Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.

RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.

Knockdown of TRIM65 suppressed the proliferation and invasiveness of gastric cancer cells by restricting the ubiquitin degradation of PPM1A.

Gastric cancer is a type of serious malignant tumors all around the world. TCGA data showed that the expression of TRIM65 (E3 ubiquitin ligase) was enhanced in the gastric cancer tissues. The role of TRIM65 in the tumorigenesis of gastric cancer remains unclear. In this study, we successfully established TRIM65-knockdown gastric cancer cells. Next, CCK-8, colony formation assays and transwell assays were performed to detect the cell proliferation and invasion. The results showed that suppression of TRIM65 inhibited the proliferation and invasion of gastric cancer cells. Interestingly, the Western blot assay confirmed that downregulation of TRIM65 increased the level of PPM1A and decreased the level of p-TBK1 in gastric cancer cells. Mechanistically, immunoprecipitation assay revealed that knockdown of TRIM65 inhibited the ubiquitin degradation of PPM1A. In rescue experiments, suppression of PPM1A promoted the proliferation and invasion of gastric cancer cells transfected with sh-TRIM65. Therefore, our results suggested that knockdown of TRIM65 inhibited the proliferation and invasion of gastric cancer cells by suppressing the ubiquitin degradation of PPM1A and phosphorylation of TBK1.

TRIM65 triggers ß-catenin signaling via ubiquitylation of Axin1 to promote hepatocellular carcinoma.

Deregulation of ubiquitin ligases contributes to the malignant progression of human cancers. Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase and has been implicated in human diseases, but its role and clinical significance in hepatocellular carcinoma (HCC) remain unknown. Here, we showed that TRIM65 expression was increased in HCC tissues and associated with poor outcome in two independent cohorts containing 888 patients. In vitro and in vivo data demonstrated that overexpression of TRIM65 promoted cell growth and tumor metastasis, whereas knockdown of TRIM65 resulted in opposite phenotypes. Further studies revealed that TRIM65 exerted oncogenic activities via ubiquitylation of Axin1 to activate the ß-catenin signaling pathway. TRIM65 directly bound to Axin1 and accelerated its degradation through ubiquitylation. Furthermore, HMGA1 was identified as an upstream regulator of TRIM65 in HCC cells. In clinical samples, TRIM65 expression was positively correlated with the expression of HMGA1 and nuclear ß-catenin. Collectively, our data indicate that TRIM65 functions as an oncogene in HCC. The newly identified HMGA1/TRIM65/ß-catenin axis serves as a promising prognostic factor and therapeutic target.

Combinatory inhibition of TRIM65 and MDM2 in lung cancer cells.

In addition to the involvement in white matter lesion, tripartite-motif protein family member 65 (TRIM65) has also been implicated in tumorigenesis as a potential oncogene. However, the underlining mechanisms of TRIM65 functions and its clinical implication still remain to be further elucidated. In the present study, we found that TRIM65 binds to the N-terminus of p53 tumor suppressor and thus competes with MDM2 for p53 binding. Intriguingly, analysis of the Cancer Genome Atlas (TCGA) gene alteration database revealed that elevated expression of TRIM65 is mutually exclusive to MDM2 up-regulation in human lung adenocarcinoma patients, indicating potential compensatory effect of one over the other. Indeed, overexpression of TRIM65 renders lung cancer cell line resistance to Nutlin-3a, an effective MDM2 inhibitor, as determined by p53 activation and cell proliferation assays. Furthermore, depletion of TRIM65 using siRNA in combination with Nutlin-3a treatment demonstrates enhanced anti-tumor effects on lung cancer cell line. Collectively, our findings provide the rationale for developing strategies to target TRIM65 for lung cancer intervention, potentially in combination with MDM2 inhibition.

TRIM65 is a potential oncogenic protein via ERK1/2 on Jurkat and Raji cells: A therapeutic target in human lymphoma malignancies.

In human lung cancer, Tripartite motif 65 (TRIM65) is documented as an important regulator in carcinogenesis. Knockdown of TRIM65 prevents the tumorigenesis of lung cancer cells, while TRIM65 overexpression presents the opposite effect. However, the roles of TRIM65 in human lymphocyte malignancies have reported little. Herein, we found that Jurkat (T-lymphocyte) and Raji (B-lymphocyte) expressed TRIM65. We aimed to investigate whether TRIM65 was a potential oncogenic protein that regulated the tumorigenesis of Jurkat and Raji cells. In our present study, cells were transfected with siRNA-TRIM65 or TRIM65 overexpression vector, Cell counting kit-8 (CCK-8), Flow cytometry and Annexin V-FITC/propidium iodide (PI) staining was carried out to detect cell viability, cell cycle profile and cell apoptosis, respectively. Extracellular signal-regulated kinases 1/2 (ERK1/2) pathway-associated proteins, such as Bcl2, cleaved-caspase 3, vascular endothelial growth factor (VEGF), and phosphorylated ERK1/2 (p-ERK1/2) were assessed. Our data indicated that knockdown of TRIM65 prevented the tumorigenesis of Jurkat and Raji cells. TRIM65 silencing inhibited cell proliferation, promoted cell apoptosis and arrested cell cycle, highly like through blocking ERK1/2 pathway. However, TRIM65 overexpression enhanced cell viability, increased the protein levels of Bcl2, VEGF, p-ERK1/2 while decreased cleaved-caspase 3 expression, suggesting the promoted effect of TRIM65 overexpression in the tumorigenesis of those two lymphoma cells. To validate the involvement of ERK1/2 pathway, ERK1/2 inhibitor AZD8330 (1 µmol/L) was introduced. We found that AZD8330 significantly prevented TRIM65 overexpression-induced tumorigenesis. We concluded that TRIM65 served as a potential oncogenic protein on Jurkat and Raji cells, and ERK1/2 pathway was the underlying mechanism. Approaches targeting TRIM65 provided a novel strategy for the treatment of lymphoma.

TRIM65 negatively regulates p53 through ubiquitination.

Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance.

Trim65: a cofactor for regulation of the microRNA pathway.

MicroRNA (miRNA) comprise a large family of non-protein coding transcripts which regulate gene expression in diverse biological pathways of both plants and animals. We recently used a systematic proteomic approach to generate a protein interactome map of the human miRNA pathway involved in miRNA biogenesis and processing. The interactome expands the number of candidate proteins in the miRNA pathway and connects the network to other cellular processes. Functional analyses identified TRIM65 and at least 3 other proteins as novel regulators of the miRNA pathway. Biochemical studies established that TRIM65 forms stable complexes with TNRC6 proteins and these molecules co-localize in P-body-like structures. Gain of function and RNAi analyses reveal that TRIM65 negatively regulates miRNA-driven suppression of mRNA translation by targeting TNRC6 proteins for ubiquitination and degradation. The potential molecular mechanisms which regulate TRIM65 catalytic activity are discussed.

Intestinal metabolite UroB alleviates cerebral ischemia/reperfusion injury by promoting competition between TRIM65 and TXNIP for binding to NLRP3 inflammasome in response to neuroinflammation.

Our previous research suggests that targeting NLRP3 inflammasomes holds promise for mitigating cerebral ischemia/reperfusion injury. The gut metabolite Urolithin B (UroB) has been shown to inhibit the neuroinflammation. However, the specific role of UroB in cerebral ischemia/reperfusion injury and its potential impact on NLRP3 inflammasome remain unclear. In this study, acute stroke was simulated using the MCAO model in male Sprague-Dawley rats. UroB was intraperitoneally administered after 1 h of reperfusion. The effects of UroB on brain tissue were evaluated, including infarct volume, brain edema, and neurobehavioral changes. Western blotting and immunofluorescence were performed to investigate the effect of UroB on inflammation-related proteins. Furthermore, TRIM65 knockdown and TXNIP overexpression experiments elucidated the role of UroB in NLRP3 inflammasome activation. The ( demonstrate the neuroprotective effect of UroB in acute stroke, reducing brain tissue damage and improving motor function. Mechanistically, UroB modulated neuroinflammation by influencing TXNIP and TRIM65 protein expression, as well as competitive binding to the NLRP3 inflammasome, attenuating cerebral ischemia/reperfusion injury. In conclusion, the potential of UroB as a protective agent against cerebral ischemia/reperfusion injury in acute stroke stands out as it regulates TRIM65 and TXNIP competitive binding to the NLRP3 inflammasome. These findings suggest that UroB is a promising drug candidate for the treatment of acute stroke.

Tripartite Motif-Containing Protein 65 (TRIM65) Inhibits Hepatitis B Virus Transcription.

Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65's antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins' capacity to inhibit HBV transcription and highlight TRIM65's pivotal role in this process.

The Magic and Mystery of TRIM65 in Diseases.

Tripartite-motif protein family member 65 (TRIM65) belongs to the tripartite motif (TRIM) protein family. Its typical structure consists of the RING, B-Box motif, and coiled-coil domains, which are highly conserved at the N-terminus and the variable SPRY domain at the C-terminus. TRIM65 is an E3 ubiquitin ligase that participates in physiological and pathological processes through the ubiquitination pathway, including intracellular signal transduction, protein degradation, cell proliferation, apoptosis, carcinogenesis, autophagy, and phenotypic transformation. Evidence shows that TRIM65 plays a remarkable and obscure role in diseases, including multisystem tumours, neurodegenerative diseases, immune system diseases, and inflammatory diseases. This review is devoted to elaborating on the relationship between TRIM65 and diseases and its pathogenic mechanism, providing a theoretical basis for TRIM65 as a possible pathogenic target of diseases and exploring the possible future research direction of TRIM65 and the challenges it may face.

TRIM65/NF2/YAP1 Signaling Coordinately Orchestrates Metabolic and Immune Advantages in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths worldwide. Significantly activated uridine nucleotide and fatty acid metabolism in HCC cells promote malignant proliferation and immune evasion. Herein, it is demonstrated that the tripartite motif 65 (TRIM65) E3 ubiquitin-protein ligase, O-GlcNAcylated via O-GlcNAcylation transferase, is highly expressed in HCC and facilitated metabolic remodeling to promote the accumulation of products related to uracil metabolism and palmitic acid, driving the progression of HCC. Mechanistically, it is showed that TRIM65 mediates ubiquitylation at the K44 residue of neurofibromatosis type 2 (NF2), the key protein upstream of classical Hippo signaling. Accelerated NF2 degradation inhibits yes-associated protein 1 phosphorylation, inducing aberrant activation of related metabolic enzyme transcription, and orchestrating metabolic and immune advantages. In conclusion, these results reveal a critical role for the TRIM family molecule TRIM65 in supporting HCC cell survival and highlight the therapeutic potential of targeting its E3 ligase activity to alter the regulation of proteasomal degradation.

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

BACKGROUND & AIMS: Tumor-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. METHODS: The 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. RESULTS: Bioinformatics 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. CONCLUSIONS: Taken 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.

TRIM65 promotes vascular smooth muscle cell phenotypic transformation by activating PI3K/Akt/mTOR signaling during atherogenesis.

BACKGROUND AND AIMS: Tripartite motif (TRIM65) is an important member of the TRIM protein family, which is a newly discovered E3 ligase that interacts with and ubiquitinates various substrates and is involved in diverse pathological processes. However, the function of TRIM65 in atherosclerosis remains unarticulated. In this study, we investigated the role of TRIM65 in the pathogenesis of atherosclerosis, specifically in vascular smooth muscle cells (VSMCs) phenotype transformation, which plays a crucial role in formation of atherosclerotic lesions. METHODS AND RESULTS: Both non-atherosclerotic and atherosclerotic lesions during autopsy were collected singly or pairwise from each individual (n = 16) to investigate the relationship between TRIM65 and the development of atherosclerosis. In vivo, Western diet-fed ApoE-/- mice overexpressing or lacking TRIM65 were used to assess the physiological function of TRIM65 on VSMCs phenotype, proliferation and atherosclerotic lesion formation. In vitro, VSMCs phenotypic transformation was induced by platelet-derived growth factor-BB (PDGF-BB). TRIM65-overexpressing or TRIM65-abrogated primary mouse aortic smooth muscle cells (MOASMCs) and human aortic smooth muscle cells (HASMCs) were used to investigate the mechanisms underlying the progression of VSMCs phenotypic transformation, proliferation and migration. Increased TRIM65 expression was detected in α-SMA-positive cells in the medial and atherosclerotic lesions of autopsy specimens. TRIM65 overexpression increased, whereas genetic knockdown of TRIM65 remarkably inhibited, atherosclerotic plaque development. Mechanistically, TRIM65 overexpression activated PI3K/Akt/mTOR signaling, resulting in the loss of the VSMCs contractile phenotype, including calponin, α-SMA, and SM22α, as well as cell proliferation and migration. However, opposite phenomena were observed when TRIM65 was deficient in vivo or in vitro. Moreover, in cultured PDGF-BB-induced TRIM65-overexpressing VSMCs, inhibition of PI3K by treatment with the inhibitor LY-294002 for 24 h markedly attenuated PI3K/Akt/mTOR activation, regained the VSMCs contractile phenotype, and blocked the progression of cell proliferation and migration. CONCLUSIONS: TRIM65 overexpression enhances atherosclerosis development by promoting phenotypic transformation of VSMCs from contractile to synthetic state through activation of the PI3K/Akt/mTOR signal pathway.

Trim65 attenuates isoproterenol-induced cardiac hypertrophy by promoting autophagy and ameliorating mitochondrial dysfunction via the Jak1/Stat1 signaling pathway.

Pathological cardiac hypertrophy is a major cause of heart failure, and there is no effective approach for its prevention or treatment. The Trim family is a recently identified family of E3 ubiquitin ligases that regulate cardiac hypertrophy. Trim65, which is a member of the Trim family, previous studies have not determined whether Trim65 affects cardiac hypertrophy. In this study, the effects of Trim65 on isoproterenol (ISO)-induced cardiac hypertrophy and the underlying mechanisms were investigated. In contrast to C57BL/6 mice, Trim65-knockout (Trim65-KO) mice developed more severe myocardial hypertrophy, fibrosis and cardiac dysfunction after being intraperitoneally injected with ISO for 2 weeks. Transmission electron microscopy (TEM) revealed that the autophagic flux was inhibited, mitochondria were swollen, and mitochondrial cristae were lost or decreased in the myocardium of Trim65-KO mice. In vitro studies demonstrated that overexpression of Trim65 inhibited ISO-induced cardiomyocyte hypertrophy by increasing mitochondrial density and membrane potential, and the Stat1 inhibitor fludarabine attenuated the effect of Trim65 knockdown on ISO-induced cardiomyocyte hypertrophy by reducing Reactive oxygen species (ROS) production and increasing the mitochondrial density and membrane potential. Our findings provide the first link between Trim65 and mitochondria, and we found for the first time that Trim65 inhibits mitochondria-dependent apoptosis and autophagy via the Jak1/Stat1 signalling pathway, ultimately attenuating ISO-induced cardiac hypertrophy; this effect of Trim65 might be mediated via the regulation of Jak1 ubiquitination. Taking these findings together, we suggest that genes that are related to mitochondria-dependent apoptosis and that are associated with Trim65 could be promising therapeutic targets for cardiac hypertrophy.

TRIM65 Suppresses oxLDL-induced Endothelial Inflammation by Interaction with VCAM-1 in Atherogenesis.

BACKGROUND AND OBJECTIVE: Endothelial cell activation, characterized by increased levels of vascular cell adhesion molecule 1 (VCAM-1), plays a crucial role in the development of atherosclerosis (AS). Therefore, inhibition of VCAM-1-mediated inflammatory response is of great significance in the prevention and treatment of AS. The tripartite motif (TRIM) protein-TRIM65 is involved in the regulation of cancer development, antivirals and inflammation. We aimed to study the functions of TRIM65 in regulating endothelial inflammation by interacting with VCAM-1 in atherogenesis. METHODS AND RESULTS: In vitro, we report that human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (oxLDL) significantly upregulate the expression of TRIM65 in a time- and dose-dependent manner. Overexpression of TRIM65 reduces oxLDL-triggered VCAM-1 protein expression, decreases monocyte adhesion to HUVECs and inhibits the production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α as well as endothelial oxLDL transcytosis. In contrast, siRNA-mediated knockdown of TRIM65 promotes the expression of VCAM-1, resulting in increased adhesion of monocytes and the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α and enhances endothelial oxLDL transcytosis. In vivo, we measured the high expression of TRIM65 in ApoE-/- mouse aortic plaques compared to C57BL/6J mouse aortic plaques. Then, we examined whether the blood levels of VCAM-1 were higher in TRIM65 knockout ApoE-/- mice than in control mice induced by a Western diet. Furthermore, Western blot results showed that the protein expression of VCAM-1 was markedly enhanced in TRIM65 knockout ApoE-/- mouse aortic tissues compared to that of the controls. Immunofluorescence staining revealed that the expression of VCAM-1 was significantly increased in atherosclerotic plaques of TRIM65-/-/ApoE-/- aortic vessels compared to ApoE-/- controls. Mechanistically, TRIM65 specifically interacts with VCAM-1 and targets it for K48-linked ubiquitination. CONCLUSION: Our studies indicate that TRIM65 attenuates the endothelial inflammatory response by targeting VCAM-1 for ubiquitination and provides a potential therapeutic target for the inhibition of endothelial inflammation in AS.

TRIM65 determines the fate of a novel subtype of pituitary neuroendocrine tumors via ubiquitination and degradation of TPIT.

BACKGROUND: Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors that are classified into seven histological subtypes, including lactotroph, somatotroph, corticotroph, thyrotroph, gonadotroph, null cell, and plurihormonal PitNETs. However, the molecular characteristics of these types of PitNETs are not completely clear. METHODS: A total of 180 consecutive cases of PitNETs were collected to perform RNA sequencing. All subtypes of PitNETs were distinguished by unsupervised clustering analysis. We investigated the regulation of TPIT by TRIM65 and its effects on ACTH production and secretion in ACTH-secreting pituitary cell lines, as well as in murine models using biochemical analyses, confocal microscopy, and luciferase reporter assays. RESULTS: A novel subtype of PitNETs derived from TPIT lineage cells was identified as with normal TPIT transcription but with lowered protein expression. Furthermore, for the first time, TRIM65 was identified as the E3 ubiquitin ligase of TPIT. Depending on the RING domain, TRIM65 ubiquitinated and degraded the TPIT protein at multiple Lys sites. In addition, TRIM65-mediated ubiquitination of TPIT inhibited POMC transcription and ACTH production to determine the fate of the novel subtype of PitNETs in vitro and in vivo. CONCLUSION: Our studies provided a novel classification of PitNETs and revealed that the TRIM65-TPIT complex controlled the fate of the novel subtype of PitNETs, which provides a potential therapy target for Cushing's disease.

TRIM65 Promotes Cervical Cancer Through Selectively Degrading p53-Mediated Inhibition of Autophagy and Apoptosis.

Tripartite motif containing 65 (TRIM65) is an E3 ubiquitin ligase that has been implicated in a variety of cellular processes as well as tumor progression, but its biological role and the underlying mechanism in cervical cancer is unclear. Here, we reported that TRIM65 expression in human cervical cancer tissues was significantly higher than that in the adjacent normal cervical tissues, and TRIM65 knockdown enhanced autophagic flux and cell apoptosis, but not cell cycle, to dramatically inhibit the proliferation and migration of cervical cancer cells. Furthermore, our experiments showed that TRIM65 exhibited oncogenic activities via directly targeting p53, a tumor suppressor and a common upsteam regulator between autophagy and apoptosis, promoting ubiquitination and proteasomal degradation of p53. Taken together, our studies demonstrated that TRIM65 knockdown promotes cervical cancer cell death through enhancing autophagy and apoptosis, suggesting that TRIM65 may be a potential therapeutic target for cervical cancer clinically.

Circ_0011292 Enhances Paclitaxel Resistance in Non-Small Cell Lung Cancer by Regulating miR-379-5p/TRIM65 Axis.

Background: Non-small cell lung cancer (NSCLC) is the most prevalent cancer in the world. Chemotherapy resistance is a major obstacle to NSCLC therapy. This study explored the role and molecular mechanism of circular RNA 0011292 (circ_0011292) in tumorigenesis and chemoresistance of NSCLC. Methods: The levels of circ_0011292, miR-379-5p, and tripartite motif-containing protein 65 (TRIM65) were measured by quantitative real-time polymerase chain reaction or Western blot assay. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was monitored by flow cytometry. Cell migration and invasion were detected by transwell assay. The levels of apoptosis-related and epithelial-mesenchymal transition-related proteins were examined by Western blot. The half-inhibition concentration (IC50) of paclitaxel (PTX) was evaluated by CCK-8 assay. Xenograft model was established to analyze the effect of circ_0011292 on PTX resistance of NSCLC in vivo. The interaction among circ_0011292, miR-379-5p, and TRIM65 was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Results: Circ_0011292 and TRIM65 were upregulated, while miR-379-5p was downregulated in NSCLC tissues and cells. Circ_0011292 knockdown hindered NSCLC progression and enhanced PTX sensitivity of NSCLC. Circ_0011292 silencing reduced PTX resistance in vivo. Besides, miR-379-5p potentiated PTX sensitivity by targeting TRIM65. Also, circ_0011292 increased PTX resistance by sponging miR-379-5p. Conclusion: Circ_0011292 facilitated tumorigenesis and PTX resistance in NSCLC by regulating the miR-379-5p/TRIM65 axis, suggesting that circ_0011292 was a promising therapeutic target for NSCLC chemotherapy.

The E3 Ubiquitin Ligase TRIM65 Negatively Regulates Inflammasome Activation Through Promoting Ubiquitination of NLRP3.

The dysregulation of NLRP3 inflammasome plays a critical role in pathogenesis of various human inflammatory diseases, thus NLRP3 inflammasome activation must be tightly controlled at multiple levels. However, the underlying mechanism regulating NLRP3 inflammasome activation remains unclear. Herein, the effects of Tripartite motif-containing protein 65 (TRIM65) on NLRP3 inflammasome activation and the underlying molecular mechanism were investigated in vitro and in vivo. Inhibition or deletion of Trim65 could significantly strengthen agonist induced NLRP3 inflammasome activation in THP-1 cells and BMDMs, indicated by increased caspase-1 activation and interleukin-1ß secretion. However, TRIM65 had no effect on poly (dA: dT)-induced AIM2 inflammasome activation or flagellin-induced IPAF inflammasome activation. Mechanistically, immunoprecipitation assays demonstrated that TRIM65 binds to NACHT domain of NLRP3, promotes lys48- and lys63- linked ubiquitination of NLRP3 and restrains the NEK7-NLRP3 interaction, thereby inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1ß secretion. In vivo, three models of inflammatory diseases were used to confirm the suppression role of TRIM65 in NLRP3 inflammasome activation. TRIM65-deficient mice had a higher production of IL-1ß induced by lipopolysaccharide in sera, and more IL-1ß secretion and neutrophil migration in the ascites, and more severity of joint swelling and associated IL-1ß production induced by monosodium urate, suggesting that TRIM65 deficiency was susceptible to inflammation. Therefore, the data elucidate a TRIM65-dependent negative regulation mechanism of NLRP3 inflammasome activation and provide potential therapeutic strategies for the treatment of NLRP3 inflammasome-related diseases.

TRIM65 Promotes Invasion of Endometrial Stromal Cells by Activating ERK1/2/C-myc Signaling via Ubiquitination of DUSP6.

BACKGROUND: Endometriosis (EM) is a benign gynecological disease that shares some characteristics with malignancy, such as proliferation and invasion. So far, the pathogenesis of EM is still unclear. In this study, we investigated whether TRIM65 can play a role in the development of EM. METHODS: TRIM65 expression levels in eutopic, ectopic, and normal endometrium were detected by quantitative real-time PCR and Western blot. Cell proliferation and invasion of primary endometrial stromal (EMS) cells were detected by CCK-8 and Transwell analysis. The interaction between TRIM65 and DUSP6 or C-myc was measured by coimmunoprecipitation, ubiquitylation, dual luciferase, and chromatin immunoprecipitation analysis. RESULTS: We found that TRIM65 was identified as an up-regulated gene in ectopic endometrial tissues and EMS cells compared with control groups without EM. TRIM65 expression was positively correlated with the levels of p-ERK1/2, C-myc, matrix metalloproteinase-2, and integrin ß1 in ectopic endometrial tissues in patients and mice. TRIM65 promoted the cell proliferation and invasion of EMS cells via the ERK1/2/C-myc pathway through ubiquitination of DUSP6. C-myc promoted TRIM65 expression through inducing TRIM65 promoter activity. Additionally, the increased expression of TRIM65, C-myc, matrix metalloproteinase-2, integrin ß1, and p-ERK1/2 and the decreased expression of DUSP6 in ectopic endometrial tissues were significantly suppressed by inhibition of ERK1/2 signaling pathway in ectopic endometrial tissues in experimental mice model. CONCLUSION: In conclusion, TRIM65 promotes invasion of ectopic EMS cells by activating a feedback loop with the ERK1/2/C-myc signaling pathway and may be a potential therapeutic target for EM.