TRAF6 Research Articles

Quantitative Proteomic Analysis Identifying and Evaluating TRAF6 and IL-8 as Potential Diagnostic Biomarkers in Neonatal Patients with Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a common gastrointestinal complication in premature infants, resulting in high morbidity and mortality, and its early detection is crucial for accurate treatment and outcome prediction. Extensive research has demonstrated a clear correlation between NEC and extremely low birth weight, degree of preterm, formula feeding, infection, hypoxic/ischemic damage, and intestinal dysbiosis. The development of noninvasive biomarkers of NEC from stool, urine, and serum has attracted a great deal of interest because to these clinical connections and the quest for a deeper knowledge of disease pathophysiology. Therefore, this study aims to identify protein expression patterns in NEC and discover innovative diagnostic biomarkers. In this study, we recruited five patients diagnosed with NEC and paired necrotic segments of intestinal tissue with adjacent normal segments of intestine to form experimental and control groups. Quantitative proteomics tandem mass tagging (TMT) labeling technique was used to detect and quantify the proteins, and the expression levels of the candidate biomarkers in the intestinal tissues were further determined by quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, Immunofluorescence methods and enzyme-linked immunosorbent assay (ELISA). A total of 6880 proteins were identified and quantified in patients with NEC. A significant disparity in protein expression was observed between necrotic and normal segments of intestinal tissue in NEC patients. A total of 55 proteins were found to be upregulated, and 40 proteins were found to be downregulated in NEC patients when using a p-value of < 0.05, and an absolute fold change of > 1.2 for analysis. GO function enrichment analysis showed the positive regulation of significant biological processes such as mitochondrial organization, vasoconstriction, rRNA catabolism, fluid shear stress response, and glycerol ether biosynthesis processes. Enrichment analysis also revealed essential functions such as ligand-gated ion channel activity, potassium channel activity, ligand-gated cation channel activity, ligand-gated ion channel activity, and ligand-gated channel activity, including molecular functions such as ligand-gated ion channel activity and mitotic events in this comparative group. Significant changes were found in endomembrane protein complex, membrane fraction, mitochondrial membrane fraction, membrane components, membrane intrinsic components, and other localized proteins. Additional validation of intestinal tissue and serum revealed a substantial increase in TRAF6 (tumor necrosis factor receptor-associated factor 6) and IL-8(Interleukin-8, CXCL8). The quantitative proteomic TMT method can effectively detect proteins with differential expression in the intestinal tissues of NEC patients. Proteins TRAF6 and CXCL8/IL-8 are significantly upregulated in the intestinal tissues and serum samples of patients and may serve as valuable predictor factors for NEC's early diagnosis.

USP2 Promotes the Proliferation and Inflammation of Fibroblast-Like Synovial Cells in Rheumatoid Arthritis Through Deubiquitination of TRAF2.

Rheumatoid arthritis (RA) is a prevalent inflammatory disorder affecting about 1% of the global population. The ubiquitin-specific protease 2 (USP2) is known to have a substantial influence on the regulation of several cellular processes. Both in vivo (using rats with collagen-induced arthritis, CIA) and in vitro (using human fibroblast-like synoviocytes, HFLS-RA) models of RA were used to examine the role of USP2 in RA. The proliferation of HFLS-RA cells was assessed using the cell counting kit 8 test and EdU staining. The technique used for the assessment of gene expression was quantitative real-time PCR. Protein expression was quantified using Western blot (WB) analysis, while the quantities of inflammatory factors and matrix metalloproteinases were assessed using an ELISA test. The co-immunoprecipitation and ubiquitination tests investigated the relationships between proteins and the underlying molecular pathways. The results of this study demonstrate an upregulation of USP2 expression in both vivo and vitro models of RA. In addition, our findings indicate that the overexpression of USP2 notably exacerbates both proliferation and inflammation. The consistent downregulation of USP2 resulted in a reduction in the secretion of inflammatory cytokines and a suppression of cellular proliferation. Furthermore, it was shown that USP2 interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2) and facilitates the removal of ubiquitination chains from TRAF2, enhancing its stability. Our findings propose that USP2 functions as a favorable modulator of proliferation and inflammatory reactions in HFLS-RA, thereby indicating its potential as a therapeutic target for the treatment of RA.

Mechanisms of Shufeng Jiedu Capsule in treating bacterial pneumonia based on network pharmacology and experimental verification

ObjectiveThis study aimed to investigate the underlying mechanism of Shufeng Jiedu Capsule (SFJD) for treating bacterial pneumonia (BP) in vivo based on network pharmacology and experimental verification study. MethodsNetwork pharmacology was used to screen the active compounds and target genes of SFJD. Then, the multi drug resistance-Pseudomonas aeruginosa (MDR-PA) mice lethal model and MDR-PA pneumonia model were established to evaluate the therapeutic effects and underlying mechanisms of SFJD. Western blot and ELISA were used to determinate the protein expression level of the IL-17 signaling pathway and JAK/STAT signaling pathway. ResultsAfter screening, 172 potential components of SFJD were generated, based on which we constructed an SFJD-component-target-BP interaction network. The Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment revealed that SFJD could regulate the IL-17 signaling pathway and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. Molecular docking showed that the potential target proteins had good combinations with the main active components. SFJD significantly reduced the mortality and prolonged survival days in lethal models. The lung index and pathological changes in the lung were also significantly decreased. SFJD could significantly decrease the expression of interleukin-17A (IL-17A), TNF receptor associated factor 6 (TRAF6), phospho-inhibitor of nuclear factor-kappa B (p-IκB)/inhibitor of NF-κB (IκB), phospho-NF-κB p65 (p-NF-κB p65), phospho-protein kinase B (p-AKT)/AKT, phospho-signal transducer and activator of transcription 3 (p-STAT3)/STAT3, phospho-signal transducer and activator of transcription 1 (p-STAT1)/STAT1, and the protein level of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β. ConclusionCombined with network pharmacology and in vivo study, it was found that SFJD exerted its therapeutic effects on BP by inhibiting the IL-17 pathway and JAK/STAT signaling pathway. This study provides new evidence for SFJD in treatment of BP.

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss

Cyathulae Radix, a traditional Chinese medicine and a common vegetable, boasts a history spanning millennia. It enhances bone density, boosts metabolism, and effectively alleviates osteoporosis-induced pain. Despite its historical use, the molecular mechanisms behind Cyathulae Radix's impact on osteoporosis remain unexplored. In this study, we investigated the effects and mechanisms of Cyathulae Radix ethanol extract (CEE) in inhibiting osteoporosis and osteoclastogenesis. Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks. Micro-computed tomography (micro-CT) assessed histomorphometric parameters, bone tissue staining observed distal femur histomorphology, and three-point bending tests evaluated tibia mechanical properties. Enzyme-linked immunosorbent assay (ELISA) measured serum estradiol (E2), receptor activator for nuclear factor B ligand (RANKL), and osteoprotegerin (OPG) levels. Osteoclastogenesis-related markers were analyzed via Western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase (TRAP) staining, qRT-PCR, and WB assay. Compared with the ovariectomy (OVX) group, CEE treatment enhanced trabecular bone density, maximal load-bearing capacity, and various histomorphometric parameters. Serum E2 and OPG levels significantly increased, while Receptor activator of nuclear factor-κB (RANK) decreased in the CEE group. CEE downregulated matrix metallopeptidase 9 (MMP-9), Cathepsin K (CTSK), and TRAP gene and protein expression. In bone marrow macrophages (BMMs), CEE reduced mature osteoclasts, bone resorption pit areas, and MMP-9, CTSK, and TRAP expression during osteoclast differentiation. Compared with DMSO treatment, CEE markedly inhibited RANK, TNF receptor associated factor 6 (TRAF6), Proto-oncogene c-Fos (c-Fos), Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expressions, and Extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), NF-kappa B-p65 (p65) phosphorylation in osteoclasts. In conclusion, CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis, potentially through modulating the Estrogen Receptor (ER)/RANK/NFATc1 signaling pathway.

Gender-related variation expressions of neuroplastin TRAF6, GluA1, GABA(A) receptor, and PMCA in cortex, hippocampus, and brainstem in an experimental epilepsy model.

Epileptic seizures are seen as a result of changing excitability balance depending on the deterioration in synaptic plasticity in the brain. Neuroplastin, and its related molecules which are known to play a role in synaptic plasticity, neurotransmitter activities that provide balance of excitability and, different neurological diseases, have not been studied before in epilepsy. In this study, a total of 34 Sprague-Dawley male and female rats, 2 months old, weighing 250-300g were used. The epilepsy model in rats was made via pentylenetetrazole (PTZ). After the completion of the experimental procedure, the brain tissue of the rats were taken and the histopathological changes in the hippocampus and cortex parts and the brain stem were investigated, as well as the immunoreactivity of the proteins related to the immunohistochemical methods. As a result of the histopathological evaluation, it was determined that neuron degeneration and the number of dilated blood vessels in the hippocampus, frontal cortex, and brain stem were higher in the PTZ status epilepticus (SE) groups than in the control groups. It was observed that neuroplastin and related proteins TNF receptor-associated factor 6 (TRAF6), Gamma amino butyric acid type A receptors [(GABA(A)], and plasma membrane Ca2+ATPase (PMCA) protein immunoreactivity levels increased especially in the male hippocampus, and only AMPA receptor subunit type 1 (GluA1) immunoreactivity decreased, unlike other proteins. We believe this may be caused by a problem in the mechanisms regulating the interaction of neuroplastin and GluA1 and may cause problems in synaptic plasticity in the experimental epilepsy model. It may be useful to elucidate this mechanism and target GluA1 when determining treatment strategies.

BZW2 promotes malignant progression in lung adenocarcinoma through enhancing the ubiquitination and degradation of GSK3β

The role of Basic leucine zipper and W2 domains 2 (BZW2) in the advancement of different types of tumors is noteworthy, but its involvement and molecular mechanisms in lung adenocarcinoma (LUAD) remain uncertain. Through this investigation, it was found that the upregulation of BZW2 was observed in LUAD tissues, which was associated with an unfavorable prognosis for individuals diagnosed with LUAD, as indicated by data from Gene Expression Omnibus and The Cancer Genome Atlas databases. Based on the clinicopathologic characteristics of LUAD patients from the tissue microarray, both univariate and multivariate analyses indicated that BZW2 functioned as an independent prognostic factor for LUAD. In terms of mechanism, BZW2 interacted with glycogen synthase kinase-3 beta (GSK3β) and enhanced the ubiquitination-mediated degradation of GSK3β through slowing down of the dissociation of the ubiquitin ligase complex, which consists of GSK3β and TNF receptor-associated factor 6. Moreover, BZW2 stimulated Wnt/β-catenin signaling pathway through GSK3β, thereby facilitating the advancement of LUAD. In conclusion, BZW2 was a significant promoter of LUAD. The research we conducted identified a promising diagnostic and therapeutic target for LUAD.

ASFV pA151R negatively regulates type I IFN production via degrading E3 ligase TRAF6.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly mortal and hemorrhagic infectious disease in pigs. Previous studies have indicated that ASFV modulates interferon (IFN) production. In this study, we demonstrated that ASFV pA151R negatively regulated type I IFN production. Ectopic expression of pA151R dramatically inhibited K63-linked polyubiquitination and Ser172 phosphorylation of TANK-binding kinase 1 (TBK1). Mechanically, we demonstrated that E3 ligase TNF receptor-associated factor 6 (TRAF6) participated in the ubiquitination of TBK1 in cGAS-STING signaling pathway. We showed that pA151R interacted with TRAF6 and degraded it through apoptosis pathway, leading to the disruption of TBK1 and TRAF6 interaction. Moreover, we clarified that the amino acids H102, C109, C132, and C135 in pA151R were crucial for pA151R to inhibit type I interferon production. In addition, we verified that overexpression of pA151R facilitated DNA virus Herpes simplex virus 1 (HSV-1) replication by inhibiting IFN-β production. Importantly, knockdown of pA151R inhibited ASFV replication and enhanced IFN-β production in porcine alveolar macrophages (PAMs). Our findings will help understand how ASFV escapes host antiviral immune responses and develop effective ASFV vaccines.

TRAF2 decrease promotes the TGF-β-mTORC1 signal in MAFLD-HCC through enhancing AXIN1-mediated Smad7 degradation.

According to recent research, metabolic-associated fatty liver disease (MAFLD) has emerged as an important underlying etiology of hepatocellular carcinoma (HCC). However, the molecular mechanism of MAFLD-HCC is still unclear. Tumor necrosis factor receptor-associated factor 2 (TRAF2) is the key molecule to mediate the signal of inflammatory NF-κB pathway. This study aims to investigate the potential dysregulation of TRAF2 and its biological function in MAFLD-HCC. Huh7 TRAF2-/- demonstrated increased tumor formation ability compared to huh7 TRAF2+/+ when stimulated with transforming growth factor-β (TGF-β). The decisive role of TGF-β in the development of MAFLD-HCC was confirmed through the specific depletion of TGF-β receptor II gene in the hepatocytes (Tgfbr2ΔHep) of mice. In TRAF2-/- cells treated with TGF-β, both the glycolysis rate and lipid synthesis were enhanced. We proved the signal of the mechanistic target of rapamycin complex 1 (mTORC1) could be activated in the presence of TGF-β, and was enhanced in TRAF2-/- cells. The coimmunoprecipitation (co-IP) experiments revealed that TRAF2 fortified the Smurf2-mediated ubiquitination degradation of AXIN1. Hence, TRAF2 depletion resulted in increased Smad7 degradation induced by AXIN1, thus promoting the TGF-β signal. We also discovered that PLX-4720 could bind with AXIN1 and restrained the tumor proliferation of TRAF2-/- in mice fed with high-fat diet (HFD). Our findings indicate that TRAF2 plays a significant role in the pathogenesis of MAFLD-HCC. The reduction of TRAF2 expression leads to the enhancement of the TGF-β-mTORC1 pathway by facilitating AXIN1-mediated Smad7 degradation.

A study on the association between gut microbiota, inflammation, and type 2 diabetes

Type 2 diabetes mellitus (T2DM) was reported to be associated with impaired immune response and alterations in microbial composition and function. However, the underlying mechanism remains elusive. To investigate the association among retinoic acid-inducible gene-I-like receptors (RLRs) signaling pathway, intestinal bacterial microbiome, microbial tryptophan metabolites, inflammation, and a longer course of T2DM, 14 patients with T2DM and 7 healthy controls were enrolled. 16S rRNA amplicon sequencing and untargeted metabolomics were utilized to analyze the stool samples. RNA sequencing (RNA-seq) was carried out on the peripheral blood samples. Additionally, C57BL/6J specific pathogen-free (SPF) mice were used. It was found that the longer course of T2DM could lead to a decrease in the abundance of probiotics in the intestinal microbiome. In addition, the production of microbial tryptophan derivative skatole declined as a consequence of the reduced abundance of related intestinal microbes. Furthermore, low abundances of probiotics, such as Bacteroides and Faecalibacterium, could trigger the inflammatory response by activating the RLRs signaling pathway. The increased level of the member of TNF receptor-associated factors (TRAF) family, nuclear factor kappa-B (NF-κB) activator (TANK), in the animal colon activated nuclear factor kappa B subunit 2 (NFκB2), resulting in inflammatory damage. In summary, it was revealed that the low abundances of probiotics could activate the RLR signaling pathway, which could in turn activate its downstream signaling pathway, NF-κB, highlighting a relationship among gut microbes, inflammation, and a longer course of T2DM.Key pointsHyperglycemia may suppress tryptophanase activity.The low abundance of Bacteroides combined with the decrease of Dopa decarboxylase (DDC) activity may lead to the decrease of the production of tryptophan microbial derivative skatole, and the low abundance of Bacteroides or reduced skatole may further lead to the increase of blood glucose by downregulating the expression of glucagon-like peptide-1 (GLP1).A low abundance of anti-inflammatory bacteria may induce an inflammatory response by triggering the RLR signaling pathway and then activating its downstream NF-κB signaling pathway in prolonged T2DM.

Functional analyses of TRAF6 gene in Argopecten scallops

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.

The Role of Rosavin in the Pathophysiology of Bone Metabolism.

Rosavin, a phenylpropanoid in Rhodiola rosea's rhizome, and an adaptogen, is known for enhancing the body's response to environmental stress. It significantly affects cellular metabolism in health and many diseases, particularly influencing bone tissue metabolism. In vitro, rosavin inhibits osteoclastogenesis, disrupts F-actin ring formation, and reduces the expression of osteoclastogenesis-related genes such as cathepsin K, calcitonin receptor (CTR), tumor necrosis factor receptor-associated factor 6 (TRAF6), tartrate-resistant acid phosphatase (TRAP), and matrix metallopeptidase 9 (MMP-9). It also impedes the nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), c-Fos, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) signaling pathways and blocks phosphorylation processes crucial for bone resorption. Moreover, rosavin promotes osteogenesis and osteoblast differentiation and increases mouse runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) expression. In vivo studies show its effectiveness in enhancing bone mineral density (BMD) in postmenopausal osteoporosis (PMOP) mice, restraining osteoclast maturation, and increasing the active osteoblast percentage in bone tissue. It modulates mRNA expressions by increasing eukaryotic translation elongation factor 2 (EEF2) and decreasing histone deacetylase 1 (HDAC1), thereby activating osteoprotective epigenetic mechanisms, and alters many serum markers, including decreasing cross-linked C-telopeptide of type I collagen (CTX-1), tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator for nuclear factor κ B ligand (RANKL), macrophage-colony-stimulating factor (M-CSF), and TRAP, while increasing alkaline phosphatase (ALP) and OCN. Additionally, when combined with zinc and probiotics, it reduces pro-osteoporotic matrix metallopeptidase 3 (MMP-3), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), and enhances anti-osteoporotic interleukin 10 (IL-10) and tissue inhibitor of metalloproteinase 3 (TIMP3) expressions. This paper aims to systematically review rosavin's impact on bone tissue metabolism, exploring its potential in osteoporosis prevention and treatment, and suggesting future research directions.

Involvement of tumor necrosis factor receptor-associated factor 6 (TRAF6) in NF-κB activation and antiviral immunity: Molecular and functional characterization of TRAF6 in red-spotted grouper (Epinephelus akaara)

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family of adaptor proteins involved in the signal transduction pathways of both TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. In this study, red-spotted grouper (Epinephelus akaara) TRAF6 (EaTraf6) was identified and characterized. The open reading frame of EaTraf6, 1713 bp in length, encodes a putative protein of 570 amino acids and has a predicted molecular weight and theoretical isoelectric point of 64.11 kDa and 6.07, respectively. EaTraf6 protein contains an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled-coil region (zf-TRAF), and a conserved C-terminal meprin and TRAF homology (MATH) domain. EaTraf6 shared the highest amino acid sequence identity with its ortholog from Epinephelus coioides, and phylogenetic analysis showed all fish TRAF6s clustered together and apart from other species. qRT-PCR results revealed that EaTraf6 was ubiquitously expressed in all examined tissues, with the highest level detected in the blood. In the immune challenge, EaTraf6 exhibited modulated mRNA expression levels in the blood and spleen. The subcellular localization analysis revealed that the EaTraf6 protein was predominantly present in the cytoplasm; however, it could translocate into the nucleus following poly (I:C) stimulation. The antiviral function of EaTraf6 was confirmed by analyzing the expression of host antiviral genes and viral genomic RNA during viral hemorrhagic septicemia virus infection. Additionally, luciferase reporter assay results indicated that EaTraf6 is involved in the activation of the NF-κB signaling pathway upon poly (I:C) stimulation. Finally, the effect of EaTraf6 on cytokine gene expression and its role in regulating macrophage M1 polarization were demonstrated. Collectively, these findings suggest that EaTraf6 is a crucial immune-related gene that significantly contributes to antiviral functions and regulation of NF-κB activity in the red-spotted grouper.

MicroRNA-146a gene transfer ameliorates senescence and senescence-associated secretory phenotypes in tendinopathic tenocytes.

Tendinopathy is influenced by multiple factors, including chronic inflammation and aging. Senescent cells exhibit characteristics such as the secretion of matrix-degrading enzymes and pro-inflammatory cytokines, collectively known as senescence-associated secretory phenotypes (SASPs). Many of these SASP cytokines and enzymes are implicated in the pathogenesis of tendinopathy. MicroRNA-146a (miR-146a) blocks senescence by targeting interleukin-1β (IL-1β) receptor-associated kinase 4 (IRAK-4) and TNF receptor-associated factor 6 (TRAF6), thus inhibiting NF-κB activity. The aims of this study were to (1) investigate miR-146a expression in tendinopathic tendons and (2) evaluate the role of miR-146a in countering senescence and SASPs in tendinopathic tenocytes. MiR-146a expression was assessed in human long head biceps (LHB) and rat tendinopathic tendons by in situ hybridization. MiR-146a over-expression in rat primary tendinopathic tenocytes was achieved by lentiviral vector-mediated precursor miR-146a transfer (LVmiR-146a). Expression of various senescence-related markers was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoblotting and immunofluorescence. MiR-146a expression showed a negative correlation with the severity of tendinopathy in human and rat tendinopathic tendons (p<0.001). Tendinopathic tenocyte transfectants overexpressing miR-146a exhibited downregulation of various senescence and SASP markers, as well as the target molecules IRAK-4 and TRAF6, and the inflammatory mediator phospho-NF-κB. Additionally, these cells showed enhanced nuclear staining of high mobility group box 1 (HMGB1) compared to LVmiR-scramble-transduced controls in response to IL-1β stimulation. We demonstrate that miR-146a expression is negatively correlated with the progression of tendinopathy. Moreover, its overexpression protects tendinopathic tenocytes from SASPs and senescence through the IRAK-4/TRAF6/NF-kB pathway.

Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction.

Potential involvement of circulating exosomal miRNA-146a in disease activity and TRAF6 gene expression in juvenile proliferative lupus nephritis

BackgroundJuvenile SLE (JSLE) is a complex autoimmune disorder that predominantly affects children and adolescents with several unique challenges, and microRNA-146a (miRNA-146a) might be an interesting anti-inflammatory molecule. Because exosomes in...

EFHD2 cooperates with E3 ubiquitin ligase Smurf1 to facilitate virus infection by promoting the degradation of TRAF6 in teleost fish.

The ubiquitin-proteasome system is one of the most important protein stability regulation systems. It can precisely regulate host immune responses by targeting signaling proteins. TRAF6 is a crucial E3 ubiquitin ligase in host antiviral signaling pathway. Here, we discovered that EF-hand domain-containing protein D2 (EFHD2) collaborated with the E3 ubiquitin ligase Smurf1 to potentiate the degradation of TRAF6, hence facilitating RNA virus Siniperca chuatsi rhabdovirus infection. The mechanism analysis revealed that EFHD2 interacted with Smurf1 and enhanced its protein stability by impairing K48-linked polyubiquitination of Smurf1, thereby promoting Smurf1-catalyzed degradation of TRAF6. This study initially demonstrated a novel mechanism by which viruses utilize host EFHD2 to achieve immune escape and provided a new perspective on the exploration of mammalian innate immunity.IMPORTANCEViruses induce host cells to activate several antiviral signaling pathways. TNF receptor-associated factor 6 (TRAF6) plays an essential role in these pathways. Numerous studies have been done on the mechanisms of TRAF6-mediated resistance to viral invasion. However, little is known about the strategies that viruses employ to antagonize TRAF6-mediated antiviral signaling pathway. Here, we discovered that EFHD2 functions as a host factor to promote viral replication. Mechanistically, EFHD2 potentiates Smurf1 to catalyze the ubiquitin-proteasomal degradation of TRAF6 by promoting the deubiquitination and stability of Smurf1, which in turn inhibits the production of proinflammatory cytokines and interferons. Our study also provides a new perspective on mammalian resistance to viral invasion.

MicroRNA-194 regulates parasitic load and IL-1β-dependent nitric oxide production in the peripheral blood mononuclear cells of dogs with leishmaniasis.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-β in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1β blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1β and NO in PBMCs from diseased dogs, and blockade of IL-1β following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1β. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.

ZC3H12A inhibits tumor growth and metastasis of breast cancer under hypoxic condition via the inactivation of IL-17 signaling pathway

ABSTRACT Hypoxia is a major contributor to tumor microenvironment (TME) and metastasis in most solid tumors. We seek to screen hypoxia-related genes affecting metastasis in breast cancer and to reveal relative potential regulatory pathway. Based on gene expression profiling of GSE17188 dataset, differential expressed genes (DEGs) were identified between highly metastatic breast cancer cells under hypoxia and samples under normoxia. The protein–protein interaction (PPI) network was utilized to determine hub genes. The gene expression profiling interactive analysis database (GEPIA2) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were employed to quantify hub genes. Moreover, overexpression of zinc finger CCCH-type containing 12A (ZC3H12A) was performed both in breast cancer cells and xenograft mouse model to determine the role of ZC3H12A. We identified 134 DEGs between hypoxic and normoxic samples. Based on PPI analysis, 5 hub genes interleukin (IL)-6, GALN (GAL), CD22 molecule (CD22), ZC3H12A and TNF receptor associated factor 1 (TRAF1) were determined; the expression levels of TRAF1, IL-6, ZC3H12A and GAL were remarkably downregulated while CD22 was upregulated in breast cancer cells. Besides, patients with higher expression of ZC3H12A had favorable prognosis. Overexpression of ZC3H12A could inhibit metastasis and tumor growth of breast cancer; overexpression of ZC3H12A downregulated the expression of IL-17 signaling pathway-related proteins such as IL-17 receptor A (IL-17RA), IL-17A and nuclear factor κB activator 1 (Act1). This study reveals ZC3H12A and IL-17 signaling pathway as potential therapeutic targets for hypoxic breast cancer.

MicroRNA-146a-loaded magnesium silicate nanospheres promote bone regeneration in an inflammatory microenvironment

Reconstruction of irregular oral-maxillofacial bone defects with an inflammatory microenvironment remains a challenge, as chronic local inflammation can largely impair bone healing. Here, we used magnesium silicate nanospheres (MSNs) to load microRNA-146a-5p (miR-146a) to fabricate a nanobiomaterial, MSN+miR-146a, which showed synergistic promoting effects on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). In addition, miR-146a exhibited an anti-inflammatory effect on mouse bone marrow-derived macrophages (BMMs) under lipopolysaccharide (LPS) stimulation by inhibiting the NF-κB pathway via targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), and MSNs could simultaneously promote M2 polarization of BMMs. MiR-146a was also found to inhibit osteoclast formation. Finally, the dual osteogenic-promoting and immunoregulatory effects of MSN+miR-146a were further validated in a stimulated infected mouse mandibular bone defect model via delivery by a photocuring hydrogel. Collectively, the MSN+miR-146a complex revealed good potential in treating inflammatory irregular oral-maxillofacial bone defects.

Hybrid nutraceutical of 2-ketoglutaric acid in improving inflammatory bowel disease: Role of prebiotics and TAK1 inhibitor

The main cause of inflammatory bowel disease (IBD) is abnormal intestinal permeability due to the disruption of the tight junction of the intestinal barrier through a pathogen-mediated inflammatory mechanism and an imbalance of the gut microbiota. This study aimed to evaluate whether 2-ketoglutaric acid alleviated permeability dysfunction with tight junction localization, activated the transforming growth factor beta-activated kinase 1 (TAK1) inflammation pathway, and regulated the homeostasis of the intestinal microbiome in vitro and in vivo IBD model. Our findings revealed that 2-ketoglutaric acid significantly suppressed abnormal intestinal permeability, delocalization of tight junction proteins from the intestinal cell, expression of inflammatory cytokines, such as TNF-α, both in vitro and in vivo. 2-Ketoglutaric acid was found to directly bind to TAK1 and inhibit the TNF receptor-associated factor 6 (TRAF6)–TAK1 interaction, which is related to the activation of nuclear factor kappa B (NF-κB) pathways, thereby regulating the expression of mitogen-activated protein kinase. Dietary 2-ketoglutaric acid also alleviated gut microbiota dysbiosis and IBD symptoms, as demonstrated by improvements in the intestine length and the abundance of Ligilactobacillus, Coriobacteriaceae_UCG_002, and Ruminococcaceae_unclassified in mice with colitis. This study indicated that 2-ketoglutaric acid binds to TAK1 for activity inhibition which is related to the NF-κB pathway and alleviates abnormal permeability by regulating tight junction localization and gut microbiome homeostasis. Therefore, 2-ketoglutaric acid is an effective nutraceutical agent and prebiotic for the treatment of IBD.