Tripartite Motif-containing Protein Research Articles

MiR-99a-3p downregulates TRIM21 to promote gastric cancer development.

Gastric cancer (GC) stands as one of the most formidable malignancies worldwide. It is well-established that miRNAs play a crucial role in the initiation and progression of various human cancers. Among these, miR-99a-3p has been implicated in the pathogenesis of GC. In the context of our study, we embarked on the comprehensive examination of miR-99a-3p expression in GC cells. Additionally, we sought to establish a correlation between miR-99a-3p expression levels and the overall survival (OS) of GC patients, and our findings hinted at its potential role in predicting an unfavorable prognosis. To further investigate the functional implications of miR-99a-3p in GC, we conducted a series of cell-based experiments after successfully knocking down miR-99a-3p. These investigations uncovered a substantial inhibition of cellular events associated with tumor progression. Moreover, employing TargetScan, we identified Tripartite motif-containing protein 21 (TRIM21) as a putative target with a binding site for miR-99a-3p. Subsequent dual-luciferase reporter gene assay confirmed the direct interaction between miR-99a-3p and TRIM21. Western blot analysis validated the alteration in TRIM21 expression levels, revealing an upregulation upon miR-99a-3p knockdown. Building on these molecular findings, we extended our investigations to human GC tissues, where we observed a downregulation of TRIM21, which, notably, correlated with shorter overall survival. Lastly, to further solidify our conclusions, we conducted a series of in vitro and in vivo rescue experiments, collectively suggesting that miR-99a-3p promoted the progression of GC cells through the downregulation of TRIM21. In summary, our study comprehensively explored the role of miR-99a-3p in GC, revealing its association with unfavorable patient outcomes, functional implications in tumor progression, and a direct regulatory relationship with TRIM21. These findings collectively underscore the significance of miR-99a-3p in the pathogenesis of GC and present a potential therapeutic avenue for further investigation.

Trim66's paternal deficiency causes intrauterine overgrowth.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

PAK1 inhibition increases TRIM21-induced PD-L1 degradation and enhances responses to anti-PD-1 therapy in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is a common malignancy with a 5-year survival <10 %. Immunosuppressive tumor microenvironment (TME) plays a critical role in the progression of PDA. In recent years, programmed death-ligand 1 (PD-L1)/programmed cell death protein-1 (PD-1) blockade has emerged as a potent anti-tumor immunotherapy, while is yet to achieve significant clinical benefits for PDA patients. P21-Activated kinase 1 (PAK1) is highly upregulated in PDA and has been reported to be involved in the regulation of anti-tumor immunity. This study aims to investigate the combined effect of PAK1 inhibition and anti-PD-1 therapy on PDA and the underlying mechanisms. We have shown that PAK1 expression positively correlated with PD-L1 in PDA patients, and that inhibition of PAK1 downregulated PD-L1 expression of PDA cells. More importantly, we have demonstrated that PAK1 competed with PD-L1 in binding to tripartite motif-containing protein 21 (TRIM21), a ubiquitin E3 ligase, resulting in less ubiquitination and degradation of PD-L1. Moreover, PAK1 inhibition promoted CD8+ T cells activation and infiltration. In a murine PDA model, the combination of PAK1 inhibition and anti-PD-1 therapy showed significant anti-tumor effects compared with the control or monotherapy. Our results indicated that the combination of PAK1 inhibition and anti-PD-1 therapy would be a more effective treatment for PDA patients.

TRIM47-CDO1 axis dictates hepatocellular carcinoma progression by modulating ferroptotic cell death through the ubiquitin‒proteasome system

Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, characterized by high morbidity and mortality rates, as well as unfavorable treatment outcomes. Tripartite motif-containing protein 47 (TRIM47) has been implicated in various diseases including tumor progression with the activity of E3 ubiquitin ligase. However, the precise regulatory mechanisms underlying the involvement of TRIM47 in HCC remain largely unexplored. Here, we provide evidence that TRIM47 exhibits heightened expression in tumor tissues, and its expression is in intimate association with clinical staging and patient prognosis. TRIM47 promotes HCC proliferation, migration, and invasion as an oncogene by in vitro gain- and loss-of-function experiments. TRIM47 knockdown results in HCC ferroptosis induction, primarily through CDO1 involvement to regulate GSH synthesis. Subsequent experiments confirm the interaction between TRIM47 and CDO1 dependent on B30.2 domain, wherein TRIM47 facilitates K48-linked ubiquitination, leading to a decrease in CDO1 protein abundance in HCC. Furthermore, CDO1 is able to counteract the promotional effect of TRIM47 on HCC biological functions. Overall, our research provides novel insight into the mechanism of TRIM47 in CDO1-mediated ferroptosis in HCC cells, highlighting its value as a potential target candidate for HCC therapeutic approaches.

Inhibition of Immunoproteasome Attenuates NLRP3 Inflammasome Response by Regulating E3 Ubiquitin Ligase TRIM31

Excessive secretion of pro-inflammatory cytokines leads to the disruption of intestinal barrier in inflammatory bowel disease (IBD). The inflammatory cytokine tumor necrosis factor alpha (TNFα) induces the assembly of the NLRP3 inflammasome, resulting in the augmented secretion of inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). TNFα has also been known to induce the formation of immunoproteasome (IP), which incorporates immunosubunits LMP2, LMP7, and MECL-1. Inhibition of IP activity using the IP subunit LMP2-specific inhibitor YU102, a peptide epoxyketone, decreased the protein levels of NLRP3 and increased the K48-linked polyubiquitination levels of NLRP3 in TNFα-stimulated intestinal epithelial cells. We observed that inhibition of IP activity caused an increase in the protein level of the ubiquitin E3 ligase, tripartite motif-containing protein 31 (TRIM31). TRIM31 facilitated K48-linked polyubiquitination and proteasomal degradation of NLRP3 with an enhanced interaction between NLRP3 and TRIM31 in intestinal epithelial cells. In addition, IP inhibition using YU102 ameliorated the symptoms of colitis in the model mice inflicted with dextran sodium sulfate (DSS). Administration of YU102 in the DSS-treated colitis model mice caused suppression of the NLRP3 protein levels and accompanied inflammatory cytokine release in the intestinal epithelium. Taken together, we demonstrated that inhibiting IP under inflammatory conditions induces E3 ligase TRIM31-mediated NLRP3 degradation, leading to attenuation of the NLRP3 inflammatory response that triggers disruption of intestinal barrier.

TRIM13 reduces cholesterol efflux and increases oxidized LDL uptake leading to foam cell formation and atherosclerosis

Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation leading to atherosclerosis. Here, we report that tripartite motif-containing protein 13 (TRIM13), a RING-type E3 ubiquitin ligase, plays a role in arterial lipid accumulation leading to atherosclerosis. Using molecular approaches and KO mouse model, we found that TRIM13 expression was induced both in the aorta and peritoneal macrophages (pMφ) of ApoE-/- mice in response to Western diet (WD) invivo. Furthermore, proatherogenic cytokine interleukin-1β also induced TRIM13 expression both in pMφ and vascular smooth muscle cells. Furthermore, we found that TRIM13 via ubiquitination and degradation of liver X receptor (LXR)α/β downregulates the expression of their target genes ABCA1/G1 and thereby inhibits cholesterol efflux. In addition, TRIM13 by ubiquitinating and degrading suppressor of cytokine signaling 1/3 (SOCS1/3) mediates signal transducer and activator of transcription 1 (STAT1) activation, CD36 expression, and foam cell formation. In line with these observations, genetic deletion of TRIM13 by rescuing cholesterol efflux and inhibiting foam cell formation protects against diet-induced atherosclerosis. We also found that while TRIM13 and CD36 levels were increased, LXRα/β, ABCA1/G1, and SOCS3 levels were decreased both in Mφ and smooth muscle cells of stenotic human coronary arteries as compared to nonstenotic arteries. More intriguingly, the expression levels of TRIM13 and its downstream signaling molecules were correlated with the severity of stenotic lesions. Together, these observations reveal for the first time that TRIM13 plays a crucial role in diet-induced atherosclerosis, and that it could be a potential drug target against this vascular lesion.

Abstract 555: Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma

Abstract Background: Esophageal adenocarcinoma (EAC) is one the deadliest cancers in United States (US) and the only major cancer in the US with increasing incidence. Gastroesophageal reflux disease (GERD) can cause Barrett’s esophagus (BE), a columnar metaplasia of the esophagus strongly associated with the development of EAC. GERD leading to BE is a common pre-occurrence in EAC patients, but the mechanism remains obscure. To explore the mechanism and its components, we compared gene expression in BE and EAC cells with normal esophageal cells and found high expression of tripartite motif-containing protein 31 (TRIM31) in BE and EAC compared to normal esophagus (NE) by transcriptomic profiling of RNA extracted from human esophageal tissues. E3 ubiquitin ligase TRIM 31 is a new member of TRIM family and it has been reported to have oncogenic potentials in some cancers. However, its role in EAC pathogenesis is yet to be understood. Methods: RNA sequencing and transcriptomic profiling were performed on human NE, BE, and EAC epithelial tissue samples. TRIM31 expression in NE cell line (Het-1A) and EAC cell lines (OE19, Flo-1, OE33, SK-GT-2, and OACM5.1C) were identified by Western blot analysis. The Het-1A cell line, after exposure to acidic pH and bile acids, was assessed for variable TRIM31 expression. Cell morphology and viability analyses of NE and EAC cell lines after exposure to acidic pH and bile acids either alone or in combinations were observed by microscopy and WST-1 assay respectively. Results: Transcriptomic analysis showed that BE and EAC are similar in their differential gene expression with upregulation of the TRIM31 compared to that in NE. Concordant with their columnar phenotype, BE and EAC groups displayed a multitude of differentially expressed genes relative to NE samples; however, less differentially expressed genes were found comparing BE and EAC groups. Western blot demonstrated frequent overexpression of TRIM31 in EAC cell lines with very low expression of TRIM31 in NE epithelial cell line. When NE epithelial cell line Het-1A was exposed to acidic pH as well as bile acids in acidic pH, we saw a change in cell morphology that may correlate with the metaplasia of these cells to more resilient forms, as seen in human BE and EAC. Interestingly, bile acids in acidic pH increased TRIM31 expression in NE cell line Het-1A. Bile acids had a greater effect on decreasing cell viability at high concentrations and that effect is enhanced when bile acids were added in acidic pH. WST-1 cell viability assay revealed that EAC cells were more resistant to bile acids as well as bile acids in acidic pH induced decreased in cell viability compared to that in NE cell line Het-1A. The sustained cell viability of EAC cell lines under these harsh conditions correlates with the upregulation of TRIM31. Conclusions: Thus, TRIM31 may be an important oncogenic factor in GERD-induced EAC development and may be an innovative therapeutic target and marker for EAC. Citation Format: Md Sazzad Hassan, Jesse Mast, Annie Ritter, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. Role of TRIM31 in the pathogenesis of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 555.

Abstract 5959: Targeting TRIM11 is a potential therapeutic strategy for malignant gliomas

Abstract Background: TRIM11 (tripartite motif-containing protein 11) belongs to the TRIM/RBCC (the RING B-box coiled-coil) family of E3 ubiquitin ligases. Emerging evidence has demonstrated that TRIM11 is a novel target against cancer and neurodegenerative diseases as it plays vital role in cellular proliferation, differentiation, tumor progression, and apoptosis. Our previous work demonstrated that TRIM11 is over-expressed in high-grade gliomas and promotes proliferation, invasion, migration and tumor growth, suggesting that TRIM11 may be used as a target for malignant glioma treatment. The tumor suppressor p53 plays critical roles in tumor prevention, and deregulated p53 pathway has been found in a majority of glioblastomas (GBMs). Recent studies have shown that TRIM proteins are involved in the regulation of p53, and TRIM proteins are also regulated by p53. Methods: To evaluate the effect of TRIM11 on GBM progression in vivo, we intracranially implanted GBM cells over-expressing TRIM11 in immunocompromised mice, and the animal survival was monitored. We have synthesized a series of TRIM11 inhibitors and evaluated their anti-GBM potential in vitro. Furthermore, we tested the crosstalk between TRIM11 and p53 by knocking down TP53 through RNA interference. Results: Our in vivo data showed that the control group mice (GL261 parent line) survived longer than the mice bearing TRIM11 over-expressing GL261 tumors (p&amp;lt;0.05), suggesting TRIM11 enhances tumor progression in vivo. In vitro data showed that the oncogenic effect of TRIM11 may be related to its influence on the apoptosis pathway since a robust induction of poly (ADP-ribose) polymerase (PARP) was observed in TRIM11 over-expressing GBM cells. When treated with Temozolomide (TMZ), TRIM11 over-expressing cells were more resistant and showed a higher survival rate compared to parental cells. We screened a small library of TRIM11 inhibitors (~ 25 compounds), and identified several compounds which significantly inhibited the growth of both established and primary GBM cells in a dose-dependent manner, implicating that TRIM11 inhibitors may serve as novel agents for GBM treatment. We found that inactivation of p53 by siRNA increased the expression of TRIM11 and modulated the sensitivities of GBM cells to chemotherapy drugs. Conclusion: Despite remarkable advances in the prognosis and multidisciplinary treatment strategies, GBM is still the most lethal primary brain tumor, and there is a pressing need for newer anti-GBM agents with novel targets and mode of action. Our preclinical studies demonstrate a significant anti-tumor effect of TRIM11 inhibitors on malignant glioma cells. Our finding that TRIM11 may be modulated by p53 adds a new layer of regulation to this complicated signaling pathway. A better understanding of the crosstalk between p53 and TRIM11 will benefit the clinical application of targeting p53 and TRIM11 signaling pathways for therapeutical indications in GBM. Citation Format: Kaijun Di, Bhaskar C. Das, Daniela Alexandru Abrams, Daniela A. Bota. Targeting TRIM11 is a potential therapeutic strategy for malignant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5959.

TRIM21 silencing inhibits the apoptosis and expedites the osteogenic differentiation of dexamethasone‑induced MC3T3‑E1 cells by activating the Keap1/Nrf2 pathway.

Steroid-induced osteonecrosis of the femoral head (ONFH) is a serious complication caused by long-term or excessive use of glucocorticoids. The present study aimed to ascertain the effects of tripartite motif-containing protein 21 (TRIM21) on the process of steroid-induced ONFH and its hidden action mechanism. TRIM21 expression in dexamethasone (Dex)-treated mouse MC3T3-E1 preosteoblast cells was examined using reverse transcription-quantitative PCR and western blotting. The Cell Counting Kit-8 (CCK-8) method and lactate dehydrogenase release assay were used to respectively measure cell viability and injury. Flow cytometry analysis was used to assay cell apoptosis. Caspase 3 activity was evaluated using a specific assay, while alkaline phosphatase and Alizarin red S staining were used to evaluate osteogenesis. 2,7-dichloro-dihydrofluorescein diacetate fluorescence probe was used to estimate reactive oxygen species generation. Specific assay kits were used to appraise oxidative stress levels. In addition, the expression of apoptosis-, osteogenic differentiation- and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling-associated proteins was assessed using western blotting. In Nrf2 inhibitor (ML385)-pretreated MC3T3-E1 cells exposed to Dex, cell apoptosis, osteogenesis and oxidative stress were detected again as aforementioned. Results revealed that TRIM21 expression was raised in Dex-induced MC3T3-E1 cells and TRIM21 deletion improved the viability and osteogenic differentiation, whereas it hampered the oxidative stress and apoptosis in MC3T3-E1 cells with Dex induction. In addition, silencing of TRIM21 activated Keap1/Nrf2 signaling. Moreover, ML385 partially abrogated the effects of TRIM21 depletion on the oxidative stress, apoptosis and osteogenic differentiation in MC3T3-E1 cells exposed to Dex. In conclusion, TRIM21 silencing might activate Keap1/Nrf2 signaling to protect against steroid-induced ONFH.

The E3 ligase TRIM7 suppresses the tumorigenesis of gastric cancer by targeting SLC7A11

Tripartite motif-containing protein 7 (TRIM7), as an E3 ligase, plays an important regulatory role in various physiological and pathological processes. However, the role of TRIM7 in gastric cancer (GC) is still undefined. Our study detected the expression of TRIM7 in clinical specimens and investigated the regulatory effect and molecular mechanism of TRIM7 on GC progression through in vitro and in vivo experiments. Our finding showed that TRIM7 was significantly downregulated in GC, and patients with high expression of TRIM7 showed long overall survival. Both in vitro and in vivo experiments showed that TRIM7 dramatically suppressed the malignant progression of GC. Further investigation showed that ferroptosis was the major death type mediated by TRIM7. Mechanistically, TRIM7 interacted with SLC7A11 through its B30.2/SPRY domain and promoted Lys48-linked polyubiquitination of SLC7A11, which effectively suppressing SLC7A11/GPX4 axis and inducing ferroptosis in GC cells. In vivo experiments and correlation analysis based on clinical specimens further confirmed that TRIM7 inhibited tumor growth through suppressing SLC7A11/GPX4 axis. In conclusion, our investigation demonstrated for the first time that TRIM7, as a tumor suppressor, induced ferroptosis via targeting SLC7A11 in GC, which provided a new strategy for the molecular therapy of GC by upregulating TRIM7.

TRIM21 Promotes Oxidative Stress and Ferroptosis through the SQSTM1-NRF2-KEAP1 Axis to Increase the Titers of H5N1 Highly Pathogenic Avian Influenza Virus.

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection.

PEDV nucleocapsid antagonizes zinc-finger antiviral protein by disrupting the interaction with its obligate co-factor, TRIM25

The genomes of many pathogens contain high-CpG content, which is less common in most vertebrate host genomes. Such a distinct di-nucleotide composition in a non-self invader constitutes a special feature recognized by its host’s immune system. The zinc-finger antiviral protein (ZAP) is part of the pattern recognition receptors (PRRs) that recognize CpG-rich viral RNA and subsequently initiate RNA degradation as an antiviral defense measure. To counteract such ZAP-mediated restriction, some viruses evolve to either suppress the CpG content in their genome or produce an antagonistic factor to evade ZAP sensing. We have previously shown that a coronavirus, Porcine epidermic diarrhea virus (PEDV), employs its nucleocapsid protein (PEDV-N) to suppress the ZAP-dependent antiviral activity. Here, we propose a mechanism by which PEDV-N suppresses ZAP function by interfering with the interaction between ZAP and its essential cofactor, Tripartite motif-containing protein 25 (TRIM25). PEDV-N was found to interact with ZAP through its N-terminal domain and with TRIM25 through its C-terminal domain. We showed that PEDV-N and ZAP compete for binding to the SPla and the RYanodine Receptor (SPRY) domain of TRIM25, resulting in PEDV-N preventing TRIM25 from interacting with and promoting ZAP. Our result also showed that the presence of PEDV-N in the complex reduces the E3 ligase activity of TRIM25 on ZAP, which is required for the antiviral activity of ZAP. The host-pathogen interaction mechanism presented herein provides an insight into the new function of this abundant and versatile viral protein from a coronavirus which could be a key target for development of antiviral interventions.

TRIM59 suppresses the brain ischaemia/reperfusion injury and pyroptosis of microglial through mediating the ubiquitination of NLRP3

Cerebral ischaemia/reperfusion (I/R) injury induces irreversible brain injury and causes functional impairment. Ubiquitination plays a crucial role in protein degradation, but its role in cerebral I/R injury remains unclear. Differentially expressed genes in stroke were identified by analysing the microarray dataset GSE119121. Cerebral I/R was simulated in vitro by treating human microglial HMC3 cells with oxygen–glucose deprivation/reperfusion (OGD/R). Cell viability was tested by Cell Counting Kit 8 (CCK-8) assays, and pyroptosis was examined by flow cytometry. Lactate dehydrogenase (LDH) and inflammatory cytokine secretion were measured by LDH cytotoxicity assays and enzyme-linked immunosorbent assay (ELISA), respectively. The cerebral I/R animal model was established by middle cerebral artery occlusion (MCAO) surgery in rats. Bioinformatic analysis indicated that tripartite motif-containing protein 59 (TRIM59) is downregulated in stroke, which was verified in cerebral I/R models. The upregulation of TRIM59 promoted viability and inhibited pyroptosis in OGD/R-treated microglia and alleviated cerebral I/R injury in vivo. TRIM59 attenuated NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expression through ubiquitination, thus degrading NLRP3 and alleviating OGD/R-induced injury. TRIM59 relieves cerebral I/R injury in vivo and in vivo. Mechanistically, TRIM59 directly interacts with NLRP3 and inhibits NLRP3 through ubiquitination. Targeting the TRIM59/NLRP3 signalling axis may be an effective therapeutic strategy for cerebral I/R.

Transcriptional responses of Crassostrea hongkongensis under high and low salinity stress

Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 ‰, S25), low (5 ‰, S5) and high (35 ‰, S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.

Microtubule association of TRIM3 revealed by differential extraction proteomics.

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of α-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

TRIM65 knockout inhibits the development of HCC by polarization tumor-associated macrophages towards M1 phenotype via JAK1/STAT1 signaling pathway

Background & aimsTumor-associated macrophages (TAMs) are main components of immune cells in tumor microenvironment (TME), and play a crucial role in tumor progression. Tripartite motif-containing protein 65 (TRIM65) has been associated with tumor progression. However, whether TRIM65 regulate the interaction of tumor cell and TAMs in HCC and the underlying mechanisms remain unknown. In this study, we investigated the role of TRIM65 in TME of HCC and explored its underlying mechanisms. MethodsThe relation of TRIM65 expression level with tumor grades, TNM stages, and worse prognosis of HCC patients was evaluated by bioinformatics analysis, as well as immune infiltration level of macrophages. TRIM65 shRNA was transfected into HepG2 cells, and TRIM65 overexpression plasmid was transfected into Huh7 cells, and the effect of TRIM65 on cell growth was examined by EdU assay. The mouse subcutaneous Hep1-6 tumor-bearing model with WT and TRIM65-/- mice was established to study the role of TRIM65 in HCC. Immunohistochemistry staining, Immunofluorescence staining, qRT-PCR and western blot were performed to evaluate the effect of TRIM65 on TAM infiltration, TAM polarization and JAK1/STAT1 signaling pathway. ResultsBioinformatics analysis revealed that TRIM65 was upregulated in 16 types of cancer especially in HCC, and high level of TRIM65 was strongly correlated with higher tumor grades, TNM stages, and worse prognosis of patients with HCC as well as immune infiltration level of macrophages (M0, M1, and M2). Moreover, we observed that TRIM65 shRNA-mediated TRIM65 knockdown significantly inhibited the HepG2 cells growth while TRIM65 overexpression highly increased the Huh7 cells growth in vitro. TRIM65 knockout significantly inhibited the tumor growth as well as macrophages polarization towards M2 but promoted macrophages polarization towards M1 in vivo. Mechanistically, the results demonstrate that TRIM65 knockout promoted macrophage M1 polarization in conditioned medium-stimulated peritoneal macrophages and in tumor tissues by activating JAK1/STAT1 signaling pathway. ConclusionsTaken together, our study suggests that tumor cells utilize TRIM65-JAK1/STAT1 axis to inhibit macrophage M1 polarization and promote tumor growth, reveals the role of TRIM65 in TAM-targeting tumor immunotherapy.

EF1α-associated protein complexes affect dendritic spine plasticity by regulating microglial phagocytosis in Fmr1 knock-out mice.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. There is no specific treatment for FXS due to the lack of therapeutic targets. We report here that Elongation Factor 1α (EF1α) forms a complex with two other proteins: Tripartite motif-containing protein 3 (TRIM3) and Murine double minute (Mdm2). Both EF1α-Mdm2 and EF1α-TRIM3 protein complexes are increased in the brain of Fmr1 knockout mice as a result of FMRP deficiency, which releases the normal translational suppression of EF1α mRNA and increases EF1α protein levels. Increased EF1α-Mdm2 complex decreases PSD-95 ubiquitination (Ub-PSD-95) and Ub-PSD-95-C1q interaction. The elevated level of TRIM3-EF1α complex is associated with decreased TRIM3-Complement Component 3 (C3) complex that inhibits the activation of C3. Both protein complexes thereby contribute to a reduction in microglia-mediated phagocytosis and dendritic spine pruning. Finally, we created a peptide that disrupts both protein complexes and restores dendritic spine plasticity and behavioural deficits in Fmr1 knockout mice. The EF1α-Mdm2 and EF1α-TRIM3 complexes could thus be new therapeutic targets for FXS.

E3 ligase TRIM65 alleviates intestinal ischemia/reperfusion injury through inhibition of TOX4-mediated apoptosis

Intestinal ischemia-reperfusion (II/R) injury is an urgent clinical disease with high incidence and mortality, and impaired intestinal barrier function caused by excessive apoptosis of intestinal cells is an important cause of its serious consequences. Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase that is recently reported to suppress the inflammatory response and apoptosis. However, the biological function and regulation of TRIM65 in II/R injury are totally unknown. We found that TRIM65 was significantly decreased in hypoxia-reoxygenation (H/R) induced intestinal epithelial cells and II/R-induced intestine tissue. TRIM65 knockout mice markedly aggravated intestinal apoptosis and II/R injury. To explore the molecular mechanism of TRIM65 in exacerbating II/R-induced intestinal apoptosis and damage, thymocyte selection-associated high mobility group box factor 4 (TOX4) was screened out as a novel substrate of TRIM65 using the yeast two-hybrid system. TRIM65 binds directly to the N-terminal of TOX4 through its coiled-coil and SPRY structural domains. Immunofluorescence confocal microscopy showed that they can co-localize both in the cytoplasm and nucleus. Furthermore, TRIM65 mediated the K48 ubiquitination and degradation of TOX4 depending on its E3 ubiquitin ligase activity. In addition, TRIM65 inhibits H/R-induced intestinal epithelial apoptosis via TOX4. In summary, our results indicated that TRIM65 promotes ubiquitination and degradation of TOX4 to inhibit apoptosis in II/R. These findings provide a promising target for the clinical treatment of II/R injury.

Degradation of AZGP1 suppresses apoptosis and facilitates cholangiocarcinoma tumorigenesis via TRIM25.

Alpha-2-Glycoprotein 1, Zinc-binding (AZGP1, ZAG) is a secreted protein that is synthesized by adipocytes and epithelial cells; it is downregulated in several malignancies such as breast, prostate, liver and lung cancers. However, its function remains unclear in cholangiocarcinoma (CCA). Here, we evaluated the impact AZGP1 in CCA using Gene Expression Omnibus (GEO) and GEPIA. In addition, we analysed AZGP1 expression using quantitative reverse transcription PCR and western blotting. Expression of AZGP1 was nearly deficient in CCA patients and cell lines and was associated with poor prognosis. AZGP1 overexpression upregulated apoptosis markers. Co-immunoprecipitation experiments showed that AZGP1 interacts with tripartite motif-containing protein 25 (TRIM25), and tissue microarray and bioinformatic analysis showed that AZGP1 is negatively correlated with TRIM25 expression in CCA. Thereafter, TRIM25 knockdown led to AZGP1 upregulation and induced cancer cell apoptosis. TRIM25 targets AZGP1 for degradation by catalysing its ubiquitination. AZGP1 overexpression significantly suppressed tumour growth in a xenograft mouse model. This study findings suggest that AZGP1 is a potential therapeutic target or a diagnostic biomarker for treating patients with CCA.

Targeted proteomics in a population-based study identifies serum PECAM-1 and TRIM21 as inflammation markers for periodontitis

ObjectivesPeriodontitis (PD) can cause systematic inflammation and is associated with various metabolic processes in the body. However, robust serum markers for these relationships are still lacking. This study aims to identify novel circulating inflammation-related proteins associated with PD using targeted proteomics.Materials and methodsWe used population-based, cross-sectional data from 619 participants of the Polish Longitudinal University Study (Bialystok PLUS). Mean pocket probing depth (mPPD) and proportion of bleeding on probing (pBOP) served as exposure variables. Fifty-two inflammation-related proteins were measured using the Olink Target 96 Cardiovascular III and the Olink Target 96 Immune Response panels. Associations between periodontal measures and proteins were tested using covariate-adjusted linear regression models.ResultsAt a false discovery rate of < 0.05, we identified associations of mPPD and pBOP with platelet-endothelial cell adhesion molecule-1 (PECAM-1) and tripartite motif–containing protein 21 (TRIM21).ConclusionThis study revealed novel associations between PD and serum levels of PECAM-1 and TRIM21. Our results suggest that these proteins might be affected by molecular processes that take place in the inflamed periodontium.Clinical relevanceNovel associations of PECAM-1 and TRIM21 with PD indicate promising serum markers for understanding the disease’s pathophysiological processes and call for further biomedical investigations.