mechanistically, NS5 interacts with the ubiquitin-like domain of TBK1 and results in less complex of TBK1 and TNF (tumor necrosis factor) receptor-associated factor 6 (TRAF6), leading to dampened TBK1 activation and IRF3 phosphorylation. Our study provides insights into the mechanism of ZIKV evasion of IFN-mediated innate immunity.

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

Reduced Annexin A1 Secretion by ABCA1 Causes Retinal Inflammation and Ganglion Cell Apoptosis in a Murine Glaucoma Model.

Variants near the ATP-binding cassette transporter A1 (ABCA1) gene are associated with elevated intraocular pressure and newly discovered risk factors for glaucoma. Previous studies have shown an association between ABCA1 deficiency and retinal inflammation. Using a mouse model of ischemia-reperfusion (IR) induced by acute intraocular pressure elevation, we found that the retinal expression of ABCA1 protein was decreased. An induction of ABCA1 expression by liver X receptor agonist TO901317 reduced retinal ganglion cell (RGC) apoptosis after IR and promoted membrane translocation and secretion of the anti-inflammatory factor annexin A1 (ANXA1). Moreover, ABCA1 and ANXA1 co-localized in cell membranes, and the interaction domain is amino acid 196 to 274 of ANXA1 fragment. TO901317 also reduced microglia migration and activation and decreased the expression of pro-inflammatory cytokines interleukin (IL)-17A and IL-1β, which could be reversed by the ANXA1 receptor blocker Boc2. Overexpression of TANK-binding kinase 1 (TBK1) increased ABCA1 degradation, which was reversed by the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132). Silencing Tbk1 with siRNA increased ABCA1 expression and promoted ANXA1 membrane translocation. These results indicate a novel IR mechanism, that leads via TBK1 activation to ABCA1 ubiquitination. This degradation decreases ANXA1 secretion, thus facilitating retinal inflammation and RGC apoptosis. Our findings suggest a potential treatment strategy to prevent RGC apoptosis in retinal ischemia and glaucoma.

STING positively regulates human ORMDL3 expression through TBK1-IRF3-STAT6 complex mediation

Orosomucoid 1-like protein 3 (ORMDL3) is an asthma candidate gene associated with virus-triggered recurrent wheeze. Stimulator of interferon gene (STING) controls TLR-independent cytosolic responses to viruses. However, the association of STING with ORMDL3 is unclear. Here, we have shown that ORMDL3 expression shows a linear correlation with STING in recurrent wheeze patients. In elucidating the molecular mechanisms of the ORMDL3-STING relationship, we found that STING promoted the transcriptional activity of ORMDL3, which was significantly associated with increased levels of interferon regulatory factor 3 (IRF3) and signal transducer and activator of transcription 6 (STAT6). Further study showed that via activation of TANK binding kinase 1 (TBK1), STING enhanced the phosphorylation and binding of IRF3 and STAT6, which upregulated ORMDL3 by binding to the promoter. Our results showed that STING positively regulated ORMDL3 through the TBK1-IRF3-STAT6 complex.

Optineurin Insufficiency Disbalances Proinflammatory and Anti-inflammatory Factors by Reducing Microglial IFN-β Responses

Mutations in a ubiquitin (Ub)-binding adaptor protein optineurin have been found in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease with a prominent neuroinflammatory component. Unlike more frequent ALS mutations which cause disease by gaining toxic properties such as aggregation, mutated optineurin is thought to cause disease by loss-of-function, highlighting its neuroprotective role. Optineurin regulates inflammatory signaling by acting as a scaffold for Tank-binding kinase 1 (TBK1) activation and interferon (IFN)-β production in peripheral immune cells. The relevance of this pathway in the CNS is unclear. To investigate IFN-β pathway as a potential mechanism of optineurin-mediated protection from neurodegeneration, we have generated a mouse model in which the Ub-binding region of optineurin was deleted (Optn470T), mimicking C-terminal truncations found in patients. Here we report reduced TBK1 activation and IFN-β production in primary microglia from Optn470T model upon Toll-like receptor (TLR) stimulation. Likewise, we found diminished expression and activation of several transcription factors that support the amplification loop for IFN-β production including STAT1, IRF7 and IRF9. Notably, although optineurin was also reported to block proinflammatory transcription factor NF-κB, normal NF-κB activation and TNF production were found in Optn470T microglia. However, expression of both proinflammatory and anti-inflammatory factors distal to IFN-β was diminished, and could be restored upon IFN-β supplementation. Taken together with the recent discoveries of TBK1 mutations as an important genetic factor in ALS, our results open up the possibility that disruption of optineurin/TBK1-mediated IFN-β axis leads to an immune failure in containing neuronal damage, which could predispose to neurodegeneration.

Kv3.3 Potassium Channels Activate the TBK1 Signaling Pathway

Mutations in KCNC3, which encodes the Kv3.3 potassium channel, are linked to degeneration of the cerebellum (SCA13, spinocerebellar ataxia type 13). We found that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies. TBK1 activity regulates binding of the channel with Hax-1, an anti-apoptotic protein required for survival of cerebellar neurons, and that embeds Kv3.3 in a stable actin cytoskeleton at the plasma membrane. TBK1 activity is elevated several-fold in the cerebellum of animals expressing a SCA13 mutant channel. Cells that express the mutant channel have increased internalization of Hax-1 and accumulate multivesicular bodies containing this cell survival protein, coupled to depolarization-induced loss of the channels themselves. Our studies indicate that the activation of Kv3.3 channels is linked directly to TBK1 activity and channel mutations enhance internalization of the cell survival protein Hax-1 to which they are bound.

ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function

Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations.

Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.

These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).

InsP3R-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca2+ elevation and TBK1 activity

BackgroundCandida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca2+ levels via the inositol 1,4,5-trisphosphate receptor (InsP3R) plays a critical role in this process. However, the molecular pathways linking the InsP3R-mediated increase in Ca2+ and immune responses remain elusive.ResultsIn the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsP3R channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca2+ concentrations ([Ca2+]c). Immunofluorescence reveals enriched InsP3R-SEC5 complex formation on phagosomes, while disruption of the InsP3R-SEC5 interaction by recombinant H1 peptides attenuates the InsP3R-mediated Ca2+ elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsP3R-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsP3R-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca2+ but also by activating the TBK1-IRF-3 pathway.ConclusionsOur data have revealed an important role of the InsP3R-SEC5 interaction in innate immune responses against C. albicans.