TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation.

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1- linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNFRSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

Enhanced ASGR2 by microplastic exposure leads to resistance to therapy in gastric cancer.

Background: Microplastics (MPs) are a new global environmental threat. Previously, we showed the biodistribution of MPs using [64Cu] polystyrene (PS) and PET in mice. Here, we aimed to identify whether PS exposure has malignant effects on the stomach and induces resistance to therapy. Methods: BALB/c nude mice were fed 1.72 × 104 particles/mL of MP. We investigated PS accumulation in the stomach using radioisotope-labeled and fluorescent-conjugated PS. Further, we evaluated whether PS exposure induced cancer stemness and multidrug resistance, and whether it affected tumor development, tumor growth, and survival rate in vivo using a 4-week PS-exposed NCI-N87 mouse model. Using RNA-Seq analysis, we analyzed whether PS exposure induced gene expression changes in gastric tissues of mice. Results: PET imaging results showed that a single dose of [64Cu]-PS remained for 24 h in the mice stomach. The 4-week daily repetitive dose of fluorescent conjugated PS was deposited in the gastric tissues of mice. When PS was exposed, a 2.9-fold increase in migration rate was observed for NCI-N87 cells. Immunocytochemistry results showed decreased E-cadherin and increased N-cadherin expression, and flow cytometry, qPCR, and western blot analysis indicated a 1.9-fold increase in N-cadherin expression after PS exposure. Further, PS-induced multidrug resistance to bortezomib, paclitaxel, gefitinib, lapatinib, and trastuzumab was observed in the NCI-N87 mouse model due to upregulated CD44 expression. RNA-seq results identified increased asialoglycoprotein receptor 2 (ASGR2) expression after PS exposure, and ASGR2 knockdown decreased cell proliferation, migration, invasion, and drug resistance. Conclusion: We demonstrated that ASGR2 enhanced cancer hallmarks on PS exposure and induced resistance to chemo- and monoclonal antibody-therapy. Our preclinical findings may provide an incentive for further epidemiological studies on the role of MP exposure and its association with gastric cancer.

Pre/post-natal exposure to microplastic as a potential risk factor for autism spectrum disorder.

In common with the increase in environmental pollution in the past 10 years, there has also been a recent increase in the prevalence of autism spectrum disorder (ASD). In this regard, we hypothesized that exposure to microplastics is a potential risk factor for ASD. To evaluate the validity of this hypothesis, we initially examined the accumulation of polyethylene (PE) in the brains of mice and then assessed the behavioral effects using mouse models at different life stages, namely, prenatal, post-weaning, puberty, and adult models. Based on typical behavioral assessments of autistic traits in the model mice, we established that ASD-like traits were induced in mice after PE feeding. In addition, we examined the induction of ASD-like traits in response to microplastic exposure using positron emission tomography, magnetic resonance spectroscopy, quantitative real-time polymerase chain reaction, microarray, and microbiome analysis. We believe these findings provide evidence in microplastics as a potential risk factor for ASD.

MST1 mediates the N-methyl-D-aspartate-induced excitotoxicity in mouse cortical neurons.

Excessive activation of the ionotropic N-methyl-D-aspartate (NMDA) receptor has been shown to cause abnormally high levels of Ca2+ influx, thereby leading to excitotoxic neuronal death. In this study, exposure of mouse primary cortical neurons to NMDA resulted in the cleavage and activation of mammalian sterile 20-like kinase-1 (MST1), both of which were mediated by calpain 1. In vitro cleavage assay data indicated that calpain 1 cleaves out the autoinhibitory domain of MST1 to generate an active form of the kinase. Furthermore, calpain 1 mediated the cleavage and activation of wild-type MST1, but not of MST1 (G339A). Intriguingly, NMDA/calpain-induced MST1 activation promoted the nuclear translocation of the kinase and the phosphorylation of histone H2B in mouse cortical neurons, leading to excitotoxicity. Thus, we propose a previously unrecognized mechanism of MST1 activation associated with NMDA-induced excitotoxic neuronal death.

UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

TRAF6-mediated ubiquitination of MST1/STK4 attenuates the TLR4-NF-κB signaling pathway in macrophages.

Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.

CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation.

The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.

Mst1-Deficiency Induces Hyperactivation of Monocyte-Derived Dendritic Cells via Akt1/c-myc Pathway.

Mst1 is a multifunctional serine/threonine kinase that is highly expressed in several immune organs. The role of Mst1 in the activation of dendritic cells (DCs), a key player of adaptive immunity, is poorly understood. In this study, we investigated the role of Mst1 in GM-CSF-induced bone marrow-derived DCs and the underlying mechanisms. Mst1-/- DCs in response to GM-CSF expressed higher levels of activation/maturation-related cell surface molecules, such as B7 and MHC class II than Mst1+/+ DCs. Furthermore, the expression of proinflammatory cytokines, such as IL-23, TNF-α, and IL-12p40, was increased in Mst1-/- DCs, indicating that Mst1-deficiency may induce the hyperactivation of DCs. Additionally, Mst1-/- DCs exhibited a stronger capacity to activate allogeneic T cells than Mst1+/+ DCs. Silencing of Mst1 in DCs promoted their hyperactivation, similar to the phenotypes of Mst1-/- DCs. Mst1-/- DCs exhibited an increase in Akt1 phosphorylation and c-myc protein levels. In addition, treatment with an Akt1 inhibitor downregulated the protein level of c-myc increased in Mst1-deficient DCs, indicating that Akt1 acts as an upstream inducer of the de novo synthesis of c-myc. Finally, Akt1 and c-myc inhibitors downregulated the increased expression of IL-23p19 observed in Mst1-knockdown DCs. Taken together, these data demonstrate that Mst1 negatively regulates the hyperactivation of DCs through downregulation of the Akt1/c-myc axis in response to GM-CSF, and suggest that Mst1 is one of the endogenous factors that determine the activation status of GM-CSF-stimulated inflammatory DCs.

MST1 Negatively Regulates TNFα-Induced NF-κB Signaling through Modulating LUBAC Activity.

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.

Yin-and-yang bifurcation of opioidergic circuits for descending analgesia at the midbrain of the mouse.

In the descending analgesia pathway, opioids are known to disinhibit the projections from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), leading to suppression of pain signals at the spinal cord level. The locus coeruleus (LC) has been proposed to engage in the descending pathway through noradrenergic inputs to the spinal cord. Nevertheless, how the LC is integrated in the descending analgesia circuit has remained unknown. Here, we show that the opioidergic analgesia pathway is bifurcated in structure and function at the PAG. A knockout as well as a PAG-specific knockdown of phospholipase C ß4 (PLCß4), a signaling molecule for G protein-coupled receptors, enhanced swim stress-induced and morphine-induced analgesia in mice. Immunostaining after simultaneous retrograde labeling from the RVM and the LC revealed two mutually exclusive neuronal populations at the PAG, each projecting either to the LC or the RVM, with PLCß4 expression only in the PAG-LC projecting cells that provide a direct synaptic input to LC-spinal cord (SC) projection neurons. The PAG-LC projection neurons in wild-type mice turned quiescent in response to opiates, but remained active in the PLCß4 mutant, suggesting a possibility that an increased adrenergic function induced by the persistent PAG-LC activity underlies the enhanced opioid analgesia in the mutant. Indeed, the enhanced analgesia in the mutant was reversed by blocking α2-noradrenergic receptors. These findings indicate that opioids suppress descending analgesia through the PAG-LC pathway, while enhancing it through the PAG-RVM pathway, i.e., two distinct pathways with opposing effects on opioid analgesia. These results point to a therapeutic target in pain control.

PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

Ataxin-1 is involved in tumorigenesis of cervical cancer cells via the EGFR-RAS-MAPK signaling pathway.

Ataxin-1 (ATXN1) is a coregulator protein within which expansion of the polyglutamine tract causes spinocerebellar ataxia type 1, an autosomal dominant neurodegenerative disorder. Previously, we reported that ATXN1 regulates the epithelial-mesenchymal transition of cervical cancer cells. In the present study, we demonstrate that ATXN1 is involved in cervical cancer tumorigenesis by promoting the proliferation of human cervical cancer cells. Chromatin immunoprecipitation assays showed that ATXN1 bound to the promoter region within cyclin D1 and activated cyclin D1 transcription, resulting in cell proliferation. ATXN1 promoted cyclin D1 expression through the EGFR-RAS-MAPK signaling pathway. Mouse xenograft tumorigenicity assays showed that ATXN1 downregulation inhibited tumorigenesis in cervical cancer cell lines in nude mice. Human cervical cancer tissue microarrays and immunohistochemical techniques showed that ATXN1 was significantly upregulated in many such tissues. Our results suggest that ATXN1 plays an important role in cervical cancer tumorigenesis and is a prognostic marker for cervical cancer.

CIB1 protects against MPTP-induced neurotoxicity through inhibiting ASK1.

Calcium and integrin binding protein 1 (CIB1) is a calcium-binding protein that was initially identified as a binding partner of platelet integrin αIIb. Although CIB1 has been shown to interact with multiple proteins, its biological function in the brain remains unclear. Here, we show that CIB1 negatively regulates degeneration of dopaminergic neurons in a mouse model of Parkinson's disease using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Genetic deficiency of the CIB1 gene enhances MPTP-induced neurotoxicity in dopaminergic neurons in CIB1-/- mice. Furthermore, RNAi-mediated depletion of CIB1 in primary dopaminergic neurons potentiated 1-methyl-4-phenyl pyrinidium (MPP+)-induced neuronal death. CIB1 physically associated with apoptosis signal-regulating kinase 1 (ASK1) and thereby inhibited the MPP+-induced stimulation of the ASK1-mediated signaling cascade. These findings suggest that CIB1 plays a protective role in MPTP/MPP+-induced neurotoxicity by blocking ASK1-mediated signaling.

Amyotrophic lateral sclerosis-related mutant superoxide dismutase 1 aggregates inhibit 14-3-3-mediated cell survival by sequestration into the JUNQ compartment.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron loss in the spinal cord and brain. Mutations in the superoxide dismutase 1 (SOD1) gene have been linked to familial ALS. To elucidate the role of SOD1 mutations in ALS, we investigated 14-3-3, a crucial regulator of cell death that was identified in patients with familial ALS. In a transgenic mouse model (SOD1-G93A) of ALS, 14-3-3 co-localized with mutant SOD1 aggregates and was more insoluble in the spinal cords of mutant SOD1 transgenic mice than in those of wild-type mice. Immunofluorescence and co-immunoprecipitation experiments showed that the 14-3-3ɛ and θ isoforms interact with mutant SOD1 aggregates in the juxtanuclear quality control compartment of N2a neuroblastoma cells. Fluorescence loss in photobleaching experiments revealed that movement of the isoforms of 14-3-3 was markedly reduced in SOD1 aggregates. Bax translocation into and cytochrome c release from the mitochondria were promoted by the sequestration of 14-3-3 into mutant SOD1 aggregates, increasing cell death. Mutant SOD1 aggregates were dissolved by the Hsp104 chaperone, which increased the interaction of 14-3-3 with Bax, reducing cell death. Our study demonstrates that mutant SOD1 inhibits 14-3-3-mediated cell survival. This information may contribute to the identification of a novel therapeutic target for ALS.

A novel conformation of the LC3-interacting region motif revealed by the structure of a complex between LC3B and RavZ.

LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel ß-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy.

Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid ß-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.

eIF4E phosphorylation by MST1 reduces translation of a subset of mRNAs, but increases lncRNA translation.

Post-transcriptional gene regulation is an important step in eukaryotic gene expression. The last step to govern production of nascent peptides is during the process of mRNA translation. mRNA translation is controlled by many translation initiation factors that are susceptible to post-translational modifications. Here we report that one of the translation initiation factors, eIF4E, is phosphorylated by Mammalian Ste20-like kinase (MST1). Upon phosphorylation, eIF4E weakly interacts with the 5' CAP to inhibit mRNA translation. Simultaneously, active polyribosome is more associated with long noncoding RNAs (lncRNAs). Moreover, the linc00689-derived micropeptide, STORM (Stress- and TNF-α-activated ORF Micropeptide), is triggered by TNF-α-induced and MST1-mediated eIF4E phosphorylation, which exhibits molecular mimicry of SRP19 and, thus, competes for 7SL RNA. Our findings have uncovered a novel function of MST1 in mRNA and lncRNA translation by direct phosphorylation of eIF4E. This novel signaling pathway will provide new platforms for regulation of mRNA translation via post-translational protein modification.

SMN1 functions as a novel inhibitor for TRAF6-mediated NF-κB signaling.

Survival motor neuron (SMN) is a 38-kDa protein, whose deficiency in humans develops spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease with muscular atrophy due to motor neuron death in the spinal cord. We now report that SMN prevents the activation of TRAF6 and IκB kinase (IKK) and thereby negatively regulates the NF-κB signaling processes. SMN physically interacted with TRAF6 and with each component of the IKK complex, IKK-α, IKK-ß, and IKK-γ in BV2 microglia cells. Moreover, SMN1 inhibited the E3 ubiquitin ligase activity of TRAF6 as well as the kinase activity of IKK. Furthermore, depletion of endogenous SMN by RNA interference enhanced the IL-1ß-induced activation of IKK and production of inflammatory mediators such as TNF-α and nitric oxide in BV2 cells. Consistently, the potentiation of IL-1ß-induced IKK activity was also found in SMA patient fibroblasts, compared with that of normal ones. Our results thus suggest that SMN functions as a natural inhibitor for IL-1ß-induced NF-κB signaling by targeting TRAF6 and the IKK complex.

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane.

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.

BAT3 negatively regulates lipopolysaccharide-induced NF-κB signaling through TRAF6.

TNF receptor-associated factor 6 (TRAF6) plays a critical role in NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, both of which mediate macrophage activation in response to pathogen-associated molecular patterns such as bacterial endotoxin, lipopolysaccharides (LPS). In this study, we investigated whether HLA-B associated transcript-3 (BAT3) regulates LPS-induced macrophage activation. BAT3 physically interacted with TRAF6 in macrophages, and this interaction was enhanced in the cells after LPS treatment. Furthermore, BAT3 inhibited the homo-oligomerization of TRAF6 as well as the interaction between TRAF6 and its downstream kinase transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing TRAF6-mediated signaling events. Intriguingly, TRAF6 mediated ubiquitination of BAT3 and this ubiquitination was crucial for its inhibitory effect on TRAF6-mediated signaling. Depletion of BAT3 by RNA interference resulted in enhancement of LPS-induced activation of the NF-κB signaling with increasing expression levels of pro-inflammatory cytokines. These findings suggest that BAT3 functions as the negative regulator of LPS-induced macrophage activation.