RÉSUMÉ
Invasion and migration are the key hallmarks of cancer, and aggressive growth is a major factor contributing to treatment failure and poor prognosis in glioblastoma. Protein arginine methyltransferase 6 (PRMT6), as an epigenetic regulator, has been confirmed to promote the malignant proliferation of glioblastoma cells in previous studies. However, the effects of PRMT6 on glioblastoma cell invasion and migration and its underlying mechanisms remain elusive. Here, we report that PRMT6 functions as a driver element for tumor cell invasion and migration in glioblastoma. Bioinformatics analysis and glioma sample detection results demonstrated that PRMT6 is highly expressed in mesenchymal subtype or invasive gliomas, and is significantly negatively correlated with their prognosis. Inhibition of PRMT6 (using PRMT6 shRNA or inhibitor EPZ020411) reduces glioblastoma cell invasion and migration in vitro, whereas overexpression of PRMT6 produces opposite effects. Then, we identified that PRMT6 maintains the protein stability of EZH2 by inhibiting the degradation of EZH2 protein, thereby mediating the invasion and migration of glioblastoma cells. Further mechanistic investigations found that PRMT6 inhibits the transcription of TRAF6 by activating the histone methylation mark (H3R2me2a), and reducing the interaction between TRAF6 and EZH2 to enhance the protein stability of EZH2 in glioblastoma cells. Xenograft tumor assay and HE staining results showed that the expression of PRMT6 could promote the invasion of glioblastoma cells in vivo, the immunohistochemical staining results of mouse brain tissue tumor sections also confirmed the regulatory relationship between PRMT6, TRAF6, and EZH2. Our findings illustrate that PRMT6 suppresses TRAF6 transcription via H3R2me2a to enhance the protein stability of EZH2 to facilitate glioblastoma cell invasion and migration. Blocking the PRMT6-TRAF6-EZH2 axis is a promising strategy for inhibiting glioblastoma cell invasion and migration.
Sujet(s)
Mouvement cellulaire , Protéine-2 homologue de l'activateur de Zeste , Glioblastome , Invasion tumorale , Stabilité protéique , Protein-arginine N-methyltransferases , Ubiquitination , Humains , Glioblastome/anatomopathologie , Glioblastome/métabolisme , Glioblastome/génétique , Protéine-2 homologue de l'activateur de Zeste/métabolisme , Protein-arginine N-methyltransferases/métabolisme , Protein-arginine N-methyltransferases/génétique , Animaux , Lignée cellulaire tumorale , Souris , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , Souris nude , Tumeurs du cerveau/anatomopathologie , Tumeurs du cerveau/métabolisme , Tumeurs du cerveau/génétique , Régulation de l'expression des gènes tumoraux , Mâle , Protéolyse , Femelle , Souris de lignée BALB C , Protéines nucléairesRÉSUMÉ
INTRODUCTION: Systemic lupus erythematosus (SLE) is a diverse autoimmune disease that arises from a combination of complex genetic factors and environmental influences. While circRNAs and miRNAs have recently been identified as promising biomarkers for disease diagnosis, their specific expression patterns, and clinical implications in SLE are not yet fully understood. AIM OF THE WORK: The aim of the present study was to determine the role of a panel of noncoding-RNAs specifically circRNAs (circ-TubD1, circ-CDC27, and circ-Med14), along with miRNA (rno-miR-146a-5p) and mRNA (TRAF6), as novel minimally invasive diagnostic biomarkers for experimentally induced SLE. Additionally, the study involved an insilico bioinformatics analysis to explore potential pathways involved in the pathogenesis of SLE, aiming to enhance our understanding of the disease, enable early diagnosis, and facilitate improved treatment strategies. MATERIALS AND METHODS: SLE was induced in rats using single IP injection of incomplete Freund's adjuvant (IFA). The Induction was confirmed by assessing the ANA and anti-ds DNA levels using ELSA technique. qPCR analysis was conducted to assess the expression of selected RNAs in sera collected from a group of 10 rats with induced SLE and a control group of 10 rats. In addition, bioinformatics and functional analysis were used to construct a circRNA-miRNA-mRNA network and to determine the potential function of these differentially expressed circRNAs. RESULTS: SLE rats demonstrated significantly higher expression levels of circ-CDC27, circ-Med14, and rno-miR-146a-5p as well as TRAF6, with lower expression level of circ-TubD1 in sera of SLE rats relative to controls. ROC curve analysis indicated that all the selected non-coding RNAs could serve as potential early diagnostic markers for SLE. In addition, the expression level of circ-TubD1 was negatively correlated with rno-miR-146a-5p, however, rno-miR-146a-5p was positively correlated with TRAF6. Bioinformatic analysis revealed the incorporation of the circRNAs targeted genes in various immune system and neurodegeneration pathways. CONCLUSIONS: Therefore, circRNAs; circ-TubD1, circ-CDC27, and circ-Med14, in addition to the miRNA (rno-miR-146a-5p) and mRNA (TRAF6) may be involved in the development of SLE and may have promising roles for future diagnosis and targeted therapy.
Sujet(s)
Marqueurs biologiques , Modèles animaux de maladie humaine , Lupus érythémateux disséminé , microARN , ARN circulaire , Animaux , Lupus érythémateux disséminé/génétique , Lupus érythémateux disséminé/sang , Lupus érythémateux disséminé/diagnostic , ARN circulaire/génétique , ARN circulaire/sang , Marqueurs biologiques/sang , Rats , microARN/génétique , microARN/sang , Facteur-6 associé aux récepteurs de TNF/génétique , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/sang , Biologie informatique , Femelle , ARN messager/génétique , ARN messager/métabolisme , ARN messager/sang , MâleRÉSUMÉ
Chronic pain has emerged as a significant public health issue, seriously affecting patients' quality of life and psychological well-being, with a lack of effective pharmacological treatments. Numerous studies have indicated that macrophages play a crucial role in inflammatory pain, and targeting neuro-immune interactions for drug development may represent a promising direction for pain management. Chilobrachys jingzhao (C. jingzhao) is used as a folk medicine of the Li nationality with the efficacy of eliminating swelling, detoxicating, and relieving pain, and the related products are widely used in the market. However, the chemical constituents of C. jingzhao have not been reported, and the pharmacodynamic substance and the precise functional mechanism are unrevealed. Here we isolated a cyclic dipeptide, cyclo(L-Pro-L-Trp) (CPT) from C. jingzhao for the first time. CPT remarkably alleviated formalin-induced inflammatory pain and significantly inhibited inflammatory responses. In vivo, CPT attenuated neutrophil infiltration and plantar tissue edema and suppressed the mRNA expression of pro-inflammatory molecules. In vitro, CPT suppressed inflammation triggered by lipopolysaccharide (LPS) in both RAW 264.7 and iBMDM cells, reducing expressions of inducible nitric oxide synthase (iNOS), superoxide, and pro-inflammatory molecules. A mechanistic study revealed that CPT exerted an anti-inflammatory activity by blocking the mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways, as well as alleviating the ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6). Our results elucidated the pharmacodynamic material basis of C. jingzhao, and CPT can be a promising lead for alleviating inflammation and inflammatory pain.
Sujet(s)
Anti-inflammatoires , Formaldéhyde , Inflammation , Facteur de transcription NF-kappa B , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Animaux , Facteur de transcription NF-kappa B/métabolisme , Souris , Facteur-6 associé aux récepteurs de TNF/métabolisme , Anti-inflammatoires/usage thérapeutique , Anti-inflammatoires/pharmacologie , Mâle , Transduction du signal/effets des médicaments et des substances chimiques , Inflammation/traitement médicamenteux , Cellules RAW 264.7 , Peptides cycliques/pharmacologie , Peptides cycliques/usage thérapeutique , Douleur/traitement médicamenteux , Douleur/induit chimiquement , Analgésiques/usage thérapeutique , Analgésiques/pharmacologie , Humains , Oedème/traitement médicamenteux , Oedème/induit chimiquement , Oedème/immunologie , Mitogen-Activated Protein Kinases/métabolisme , Macrophages/effets des médicaments et des substances chimiques , Macrophages/immunologieRÉSUMÉ
BACKGROUND: The neuropathic pain with complex networks of neuroinflammatory activation severely limits clinical therapeutic research. TNF receptor-associated factor 6 (TRAF6) is associated with multiple inflammatory diseases. However, there remains confusion about the effects and mechanisms of TRAF6 in neuropathic pain. METHODS: A chronic constriction injury (CCI) model was developed to simulate neuralgia in vivo. We overexpressed or knocked down TRAF6 in CCI mice, respectively. Activation of microglia by TRAF6, the inflammatory response, and disease progression were inspected using WB, qRT-PCR, immunofluorescence, flow cytometry, and ELISA assays. Moreover, the mechanism of M1/M2 polarization activation of microglia by TRAF6 was elaborated in BV-2 cells. RESULTS: TRAF6 was enhanced in the spinal neurons and microglia of the CCI mice model compared with the sham operation group.. Down-regulation of TRAF6 rescued the expression of Iba-1. In response to mechanical and thermal stimulation, PWT and PWL were improved after the knockdown of TRAF6. Decreased levels of pro-inflammatory factors were observed in TRAF6 knockdown groups. Meanwhile, increased microglial M1 markers induced by CCI were limited in mice with TRAF6 knockdown. In addition, TRAF6 overexpression has the precise opposite effect on CCI mice or microglia polarization. We also identifed that TRAF6 activated the c-JUN/NF-kB pathway signaling; the inhibitor of c-JUN/NF-kB could effectively alleviate the neuropathic pain induced by upregulated TRAF6 in the CCI mice model. CONCLUSION: In summary, this study indicated that TRAF6 was concerned with neuropathic pain, and targeting the TRAF6/c-JUN/NF-kB pathway may be a prospective target for treating neuropathic pain.
Sujet(s)
Microglie , Facteur de transcription NF-kappa B , Névralgie , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Animaux , Mâle , Souris , Lignée cellulaire , Polarité de la cellule , Modèles animaux de maladie humaine , Souris de lignée C57BL , Microglie/métabolisme , Névralgie/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Protéines proto-oncogènes c-jun/métabolisme , Moelle spinale/métabolisme , Moelle spinale/anatomopathologie , Facteur-6 associé aux récepteurs de TNF/métabolismeRÉSUMÉ
The pathogenesis of membranous nephropathy (MN) involves podocyte injury that is attributed to inflammatory responses induced by local immune deposits. Astragaloside IV (AS-IV) is known for its robust anti-inflammatory properties. Here, we investigated the effects of AS-IV on passive Heymann nephritis (PHN) rats and TNF-α-induced podocytes to determine the underlying molecular mechanisms of MN. Serum biochemical parameters, 24-h urine protein excretion and renal histopathology were evaluated in PHN and control rats. The expression of tumor necrosis factor receptor associated factor 6 (TRAF6), the phosphorylation of nuclear factor kappa B (p-NF-κB), the expression of associated proinflammatory cytokines (TNF-α, IL-6 and IL-1ß) and the ubiquitination of TRAF6 were measured in PHN rats and TNF-α-induced podocytes. We detected a marked increase in mRNA expression of TNF-α, IL-6 and IL-1ß and in the protein abundance of p-NF-κB and TRAF6 within the renal tissues of PHN rats and TNF-α-induced podocytes. Conversely, there was a reduction in the K48-linked ubiquitination of TRAF6. Additionally, AS-IV was effective in ameliorating serum creatinine, proteinuria, and renal histopathology in PHN rats. This effect was concomitant with the suppression of NF-κB pathway activation and decreased expression of TNF-α, IL-6, IL-1ß and TRAF6. AS-IV decreased TRAF6 levels by promoting K48-linked ubiquitin conjugation to TRAF6, which triggered ubiquitin-mediated degradation. In summary, AS-IV averted renal impairment in PHN rats and TNF-α-induced podocytes, likely by modulating the inflammatory response through the TRAF6/NF-κB axis. Targeting TRAF6 holds therapeutic promise for managing MN.
Sujet(s)
Glomérulonéphrite extra-membraneuse , Facteur de transcription NF-kappa B , Podocytes , Saponines , Facteur-6 associé aux récepteurs de TNF , Triterpènes , Animaux , Podocytes/effets des médicaments et des substances chimiques , Podocytes/anatomopathologie , Podocytes/métabolisme , Rats , Triterpènes/pharmacologie , Triterpènes/usage thérapeutique , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Saponines/pharmacologie , Saponines/usage thérapeutique , Mâle , Glomérulonéphrite extra-membraneuse/traitement médicamenteux , Glomérulonéphrite extra-membraneuse/anatomopathologie , Glomérulonéphrite extra-membraneuse/métabolisme , Rat Sprague-Dawley , Transduction du signal/effets des médicaments et des substances chimiques , Facteur de nécrose tumorale alpha/métabolisme , Modèles animaux de maladie humaine , Ubiquitination/effets des médicaments et des substances chimiques , Rein/anatomopathologie , Rein/effets des médicaments et des substances chimiques , Rein/métabolisme , Cytokines/métabolisme , Anti-inflammatoires/pharmacologieRÉSUMÉ
Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.
Sujet(s)
Facteur-3 de régulation d'interféron , Interférons , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Humains , Interférons/métabolisme , Cellules HEK293 , Facteur-3 de régulation d'interféron/métabolisme , Facteur-3 de régulation d'interféron/génétique , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , I-kappa B Kinase/métabolisme , I-kappa B Kinase/génétique , Domaines protéiques , Animaux , Protéines et peptides de signalisation intracellulaire/métabolisme , Protéines et peptides de signalisation intracellulaire/génétique , Motifs d'acides aminés , Mitogen-Activated Protein Kinases/métabolismeRÉSUMÉ
AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.
Sujet(s)
Noyau de la cellule , Insuline , Protéines proto-oncogènes c-akt , Facteur-6 associé aux récepteurs de TNF , Ubiquitination , bêta-Arrestine 2 , Protéines proto-oncogènes c-akt/métabolisme , Ubiquitination/physiologie , Ubiquitination/effets des médicaments et des substances chimiques , Insuline/métabolisme , Insuline/pharmacologie , Animaux , Facteur-6 associé aux récepteurs de TNF/métabolisme , Noyau de la cellule/métabolisme , Souris , Humains , bêta-Arrestine 2/métabolisme , bêta-Arrestine 2/génétique , Cellules HEK293RÉSUMÉ
OBJECTIVES: To investigate the possible roles of Interleukin 17A (IL-17A) and IL-17A neutralizing antibodies (IL-17Ab) in glaucoma and the potential mechanisms. METHODS: The two glaucoma animal models, chronic ocular hypertension (COH) and N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage, were established and treated with intravitreal injection of IL-17A or IL-17Ab. Intraocular pressure (IOP) was measured by a rebound tonometer. The retina and RGC injury were evaluated by HE staining, TUNLE assay and Brn3a immunofluorescence staining. The frequency of IL-17A+CD4+T cells in peripheral blood was detected by flow cytometry. The expression of glial fibrillary acidic protein (GFAP) was detected by immunofluorescence staining, Western Blot and qPCR in retina. The RNA and protein expression of Act1/TRAF6/NF-κB were detected by Western Blot and qPCR in retina. RESULTS: The expression of IL-17A increased in glaucoma models. After intravitreal injection of IL-17A, in the retina, the number of RGCs decreased, the apoptosis of RGCs increased, the Müller cell gliosis was more obvious. In addition, peripheral inflammation aggravated. Whereas the intravitreal injection of IL-17Ab alleviated the relevant manifestations and peripheral inflammation, reduced the gliosis of Müller cells. In the COH model, IOP increased after the injection of IL-17A, while the intravitreal injection of IL-17Ab led to a decrease in IOP. Furthermore, IL-17A promotes the apoptosis of RGCs by binding to IL-17A receptor, activating Act1/TRAF6/NF-κB pathways. CONCLUSION: IL-17A plays a role in and aggravates RGC damage in glaucoma. IL-17Ab can neutralize the pro-inflammatory effect of IL-17A and have a protective function in glaucoma. These findings reveal the importance of IL-17A in the pathogenesis of glaucoma, which will shed light on a novel direction for the prevention and treatment of glaucoma, and also provide a reference for further research on other retinal diseases.
Sujet(s)
Glaucome , Interleukine-17 , Facteur de transcription NF-kappa B , Facteur-6 associé aux récepteurs de TNF , Interleukine-17/métabolisme , Animaux , Glaucome/métabolisme , Glaucome/anatomopathologie , Facteur de transcription NF-kappa B/métabolisme , Facteur-6 associé aux récepteurs de TNF/métabolisme , Inflammation/métabolisme , Inflammation/anatomopathologie , Souris , Souris de lignée C57BL , Transduction du signal/physiologie , Transduction du signal/effets des médicaments et des substances chimiques , Mâle , Cellules ganglionnaires rétiniennes/métabolisme , Cellules ganglionnaires rétiniennes/anatomopathologie , Cellules ganglionnaires rétiniennes/effets des médicaments et des substances chimiques , Rats , Protéines adaptatrices de la transduction du signal/métabolisme , Pression intraoculaire/physiologie , Pression intraoculaire/effets des médicaments et des substances chimiques , Rat Sprague-DawleyRÉSUMÉ
IL-1R-associated kinases (IRAKs) are signal transducers of the TLR/IL-1R-MyD88-TRAF6 pathways. Vertebrates possess two IRAK lineages, IRAK1/2/3 and IRAK4. In mammals, IRAK4/IRAK1 and IRAK4/IRAK2 are pathway enhancers, whereas IRAK3 is a repressor. However, in bony fish, IRAK2 is absent, and it remains elusive how fish IRAK1/3/4 functionally differ from their mammalian counterparts. In this study, we explored this using the zebrafish model. First, we showed that in human 293T cells, zebrafish IRAK1 and IRAK4 were components of the Myddosome (MyD88-IRAK4-IRAK1) complex, with IRAK1 serving as a potent pathway enhancer. Then, we discovered two zebrafish IRAK3 variants: one (IRAK3a) contains an N-terminal Death domain, a middle pseudokinase domain, and a C-terminal TRAF6-binding domain, whereas the other (IRAK3b) lost both the kinase and TRAF6-binding domains. This truncation of IRAK3 variants could be a conserved phenomenon in fish, because it is also observed in trout and grass carp. We proceeded to show that zebrafish IRAK3a acts as a pathway enhancer by binding with MyD88 and TRAF6, but its activity is milder than IRAK1, possibly because it has no kinase activity. Zebrafish IRAK3b, however, plays a sheer negative role, apparently because of its lack of kinase and TRAF6-binding domains. Moreover, zebrafish IRAK3a/3b inhibit the activity of IRAK1/4, not by interacting with IRAK1/4 but possibly by competing for MyD88 and TRAF6. Finally, we have verified the essential activities of zebrafish IRAK1/3a/3b/4 in zebrafish cells and embryos. In summary, to our knowledge, our findings provide new insights into the molecular functions of fish IRAKs and the evolution of the IRAK functional modes in vertebrates.
Sujet(s)
Interleukin-1 Receptor-Associated Kinases , Facteur de différenciation myéloïde-88 , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Protéines de poisson-zèbre , Danio zébré , Animaux , Interleukin-1 Receptor-Associated Kinases/métabolisme , Interleukin-1 Receptor-Associated Kinases/génétique , Facteur de différenciation myéloïde-88/métabolisme , Facteur de différenciation myéloïde-88/génétique , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , Humains , Transduction du signal/immunologie , Cellules HEK293 , Protéines de poisson-zèbre/métabolisme , Protéines de poisson-zèbre/génétiqueRÉSUMÉ
Colorectal cancer (CRC) remains a major global cause of cancer-related mortality, lacking effective biomarkers and therapeutic targets. Revealing the critical pathogenic factors of CRC and the underlying mechanisms would offer potential therapeutic strategies for clinical application. G protein signaling (RGS) protein family modulators play essential role within regulating downstream signaling of GPCR receptors, with function in cancers unclear. Our study focused on the expression patterns of RGS proteins in CRC, identifying Regulator of G protein signaling 16 (RGS16) as a prospective diagnostic and therapeutic target. Analyzing 899 CRC tissues revealed elevated RGS16 levels, correlating with clinicopathological features and CRC prognosis by immunohistochemistry (IHC) combined with microarray. We confirmed the elevated RGS16 protein level in CRC, and found that patients with RGS16-high tumors exhibited decreased disease-specific survival (DSS) and disease-free survival (DFS) compared to those with low RGS16 expression. Functional assays demonstrated that RGS16 promoted the CRC progression, knockdown of RGS16 led to significantly increased apoptosis rates of CRC in vitro and in vivo. Notably, we also confirmed these phenotypes of RGS16 in organoids originated from resected primary human CRC tissues. Mechanistically, RGS16 restrained JNK/P38-mediated apoptosis in CRC cells through disrupting the recruitment of TAB2/TAK1 to TRAF6. This study provides insights into addressing the challenges posed by CRC, offering avenues for clinical translation.
Sujet(s)
Apoptose , Tumeurs colorectales , MAP Kinase Kinase Kinases , Protéines RGS , Animaux , Femelle , Humains , Mâle , Souris , Adulte d'âge moyen , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/génétique , Lignée cellulaire tumorale , Tumeurs colorectales/anatomopathologie , Tumeurs colorectales/métabolisme , Tumeurs colorectales/génétique , Protéines et peptides de signalisation intracellulaire , MAP Kinase Kinase Kinases/métabolisme , MAP Kinase Kinase Kinases/génétique , Système de signalisation des MAP kinases , Souris nude , p38 Mitogen-Activated Protein Kinases/métabolisme , Protéines RGS/métabolisme , Protéines RGS/génétique , Transduction du signal , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétiqueRÉSUMÉ
CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IκB kinase (IKK)/NF-κB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other non-hematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates.
Sujet(s)
Protéine-1 de translocation de lymphome du tissu lymphoïde associé aux muqueuses , Transduction du signal , Protéine-1 de translocation de lymphome du tissu lymphoïde associé aux muqueuses/métabolisme , Protéine-1 de translocation de lymphome du tissu lymphoïde associé aux muqueuses/génétique , Humains , Animaux , Spécificité du substrat , Protéine-10 du lymphome LLC à cellules B/métabolisme , Protéine-10 du lymphome LLC à cellules B/génétique , Protéines adaptatrices de signalisation CARD/métabolisme , Protéines adaptatrices de signalisation CARD/génétique , Protéolyse , Facteur-6 associé aux récepteurs de TNF/métabolismeRÉSUMÉ
Introduction: Pathological changes in the articular cartilage (AC) and synovium are major manifestations of osteoarthritis (OA) and are strongly associated with pain and functional limitations. Exosome-derived microRNAs (miRNAs) are crucial regulatory factors in intercellular communication and can influence the progression of OA by participating in the degradation of chondrocytes and the phenotypic transformation in the polarization of synovial macrophages. However, the specific relationships and pathways of action of exosomal miRNAs in the pathological progression of OA in both cartilage and synovium remain unclear. Methods: This study evaluates the effects of fibroblast-like synoviocyte (FLS)-derived exosomes (FLS-Exos), influenced by miR-146a, on AC degradation and synovial macrophage polarization. We investigated the targeted relationship between miR-146a and TRAF6, both in vivo and in vitro, along with the involvement of the NF-κB signaling pathway. Results: The expression of miR-146a in the synovial exosomes of OA rats was significantly higher than in healthy rats. In vitro, the upregulation of miR-146a reduced chondrocyte apoptosis, whereas its downregulation had the opposite effect. In vivo, exosomes derived from miR-146a-overexpressing FLSs (miR-146a-FLS-Exos) reduced AC injury and chondrocyte apoptosis in OA. Furthermore, synovial proliferation was reduced, and the polarization of synovial macrophages shifted from M1 to M2. Mechanistically, the expression of TRAF6 was inhibited by targeting miR-146a, thereby modulating the Toll-like receptor 4/TRAF6/NF-κB pathway in the innate immune response. Discussion: These findings suggest that miR-146a, mediated through FLS-Exos, may alleviate OA progression by modulating cartilage degradation and macrophage polarization, implicating the NF-κB pathway in the innate immune response. These insights highlight the therapeutic potential of miR-146a as a protective agent in OA, underscoring the importance of exosomal miRNAs in the pathogenesis and potential treatment of the disease.
Sujet(s)
Exosomes , Macrophages , microARN , Arthrose , Cellules synoviales , Facteur-6 associé aux récepteurs de TNF , microARN/génétique , Animaux , Exosomes/métabolisme , Arthrose/métabolisme , Arthrose/anatomopathologie , Arthrose/immunologie , Rats , Macrophages/immunologie , Macrophages/métabolisme , Cellules synoviales/métabolisme , Cellules synoviales/anatomopathologie , Mâle , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , Cartilage articulaire/métabolisme , Cartilage articulaire/anatomopathologie , Chondrocytes/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Transduction du signal , Rat Sprague-Dawley , Fibroblastes/métabolisme , Membrane synoviale/métabolisme , Membrane synoviale/anatomopathologie , Membrane synoviale/immunologie , Cellules cultivées , Apoptose , Récepteur de type Toll-4/métabolisme , Récepteur de type Toll-4/génétique , Activation des macrophagesRÉSUMÉ
TRAF6 is an E3 ubiquitin ligase that plays a crucial role in cell signaling. It is known that MMP is involved in tumor metastasis, and TRAF6 induces MMP-9 expression by binding to BSG. However, inhibiting TRAF6's ubiquitinase activity without disrupting the RING domain is a challenge that requires further research. To address this, we conducted computer-based drug screening to identify potential TRAF6 inhibitors. Using a ligand-receptor complex pharmacophore based on the inhibitor EGCG, known for its anti-tumor properties, we screened 52,765 marine compounds. After the molecular docking of 405 molecules with TRAF6, six compounds were selected for further analysis. By replacing fragments of non-binding compounds and conducting second docking, we identified two promising molecules, CMNPD9212-16 and CMNPD12791-8, with strong binding activity and favorable pharmacological properties. ADME and toxicity predictions confirmed their potential as TRAF6 inhibitors. Molecular dynamics simulations showed that CMNPD12791-8 maintained a stable structure with the target protein, comparable to EGCG. Therefore, CMNPD12791-8 holds promise as a potential inhibitor of TRAF6 for inhibiting tumor growth and metastasis.
Sujet(s)
Simulation de docking moléculaire , Simulation de dynamique moléculaire , Facteur-6 associé aux récepteurs de TNF , Humains , Facteur-6 associé aux récepteurs de TNF/antagonistes et inhibiteurs , Facteur-6 associé aux récepteurs de TNF/métabolisme , Organismes aquatiques , Animaux , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Évaluation préclinique de médicament/méthodes , Catéchine/analogues et dérivés , Catéchine/pharmacologie , Catéchine/composition chimique , Pharmacophore , Protéines et peptides de signalisation intracellulaireRÉSUMÉ
Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κß ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and YwhazKO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.
Sujet(s)
Protéines 14-3-3 , Ostéoclastes , Ligand de RANK , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Animaux , Souris , Protéines 14-3-3/métabolisme , Protéines 14-3-3/génétique , Résorption osseuse/métabolisme , Résorption osseuse/génétique , Résorption osseuse/anatomopathologie , Macrophages/métabolisme , Souris knockout , Ostéoclastes/métabolisme , Ostéoclastes/cytologie , Stabilité protéique , Ligand de RANK/métabolisme , Ligand de RANK/génétique , Cellules RAW 264.7 , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , UbiquitinationRÉSUMÉ
Macrophage-orchestrated inflammation contributes to multiple diseases including sepsis. However, the underlying mechanisms remain to be defined clearly. Here, we show that macrophage TP53-induced glycolysis and apoptosis regulator (TIGAR) is up-regulated in murine sepsis models. When myeloid Tigar is ablated, sepsis induced by either lipopolysaccharide treatment or cecal ligation puncture in male mice is attenuated via inflammation inhibition. Mechanistic characterizations indicate that TIGAR directly binds to transforming growth factor ß-activated kinase (TAK1) and promotes tumor necrosis factor receptor-associated factor 6-mediated ubiquitination and auto-phosphorylation of TAK1, in which residues 152-161 of TIGAR constitute crucial motif independent of its phosphatase activity. Interference with the binding of TIGAR to TAK1 by 5Z-7-oxozeaenol exhibits therapeutic effects in male murine model of sepsis. These findings demonstrate a non-canonical function of macrophage TIGAR in promoting inflammation, and confer a potential therapeutic target for sepsis by disruption of TIGAR-TAK1 interaction.
Sujet(s)
Protéines régulatrices de l'apoptose , Modèles animaux de maladie humaine , Lipopolysaccharides , MAP Kinase Kinase Kinases , Macrophages , Sepsie , Animaux , Sepsie/immunologie , Sepsie/traitement médicamenteux , Sepsie/métabolisme , MAP Kinase Kinase Kinases/métabolisme , MAP Kinase Kinase Kinases/génétique , Mâle , Souris , Macrophages/métabolisme , Macrophages/immunologie , Macrophages/effets des médicaments et des substances chimiques , Protéines régulatrices de l'apoptose/métabolisme , Protéines régulatrices de l'apoptose/génétique , Souris de lignée C57BL , Phosphorylation , Humains , Ubiquitination , Zéaralénone/analogues et dérivés , Zéaralénone/pharmacologie , Zéaralénone/administration et posologie , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , Inflammation/métabolisme , Inflammation/anatomopathologie , Phosphoric monoester hydrolases/métabolisme , Souris knockout , Lactones , RésorcinolRÉSUMÉ
BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.
Sujet(s)
Canal sodique voltage-dépendant NAV1.3 , Rat Sprague-Dawley , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Récepteur de type Toll-4 , Animaux , Récepteur de type Toll-4/métabolisme , Facteur-6 associé aux récepteurs de TNF/métabolisme , Mâle , Canal sodique voltage-dépendant NAV1.3/métabolisme , Transduction du signal/physiologie , Facteur de transcription NF-kappa B/métabolisme , Névralgie essentielle du trijumeau/métabolisme , Rats , Modèles animaux de maladie humaine , Facteur de transcription RelA/métabolisme , Facteur de transcription ATF-3/métabolisme , Seuil nociceptif/physiologieRÉSUMÉ
TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that has been implicated in myeloid malignancies. Although altered TRAF6 expression is observed in human acute myeloid leukemia (AML), its role in the AML pathogenesis remains elusive. In this study, we showed that the loss of TRAF6 in AML cells significantly impairs leukemic function in vitro and in vivo, indicating its functional importance in AML subsets. Loss of TRAF6 induces metabolic alterations, such as changes in glycolysis, TCA cycle, and nucleic acid metabolism as well as impaired mitochondrial membrane potential and respiratory capacity. In leukemic cells, TRAF6 expression shows a positive correlation with the expression of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of O-GlcNAc to target proteins involved in metabolic regulation. The restoration of growth capacity and metabolic activity in leukemic cells with TRAF6 loss, achieved through either forced expression of OGT or pharmacological inhibition of O-GlcNAcase (OGA) that removes O-GlcNAc, indicates the significant role of O-GlcNAc modification in the TRAF6-related cellular and metabolic dynamics. Our findings highlight the oncogenic function of TRAF6 in leukemia and illuminate the novel TRAF6/OGT/O-GlcNAc axis as a potential regulator of metabolic reprogramming in leukemogenesis.
Sujet(s)
Évolution de la maladie , Protéines et peptides de signalisation intracellulaire , Leucémie aigüe myéloïde , Humains , Leucémie aigüe myéloïde/métabolisme , Leucémie aigüe myéloïde/anatomopathologie , Leucémie aigüe myéloïde/génétique , Animaux , Souris , Facteur-6 associé aux récepteurs de TNF/métabolisme , N-acetylglucosaminyltransferase/métabolisme , N-acetylglucosaminyltransferase/génétique , Glycolyse , Lignée cellulaire tumorale ,RÉSUMÉ
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and characterized by dysregulated immune response. Studies have shown that the SARS-CoV-2 accessory protein ORF7b induces host cell apoptosis through the tumor necrosis factor alpha (TNF-α) pathway and blocks the production of interferon beta (IFN-ß). The underlying mechanism remains to be investigated. In this study, we found that ORF7b facilitated viral infection and production, and inhibited the RIG-I-like receptor (RLR) signaling pathway through selectively interacting with mitochondrial antiviral-signaling protein (MAVS). MAVS439-466 region and MAVS Lys461 were essential for the physical association between MAVS and ORF7b, and the inhibition of the RLR signaling pathway by ORF7b. MAVSK461/K63 ubiquitination was essential for the RLR signaling regulated by the MAVS-ORF7b complex. ORF7b interfered with the recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) and the activation of the RLR signaling pathway by MAVS. Furthermore, interfering peptides targeting the ORF7b complex reversed the ORF7b-suppressed MAVS-RLR signaling pathway. The most potent interfering peptide V disrupts the formation of ORF7b tetramers, reverses the levels of the ORF7b-inhibited physical association between MAVS and TRAF6, leading to the suppression of viral growth and infection. Overall, this study provides a mechanism for the suppression of innate immunity by SARS-CoV-2 infection and the mechanism-based approach via interfering peptides to potentially prevent SARS-CoV-2 infection.IMPORTANCEThe pandemic coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and continues to be a threat to public health. It is imperative to understand the biology of SARS-CoV-2 infection and find approaches to prevent SARS-CoV-2 infection and ameliorate COVID-19. Multiple SARS-CoV-2 proteins are known to function on the innate immune response, but the underlying mechanism remains unknown. This study shows that ORF7b inhibits the RIG-I-like receptor (RLR) signaling pathway through the physical association between ORF7b and mitochondrial antiviral-signaling protein (MAVS), impairing the K63-linked MAVS polyubiquitination and its recruitment of tumor necrosis factor receptor-related factor 6 (TRAF6) to MAVS. The most potent interfering peptide V targeting the ORF7b-MAVS complex may reverse the suppression of the MAVS-mediated RLR signaling pathway by ORF7b and prevent viral infection and production. This study may provide new insights into the pathogenic mechanism of SARS-CoV-2 and a strategy to develop new drugs to prevent SARS-CoV-2 infection.
Sujet(s)
Protéines adaptatrices de la transduction du signal , COVID-19 , SARS-CoV-2 , Transduction du signal , Animaux , Humains , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/génétique , Apoptose , COVID-19/virologie , COVID-19/immunologie , COVID-19/métabolisme , Protéine-58 à domaine DEAD/métabolisme , Cellules HEK293 , Immunité innée , Interféron bêta/métabolisme , Récepteurs immunologiques/métabolisme , SARS-CoV-2/immunologie , SARS-CoV-2/métabolisme , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur de nécrose tumorale alpha/métabolisme , Ubiquitination , Protéines virales régulatrices ou accessoires/métabolisme , Protéines virales régulatrices ou accessoires/génétiqueRÉSUMÉ
Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.
Sujet(s)
Protéines adaptatrices de la transduction du signal , Antigène CD274 , Mélanome , Transduction du signal , Facteur-6 associé aux récepteurs de TNF , Facteurs de transcription , Protéines de signalisation YAP , Humains , Antigène CD274/métabolisme , Antigène CD274/génétique , Mélanome/métabolisme , Mélanome/génétique , Mélanome/traitement médicamenteux , Mélanome/anatomopathologie , Mélanome/immunologie , Protéines de signalisation YAP/génétique , Protéines de signalisation YAP/métabolisme , Protéines adaptatrices de la transduction du signal/métabolisme , Protéines adaptatrices de la transduction du signal/génétique , Facteurs de transcription/génétique , Facteurs de transcription/métabolisme , Animaux , Lignée cellulaire tumorale , Souris , Facteur-6 associé aux récepteurs de TNF/métabolisme , Facteur-6 associé aux récepteurs de TNF/génétique , Régulation de l'expression des gènes tumoraux , Ubiquitination , Protéines et peptides de signalisation intracellulaire/génétique , Protéines et peptides de signalisation intracellulaire/métabolismeRÉSUMÉ
Bone fracture as a consequence of colorectal cancer (CRC) and associated osteoporosis (OP) is considered a risk factor for increasing the mortality rate among CRC patients. SNHG16/ miRNA-146a/ TRAF6 signaling pathway is a substantial contributor to neoplastic evolution, progression, and metastasis. Here, we investigated the effect of zoledronate (ZOL) on the growth of CRC and associated OP in a mouse model. Thirty Balb/c mice were divided into Naïve, azoxymethane (AOM)/dextran sodium sulfate (DSS), and ZOL groups. Body weight and small nucleolar RNA host gene 16 (SNHG16) expression, microRNA-146a, and TRAF6 in bone, colon, and stool were investigated. Samples of colon and bone were collected and processed for light microscopic, immunohistochemical staining for cytokeratin 20 (CK20), nuclear protein Ki67 (pKi-67), and caudal type homeobox transcription factor 2 (CDx2) in colon and receptor activator of nuclear factor kB (RANK) and osteoprotegerin (OPG) in bone. A computerized tomography (CT) scan of the femur and tibia was studied. ZOL produced a significant decrease in the expression of SNHG16 and TRAF6 and an increase in miRNA-146a in the colon and bone. ZOL administration improved the histopathological changes in the colon, produced a significant decrease in CK20 and Ki-67, and increased CDx2 expressions. In bone, ZOL prevented osteoporotic changes and tumour cell invasion produced a significant decrease in RANK and an increase in OPG expressions, alongside improved bone mineral density in CT scans. ZOL could be a promising preventive therapy against colitis-induced cancer and associated OP via modulation expression of SNHG16, miRNA-146a, and TRAF6.