UBD participates in neutrophilic asthma by promoting the activation of IL-17 signaling.

Neutrophilic asthma is a persistent and severe inflammatory lung disease characterized by neutrophil activation and the mechanisms of which are not completely elucidated. Ubiquitin D (UBD) is a ubiquitin-like modifier participating in infections, immune responses, and tumorigenesis, while whether UBD involves in neutrophilic asthma needs further study. In this study, we initially found that UBD expression was significantly elevated and interleukin 17 (IL-17) signaling was enriched in the endobronchial biopsies of severe asthma along with neutrophils increasing by bioinformatics analysis. We further confirmed that UBD was upregulated in the lung tissues of neutrophilic asthma mouse model. UBD overexpression promoted IL-17 signaling activation. Knockdown of UBD suppressed the activation of IL-17 signaling. UBD interacted with TRAF2 and reduced the total and the K48-linked ubiquitination of TRAF2. However, IL-17 A stimulation increased both the total and the K48-linked ubiquitination of TRAF2. Together, these findings indicated that UBD was upregulated and played a critical role in IL-17 signaling which contributed to a better understanding of the complex mechanisms in neutrophilic asthma.

Signaling via a CD27-TRAF2-SHP-1 axis during naive T cell activation promotes memory-associated gene regulatory networks.

The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.

TRAF2 decrease promotes the TGF-ß-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-ß (TGF-ß). The decisive role of TGF-ß in the development of MAFLD-HCC was confirmed through the specific depletion of TGF-ß receptor II gene in the hepatocytes (Tgfbr2ΔHep) of mice. In TRAF2-/- cells treated with TGF-ß, 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-ß, 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-ß 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-ß-mTORC1 pathway by facilitating AXIN1-mediated Smad7 degradation.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

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.

Unlocking hepatocellular carcinoma aggression: STAMBPL1-mediated TRAF2 deubiquitination activates WNT/PI3K/NF-kb signaling pathway.

STAM Binding Protein Like 1 (STAMBPL1), functions as a deubiquitinase (DUB) and plays a significant role in various types of cancers. However, its effect as a DUB participating in the HCC tumorigenesis and progression still unknown. In the study, the upregulation and strong prognosis value of STAMBPL1 were identified in HCC patients. Functionally, STAMBPL1 significantly promoted HCC cells proliferation and metastasis, and it interacts with TRAF2 and stabilize it via the deubiquitination at the K63 residue. The TRAF2 upregulation stabilized by STAMBPL1 overexpression transfers of P65 protein into the nucleus and activates the WNT/PI3K/ NF-kb signaling pathway. The 251-436 sites of STAMBPL1 particularly interact with the 294-496 sites of TRAF2, thereby exerting the function of DUB and removing the ubiquitin molecules attached to TRAF2. Our research unveiled a new function of STAMBPL1 in mediating TRAF2 deubiquitination and stabilization, thereby activating the WNT/PI3K/NF-kb signaling pathway, suggesting its potential as a novel biomarker and therapeutic target for HCC.

[Xinfeng Capsule alleviates interleukin-1ß-induced chondrocyte inflammation and extracellular matrix degradation by regulating the miR-502-5p/TRAF2/NF-κB axis].

OBJECTIVE: To investigate the mechanism that mediates the inhibitory effect of Xinfeng Capsule (XFC) on interleukin (IL)-1ß-induced impairment of chondrocytes. METHODS: XFC-medicated serum was collected from SD rats with XFC gavage, and its optimal concentration for chondrocyte treatment was determined using Cell Counting Kit-8 assay and flow cytometry. Dual luciferase reporter analysis was performed to analyze the targeting relationship between miR-502-5p and TRAF2. In cultured human chondrocytes induced with IL-1ß, the effects of transfection with miR-502-5p inhibitor and XFC-medicated serum, alone or in combination, on expression levels of IL-1ß, tumor necrosis factor-α (TNF-α), IL-4, and IL-10 were examined with ELISA, and the changes in the expressions of collagen type Ⅱ alpha 1 (COL2A1), matrix metalloproteinase 13 (MMP13), adisintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and miR-502-5p/TRAF2/NF-κB axis gene expression were detected using RT-qPCR, Western blotting, and immunofluorescence assay. RESULTS: In cultured human chondrocytes, treatment with IL-1ß significantly decreased the cell viability, increased cell apoptosis rate, lowered miR-502-5p, IL-4, IL-10, and COL2A1 expressions, and enhanced IL-1ß, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 expressions (P < 0.05), and these changes were significantly improved by treatment with XFC-medicated serum at the optimal concentration of 20% (P < 0.05). Transfection of the chondrocytes with miR-502-5p inhibitor resulted in elevated expressions of IL-1ß, TNF-α, ADAMTS5, MMP13, TRAF2, and NF-κB p65 and lowered expressions of miR-502-5p, IL-4, IL-10, and COL2A1, and XFC-medicated serum obviously reversed the effects of miR-502-5p inhibitor. CONCLUSION: XFC can inhibit IL-1ß-induced inflammatory response and ECM degradation in cultured human chondrocytes possibly by regulating the miR-502-5p/TRAF2/NF-κB axis.

Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells.

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.

TRAIP suppressed apoptosis and cell cycle to promote prostate cancer proliferation via TRAF2-PI3K-AKT pathway activation.

BACKGROUND: TRAF-interacting protein (TRAIP) is a RING-type E3 ubiquitin ligase, which has been implicated in various cellular processes and participated in various cancers as an oncogene. However, the function and potential mechanism of TRAIP in prostate cancer (PCa) have not been investigated so far. METHODS: Public TGCA data were used to evaluate the expression profile of TRAIP in prostatic tumors. The relative expression of TRAIP and TRAF2 in PCa tissues and tumor cell lines was detected by qPCR, western blot, and IHC staining. Next, TRAIP knockdown and overexpression plasmids were constructed and transfected into PCa cell lines. Moreover, cell proliferation, invasion, migration, and apoptosis were measured by colony formation, Transwell, wound healing, and flow cytometry assays. Subsequently, cell cycle and signaling pathway-related proteins were tested by western blot. Finally, the effect of TRAIP on PCa was measured based on the nude mouse xenograft model. RESULTS: TRAIP was significantly upregulated in PCa tissues and tumor cell lines. In addition, TRAIP promoted cell proliferation, invasion, and migration of PCa cell lines. Such an oncogenic property was mediated by the cell cycle arrest and the inhibition of apoptosis, as indicated by different functional assays and the expression of cell cycle and apoptosis regulatory proteins in cultured cells. Moreover, TRAIP combined with TRAF2 to activate PI3K/AKT pathway. Finally, TRAIP depletion suppressed the growth of tumors and cell proliferation in vivo. CONCLUSIONS: Our study first revealed that TRAIP promoted tumor progression and identified it as a potential therapeutic target for PCa patients in the future.

Xinfeng capsule inhibits lncRNA NONHSAT227927.1/TRAF2 to alleviate NF-κB-p65-induced immuno-inflammation in ankylosing spondylitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ankylosing spondylitis (AS) is a chronic rheumatic disease known for its insidious and refractory symptoms, primarily associated with immuno-inflammation in its early stages, that affects the self-perception of patients (SPP). The exploration of long noncoding RNA (lncRNA) in immuno-inflammation of AS has garnered considerable interest. Additionally, the effectiveness of traditional Chinese medicine Xinfeng Capsule (XFC) in mitigating immuno-inflammation in AS has also been observed. However, the specific mechanisms still need to be characterized. AIM OF THE STUDY: This study elucidated the mechanism of the lncRNA NONHSAT227927.1/TRAF2/NF-κB axis in the immuno-inflammation of AS and XFC in AS treatment. METHODS: LncRNA NONHSAT227927.1 and mRNA expression were assessed utilizing real-time fluorescence quantitative PCR. Protein level was determined using Western blot, and cytokine expression was measured using ELISA. Furthermore, mass spectrometry was used to analyze the binding proteins of lncRNA and rescue experiments were conducted to validate the findings. Inconsistencies in clinical baseline data were addressed using propensity score matching. The association between the XFC effect and indicator changes was evaluated using the Apriori algorithm. RESULTS: The study revealed a substantial elevation in the expression of lncRNA NONHSAT227927.1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) in AS-peripheral blood mononuclear cells. Its expression was also notably reduced after XFC treatment. In addition to this, there was a positive correlation between lncRNA NONHSAT227927.1 and TRAF2 with clinical immuno-inflammatory indicators. On the other hand, they showed a negative association with the SPP indicators. In vitro experiments have demonstrated that lncRNA NONHSAT227927.1 activated the nuclear factor (NF)-κB-p65 pathway by promoting TRAF2 expression. This activation resulted in enhanced IL-6 and TNF-α levels and reduced IL-10 and IL-4 levels. Conversely, XFC decreased the expression of lncRNA NONHSAT227927.1 and TRAF2, inhibiting the stimulation of the NF-κB-p65 cascade and restoring balance to the cytokines. The association rule analysis results indicated a strong association between XFC and decreased levels of C-reactive protein, erythrocyte sedimentation rate, and immunoglobulin A. Furthermore, XFC was strongly associated with improved SPP indicators, including general health, vitality, mental health, and role-emotional. CONCLUSIONS: LncRNA NONHSAT227927.1 plays a pro-inflammatory role in AS. XFC treatment may reverse lncRNA NONHSAT227927.1 to suppress TRAF2-mediated NF-κB-p65 activation, which in turn suppresses immuno-inflammation and improves SPP, thereby making XFC a promising candidate for therapeutic applications in AS management.

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.

The natural compound Sanggenon C inhibits PRRSV infection by regulating the TRAF2/NF-κB signalling pathway.

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease and one of the major causes of death in the global pig industry. PRRS virus (PRRSV) strains have complex and diverse genetic characteristics and cross-protection between strains is low, which complicates vaccine selection; thus, the current vaccination strategy has been greatly compromised. Therefore, it is necessary to identify effective natural compounds for the clinical treatment of PRRS. A small molecule library composed of 720 natural compounds was screened in vitro, and we found that Sanggenon C (SC) was amongst the most effective natural compound inhibitors of PRRSV infection. Compared with ribavirin, SC more significantly inhibited PRRSV infection at both the gene and protein levels and reduced the viral titres and levels of protein expression and inflammatory cytokine secretion to more effectively protect cells from PRRSV infection and damage. Mechanistically, SC inhibits activation of the NF-κB signalling pathway by promoting TRAF2 expression, thereby reducing PRRSV replication. In conclusion, by screening natural compounds, we found that SC suppresses PRRSV infection by regulating the TRAF2/NF-κB signalling pathway. This study contributes to a deeper understanding of the therapeutic targets and pathogenesis of PRRSV infection. More importantly, our results demonstrate that SC has potential as a candidate for the treatment of PRRS.

DNAJA4 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma via PSMD2-mediated MYH9 degradation.

Emerging evidence indicates that DNA methylation plays an important role in the initiation and progression of nasopharyngeal carcinoma (NPC). DNAJA4 is hypermethylated in NPC, while its role in regulating NPC progression remains unclear. Here, we revealed that the promoter of DNAJA4 was hypermethylated and its expression was downregulated in NPC tissues and cells. Overexpression of DNAJA4 significantly suppressed NPC cell migration, invasion, and EMT in vitro, and markedly inhibited the inguinal lymph node metastasis and lung metastatic colonization in vivo, while it did not affect NPC cell viability and proliferation capability. Mechanistically, DNAJA4 facilitated MYH9 protein degradation via the ubiquitin-proteasome pathway by recruiting PSMD2. Furthermore, the suppressive effects of DNAJA4 on NPC cell migration, invasion, and EMT were reversed by overexpression of MYH9 in NPC cells. Clinically, a low level of DNAJA4 indicated poor prognosis and an increased probability of distant metastasis in NPC patients. Collectively, DNAJA4 serves as a crucial driver for NPC invasion and metastasis, and the DNAJA4-PSMD2-MYH9 axis might contain potential targets for NPC treatments.

RACK1 facilitates breast cancer progression by competitively inhibiting the binding of ß-catenin to PSMD2 and enhancing the stability of ß-catenin.

The receptor for activated C kinase 1 (RACK1) is a key scaffolding protein with multifunctional and multifaceted properties. By mediating protein-protein interactions, RACK1 integrates multiple intracellular signals involved in the regulation of various physiological and pathological processes. Dysregulation of RACK1 has been implicated in the initiation and progression of many tumors. However, the exact function of RACK1 in cancer cellular processes, especially in proliferation, remains controversial. Here, we show that RACK1 is required for breast cancer cell proliferation in vitro and tumor growth in vivo. This effect of RACK1 is associated with its ability to enhance ß-catenin stability and activate the canonical WNT signaling pathway in breast cancer cells. We identified PSMD2, a key component of the proteasome, as a novel binding partner for RACK1 and ß-catenin. Interestingly, although there is no interaction between RACK1 and ß-catenin, RACK1 binds PSMD2 competitively with ß-catenin. Moreover, RACK1 prevents ubiquitinated ß-catenin from binding to PSMD2, thereby protecting ß-catenin from proteasomal degradation. Collectively, our findings uncover a novel mechanism by which RACK1 increases ß-catenin stability and promotes breast cancer proliferation.

Ginsenoside F4 Alleviates Skeletal Muscle Insulin Resistance by Regulating PTP1B in Type II Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with increasing morbidity. Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling cascade and has attracted intensive investigation in the T2DM study. Ginseng is widely used to treat metabolic diseases, while the effects of ginsenoside F4 (F4) on T2DM have remained unknown. Here, we identify F4 as an inhibitor of skeletal muscle insulin resistance. The results showed that F4 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the inhibition of the PTP1B activity. On the one hand, the inhibition of PTP1B activity by F4 resulted in increased insulin receptor (INSR) and insulin receptor substrate 1 tyrosine phosphorylation and enhanced insulin sensitivity. On the other hand, F4 as a PTP1B inhibitor inhibited the inositol-requiring enzyme 1 (IRE-1)/recombinant TNF receptor associated factor 2 (TRAF2)/c-Jun N-terminal kinase signaling pathway and alleviated skeletal muscle endoplasmic reticulum (ER) stress, thereby reducing IRS-1 serine phosphorylation. Both finally activated the PI3K/AKT signaling pathway and promoted glucose transporter protein 4 translocation to the cell membrane for glucose uptake. Taken together, our experiments demonstrate that F4 activates the insulin signaling pathway by inhibiting the activity of PTP1B while inhibiting the IRE-1/TRAF2/JNK signaling pathway, enhancing insulin sensitivity, and alleviating ER stress in the skeletal muscle of db/db mice. Our results indicate that F4 can be used as a PTP1B inhibitor for the treatment of T2DM.

NUMA1 modulates apoptosis of esophageal squamous cell carcinoma cells through regulating ASK1-JNK signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy worldwide with a low survival rate due to a lack of therapeutic targets. Here, our results showed that nuclear mitotic apparatus protein 1 (NUMA1) transcript and protein levels are significantly upregulated in ESCC patient samples and its high expression predicated poor prognosis. Knock-down of NUMA1 promoted cell apoptosis and suppressed cell proliferation and colony formation. By using cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mice models, we found silencing the NUMA1 expression suppressed tumor progression. In addition, conditional knocking-out of NUMA1 reduced 4NQO-induced carcinogenesis in mice esophagus, which further confirmed the oncogenic role of NUMA1 in ESCC. Mechanistically, from the immunoprecipitation assay we revealed that NUMA1 interacted with GSTP1 and TRAF2, promoted the association of TRAF2 with GSTP1 while inhibited the interaction of TRAF2 and ASK1, thus to regulate sustained activation of JNK. In summary, our findings suggest that NUMA1 plays an important role during ESCC progression and it functions through regulating ASK1-MKK4-SAPK/JNK signaling pathway.

[Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway].

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1ß, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

Diosmetin inhibits the growth and invasion of gastric cancer by interfering with M2 phenotype macrophage polarization.

Overturning M2 phenotype macrophage polarization is a promising therapeutic strategy for gastric cancer (GC). Diosmetin (DIO) is a natural flavonoid with antitumor effect. The aim of this study was to investigate the effect of DIO on polarization of M2 phenotype macrophages in GC. THP-1 cells were induced to M2 phenotype macrophages and co-cultured with AGS cells. The effects of DIO were determined by flow cytometry, qRT-PCR, CCK-8, Transwell, and western blot. To explore the mechanisms, THP-1 cells were transfected with adenoviral vectors containing tumor necrosis factor receptor-associated factor 2 (TRAF2) or si-TRAF2. DIO (0, 5, 10, and 20 µM) restrained the M2 phenotype macrophage polarization. In addition, DIO (20 µM) reversed the increased viability and invasion of AGS cells induced by the co-culture of M2 macrophages. Mechanistically, TRAF2 knockdown inhibited the effect of M2 phenotype macrophages on AGS cells' growth and invasion. Furthermore, DIO (20 µM) was found to decrease TRAF2/NF-κB activity in GC cells. However, TRAF2 overexpressed reversed the inhibitory effect of DIO on the co-culture system. The in vivo study confirmed that DIO treatment (50 mg/kg) could repress the growth of GC. DIO treatment markedly reduced the expressions of Ki-67 and N-cadherin, and decreased the protein levels of TRAF2 and p-NF-κB/NF-κB. In conclusion, DIO inhibited the growth and invasion of GC cells by interfering with M2 phenotype macrophage polarization through repression of the TRAF2/NF-κB signaling pathway.