Tripartite Motif-containing 21 Research Articles

TRIM21-mediated ubiquitylation of TAT suppresses liver metastasis in gallbladder cancer

Liver metastasis is common in patients with gallbladder cancer (GBC), imposing a significant challenge in clinical management and serving as a poor prognostic indicator. However, the mechanisms underlying liver metastasis remain largely unknown. Here, we report a crucial role of tyrosine aminotransferase (TAT) in liver metastasis of GBC. TAT is frequently up-regulated in GBC tissues. Increased TAT expression is associated with frequent liver metastasis and poor prognosis of GBC patients. Overexpression of TAT promotes GBC cell migration and invasion in vitro, as well as liver metastasis in vivo. TAT knockdown has the opposite effects. Intriguingly, TAT promotes liver metastasis of GBC by potentiating cardiolipin-dependent mitophagy. Mechanistically, TAT directly binds to cardiolipin and leads to cardiolipin externalization and subsequent mitophagy. Moreover, TRIM21 (Tripartite Motif Containing 21), an E3 ubiquitin ligase, interacts with TAT. The histine residues 336 and 338 at TRIM21 are essential for this binding. TRIM21 preferentially adds the lysine 63 (K63)-linked ubiquitin chains on TAT principally at K136. TRIM21-mediated TAT ubiquitination impairs its dimerization and mitochondrial location, subsequently inhibiting tumor invasion and migration of GBC cells. Therefore, our study identifies TAT as a novel driver of GBC liver metastasis, emphasizing its potential as a therapeutic target.

SPARC stabilizes ApoE to induce cholesterol-dependent invasion and sorafenib resistance in hepatocellular carcinoma.

Dysregulation of cholesterol homeostasis is implicated in the development and progression of hepatocellular carcinoma (HCC) that is characterized by intrahepatic and early extrahepatic metastasis. A better understanding of the underlying mechanisms regulating cholesterol metabolism in HCC could help identify strategies to circumvent the aggressive phenotype. Here, we found that high expression of intracellular SPARC was significantly associated with elevated cholesterol levels and an enhanced invasive phenotype in HCC. SPARC potentiated cholesterol accumulation in HCC cells during tumor progression by stabilizing the ApoE protein. Mechanistically, SPARC competitively bound to ApoE, impairing its interaction with the E3 ligase tripartite motif containing 21 (TRIM21) and preventing its ubiquitylation and subsequent degradation. ApoE accumulation led to cholesterol enrichment in HCC cells, stimulating PI3K-AKT signaling and inducing epithelial-mesenchymal transition (EMT). Importantly, sorafenib-resistant HCC cells were characterized by increased expression of intracellular SPARC, elevated cholesterol levels, and enhanced invasive capacity. Inhibiting SPARC expression or reducing cholesterol levels enhanced the sensitivity of HCC cells to sorafenib treatment. Together, these findings unveil interplay between SPARC and cholesterol homeostasis. Targeting SPARC-triggered cholesterol-dependent oncogenic signaling is a potential therapeutic strategy for advanced HCC.

UBE2N Regulates Oncoprotein Networks in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate from clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). Ubiquitination is an essential process for various cellular maintenance functions, including protein degradation and scaffolding, which is often perturbed in a variety of cancers. Ubiquitination of protein substrates involves three related enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase. In humans, there are up to 40 E2 conjugating enzymes, among which Ubiquitin conjugating enzyme E2 N (UBE2N) stands out as one of the most crucial and overexpressed E2 enzymes in AML cells. Our recent study reported that UBE2N is a druggable target in MDS and AML (Barreyro et al., Science Translational Medicine, 2022), and we found that its catalytic active site cysteine 87 is required for various AML mouse models, including MLL-AF9, FLT3-ITD, AML1-ETO9a, and MN1 (unpublished). These observations indicate that the catalytic function of UBE2N is critical for AML cells but dispensable for normal hematopoiesis. To examine the active state of UBE2N, we investigated the “charged” version of UBE2N, which contains a ubiquitin bound to cysteine 87, in normal hematopoietic cells and in a AML cell line panels. Our findings confirmed that the activated form of UBE2N is highly expressed in human AML cells compared to normal hematopoietic cells. UBE2N exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in conjunction with selected E3 ligases, leading to the stabilization and/or activation of protein substrates. To identify the relevant targets of UBE2N in AML, we conducted a ubiquitin-enrichment followed by a proteomic analysis in isogenic UBE2N-deficient AML cells compared to WT AML cells. We identified ~300 proteins that are ubiquitinated by UBE2N. Importantly, UBE2N regulates a network of leukemia-associated oncoproteins, including SYNCRIP, HUWE1, NPM1, TYK2, IKBKG, VAV1, BAX, IGF2BP2, IRF4, IRAK4, RBMX, STAT3, and BTK. UBE2N can interact with various E3 ligases in a cell- and context-dependent manner. To gain insight into the major E3 ligase that cooperates with activated UBE2N in AML, we performed a proximity-based labeling assay followed by mass spectrometry. This interactome screen identified Tripartite motif containing 21 (TRIM21) as the top E3 ligase that partners with UBE2N in AML cells. TRIM21 is overexpressed in AML patients compared to healthy controls. Moreover, deletion of TRIM21 in AML cells impaired cell viability and proliferation in vitro and prolonged survival in a xenograft model in vivo, which phenocopies the loss of UBE2N. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. To prioritize the UBE2N-dependent oncoprotein network, we focused on UBE2N-TRIM21 regulated signaling in AML. For this, a gene expression analysis was performed in UBE2N-deficient AML cells that were restored with TRIM21. The top UBE2N-TRIM21 oncoprotein network that emerged was related to interferon-STAT signaling. Additionally, both UBE2N and TRIM21 were found to regulate STAT3 ubiquitination. Deletion or inhibition of UBE2N catalytic activity significantly reduced total and activated STAT3 in AML cells. Finally, the expression of constitutively active STAT3 partially restored the functional defect of AML cells treated with a UBE2N inhibitor, indicating that STAT3 function, a known driver of LSPCs, is regulated via the UBE2N-TRIM21 axis in AML. In summary, our data reveal that UBE2N regulates oncoprotein networks in AML and that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, effectively suppresses LSPCs.

O-GlcNAcylation of melanophilin enhances radiation resistance in glioblastoma via suppressing TRIM21 mediated ubiquitination.

The molecular mechanism of glioblastoma (GBM) radiation resistance remains poorly understood. The aim of this study was to elucidate the potential role of Melanophilin (MLPH) O-GlcNAcylation and the specific mechanism through which it regulates GBM radiotherapy resistance. We found that MLPH was significantly upregulated in recurrent GBM tumor tissues after ionizing radiation (IR). MLPH induced radiotherapy resistance in GBM cells and xenotransplanted human tumors through regulating the NF-κB pathway. MLPH was O-GlcNAcylated at the conserved serine 510, and radiation-resistant GBM cells showed higher levels of O-GlcNAcylation of MLPH. O-GlcNAcylation of MLPH protected its protein stability and tripartite motif containing 21(TRIM21) was identified as an E3 ubiquitin ligase promoting MLPH degradation whose interaction with MLPH was affected by O-GlcNAcylation. Our data demonstrate that MLPH exerts regulatory functions in GBM radiation resistance by promoting the NF-κB signaling pathway and that O-GlcNAcylation of MLPH both stabilizes and protects it from TRIM21-mediated ubiquitination. These results identify a potential mechanism of GBM radiation resistance and suggest a potential therapeutic strategy for GBM treatment.

Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.

TRIM21 mediates the synergistic effect of Olaparib and Sorafenib by degrading BRCA1 through ubiquitination in TNBC

Triple-negative breast cancer (TNBC) is a heterogeneous and aggressive type of breast cancer with a poor prognosis and a high recurrence rate. Chemotherapy is still the mainstay of treatment for cancer patients without a genetic BRCA mutation, despite the approval of Olaparib, an inhibitor of the poly (ADP-ribose) polymerase (PARP) enzyme. Tripartite motif containing-21 (TRIM21) is one of the TRIM family members that has been investigated in various types of cancer. Here, we found that a low TRIM21 expression level was correlated with poor overall survival of TNBC patients. Knockout of TRIM21 promoted the proliferation of TNBC cells in vivo and in vitro, as well as migratory and invasive capabilities in vitro. Importantly, breast cancer susceptibility gene 1 (BRCA1) was identified as a ubiquitination substrate of TRIM21. It was confirmed that BRCA1 was upregulated after Olaparib treatment, which may explain the relative resistance of BRCA1-proficient TNBC cells to Olaparib. Moreover, Sorafenib, a standard treatment for hepatocellular carcinoma, increased the sensitivity of TNBC cells to Olaparib by promoting TRIM21-mediated ubiquitination degradation of BRCA1. Thus, a synergic effect of Olaparib and Sorafenib was found in vitro and in vivo. This combined treatment also aggravated DNA damage, cell cycle arrest, and apoptosis of TNBC cells. In summary, the findings verified the synergistic effect of Olaparib and Sorafenib and revealed TRIM21 as a potential target for TNBC therapy.

FGF19 promotes nasopharyngeal carcinoma progression by inducing angiogenesis via inhibiting TRIM21-mediated ANXA2 ubiquitination

PurposeNasopharyngeal carcinoma (NPC) has characteristics of high invasion and early metastasis. Most NPC patients present with locoregionally advanced illness when first diagnosed. Therefore, it is urgent to discover NPC biomarkers. Fibroblast growth Factor 19 (FGF19) plays a role in various physiological or pathological processes, including cancer. In this research, we discovered the importance of FGF19 in NPC, and clarified its role in tumour angiogenesis.MethodsWestern blotting, immunohistochemistry and ELISA were used to investigate FGF19 expression in NPC. Then we took CCK8, colony formation, Transwell and wound healing assays to identify the influence of FGF19 on NPC malignant behaviours. The proliferative and metastatic capacity of FGF19 were evaluated in nude mice and zebrafish. The role of FGF19 in angiogenesis was investigated by tube formation and Matrigel plug angiogenesis assays. We then evaluated the variation in Annexin A2(ANXA2) levels with the treatment of FGF19. Lastly, co-immunoprecipitation and ubiquitination assays were performed to identify the mechanisms involved.ResultsFGF19 levels were elevated in tissues and serum of NPC patients and were associated with poor clinical stages. High expression of FGF19 promoted NPC malignant behaviours. In particular, FGF19 expression was correlated with microvessel density in tissues and NPC-derived FGF19 could accelerate angiogenesis in vitro and in vivo. Mechanistically, FGF19 influenced ANXA2 expression to promote angiogenesis. Moreover, tripartite motif-containing 21(TRIM21) interacted with ANXA2 and was responsible for ANXA2 ubiquitination.ConclusionFGF19 promoted NPC angiogenesis by inhibiting TRIM21-mediated ANXA2 ubiquitination. It may serve as a noninvasive biomarker for NPC and provides new insights for therapy.

DUSP2 recruits CSNK2A1 to suppress AKT1-mediated apoptosis resistance under hypoxic microenvironment in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic tumor microenvironment (TME), which aids tumor progression, drug resistance, and immune evasion. Dual-specificity phosphatase 2 (DUSP2), a member of the mitogen-activated protein kinase phosphatase family, regulates pancreatic cancer metastasis. However, its role in the hypoxic TME in PDAC remains unknown. We explored the role of DUSP2 by simulating the hypoxic TME. DUSP2 significantly promoted apoptosis in PDAC both in vitro and in vivo, mainly through AKT1 rather than ERK1/2. Mechanistically, DUSP2 competed with AKT1 to bind to casein kinase 2 alpha 1 (CSNK2A1) and inhibited the phosphorylation of AKT1, which plays a crucial role in apoptosis resistance. Interestingly, aberrant activation of AKT1 resulted in an increase in the ubiquitin E3 ligase tripartite motif-containing 21 (TRIM21), which binds to and mediates the ubiquitination-dependent proteasomal degradation of DUSP2. Overall, we identified CSNK2A1 as a novel binding partner of DUSP2 that promotes PDAC apoptosis through CSN2KA1/AKT1 in an ERK1/2-independent manner. Activation of AKT1 also mediated proteasomal degradation of DUSP2 via the AKT1/TRIM21 positive feedback loop. We propose increasing the level of DUSP2 as a potential therapeutic strategy for PDAC.

TRIM21 ubiquitylates GPX4 and promotes ferroptosis to aggravate ischemia/reperfusion-induced acute kidney injury

AimsThis study aims to verify the molecular mechanism that Tripartite motif containing 21 (TRIM21) promotes ubiquitination degradation of glutathione peroxidase 4 (GPX4) by regulating ferroptosis, and to discuss the feasibility of TRIM21 as a new therapeutic target for acute kidney injury (AKI). Materials and methodsIschemia-reperfusion (I/R)-AKI model was constructed using Trim21+/+ and Trim21−/− mice, and the expression of markers associated with kidney injury and ferroptosis were evaluated. HK-2 cells were treated by RSL3 and Erastin, and a hypoxia/reoxygenation (H/R) model was constructed to simulate I/R injury in vivo. Key findingsIn vivo, TRIM21 is highly expressed in I/R kidney tissues. Loss of TRIM21 alleviated I/R-AKI and improved renal function. The upregulation of GPX4, a key ferroptosis regulator, and the mild mitochondrial damage suggested that loss of TRIM21 had a negative regulation of ferroptosis. In vitro, TRIM21 was highly expressed in H/R models, and overexpression of TRIM21 in HK-2 cells increased ROS production, promoted intracellular iron accumulation, and boosted cellular sensitivity to RSL3 and Erastin. Mechanistically, we confirmed that GPX4 is a substrate of TRIM21 and can be degraded by TRIM21-mediated ubiquitination, suggesting that inhibiting TRIM21 attenuates ferroptosis. A JAK2 inhibitor Fedratinib downregulated TRIM21 expression and reduced damage both in vivo and in vitro, which is correlated with the upregulation of GPX4. SignificanceOur study showed that loss of TRIM21 could alleviate ferroptosis induced by I/R, revealed the mechanism of ubiquitination degradation of GPX4 by TRIM21 and suggested TRIM21 is a potential target for the treatment of AKI.

TRIM21 inhibits irradiation-induced mitochondrial DNA release and impairs antitumour immunity in nasopharyngeal carcinoma tumour models

Although radiotherapy can promote antitumour immunity, the mechanisms underlying this phenomenon remain unclear. Here, we demonstrate that the expression of the E3 ubiquitin ligase, tumour cell-intrinsic tripartite motif-containing 21 (TRIM21) in tumours, is inversely associated with the response to radiation and CD8+ T cell-mediated antitumour immunity in nasopharyngeal carcinoma (NPC). Knockout of TRIM21 modulates the cGAS/STING cytosolic DNA sensing pathway, potentiates the antigen-presenting capacity of NPC cells, and activates cytotoxic T cell-mediated antitumour immunity in response to radiation. Mechanistically, TRIM21 promotes the degradation of the mitochondrial voltage-dependent anion-selective channel protein 2 (VDAC2) via K48-linked ubiquitination, which inhibits pore formation by VDAC2 oligomers for mitochondrial DNA (mtDNA) release, thereby inhibiting type-I interferon responses following radiation exposure. In patients with NPC, high TRIM21 expression was associated with poor prognosis and early tumour relapse after radiotherapy. Our findings reveal a critical role of TRIM21 in radiation-induced antitumour immunity, providing potential targets for improving the efficacy of radiotherapy in patients with NPC.

Stress granule homeostasis is modulated by TRIM21-mediated ubiquitination of G3BP1 and autophagy-dependent elimination of stress granules

ABSTRACT Eukaryotic stress granules (SGs) are highly dynamic assemblies of untranslated mRNAs and proteins that form through liquid-liquid phase separation (LLPS) under cellular stress. SG formation and elimination process is a conserved cellular strategy to promote cell survival, although the precise regulation of this process is poorly understood. Here, we screened six E3 ubiquitin ligases present in SGs and identified TRIM21 (tripartite motif containing 21) as a central regulator of SG homeostasis that is highly enriched in SGs of cells under arsenite-induced oxidative stress. Knockdown of TRIM21 promotes SG formation whereas overexpression of TRIM21 inhibits the formation of physiological and pathological SGs associated with neurodegenerative diseases. TRIM21 catalyzes K63-linked ubiquitination of the SG core protein, G3BP1 (G3BP stress granule assembly factor 1), and G3BP1 ubiquitination can effectively inhibit LLPS, in vitro. Recent reports suggested the involvement of macroautophagy/autophagy, as a stress response pathway, in the regulation of SG homeostasis. We systematically investigated well-defined autophagy receptors and identified SQSTM1/p62 (sequestosome 1) and CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) as the primary receptors that directly interact with G3BP1 during arsenite-induced stress. Endogenous SQSTM1 and CALCOCO2 localize to the periphery of SGs under oxidative stress and mediate SG elimination, as single knockout of each receptor causes accumulation of physiological and pathological SGs. Collectively, our study broadens the understanding in the regulation of SG homeostasis by showing that TRIM21 and autophagy receptors modulate SG formation and elimination respectively, suggesting the possibility of clinical targeting of these molecules in therapeutic strategies for neurodegenerative diseases. Abbreviations: ACTB: actin beta; ALS: amyotrophic lateral sclerosis; BafA1: bafilomycin A1; BECN1: beclin 1; C9orf72: C9orf72-SMCR8 complex subunit; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Co-IP: co-immunoprecipitation; DAPI: 4’,6-diamidino-2-phenylindole; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; G3BP1: G3BP stress granule assembly factor 1; GFP: green fluorescent protein; LLPS: liquid-liquid phase separation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; NES: nuclear export signal; OPTN: optineurin; RFP: red fluorescent protein; SQSTM1/p62: sequestosome 1; SG: stress granule; TAX1BP1: Tax1 binding protein 1; TOLLIP: toll interacting protein; TRIM21: tripartite motif containing 21; TRIM56: tripartite motif containing 56; UB: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Targeting VPS72 inhibits ACTL6A/MYC axis activity in HCC progression.

HCC is a highly heterogeneous disease that is caused largely by genomic copy number variations. Herein, the mechanistic and therapeutically targeted role of vacuolar protein sorting 72 homologue (VPS72), a novel copy number variation cis-driven gained gene identified by genome-wide copy number variation and transcriptome analyses in HCC, is not well understood. First, overexpression of VPS72 enhanced the initiation and progression of HCC in vitro and in vivo . Mechanistically, VPS72 interacted with the oncoproteins MYC and actin-like 6A (ACTL6A) and promoted the formation of the ACTL6A/MYC complex. Furthermore, ACTL6A regulated VPS72 protein stability by weakening the interaction between tripartite motif containing 21 (TRIM21) and VPS72. Thus, the interaction between VPS72 and ACTL6A enhanced the affinity of MYC for its target gene promoters and promoted their transcription, thereby contributing to HCC progression, which was inhibited by adeno-associated virus serotype 8 (AAV8)-mediated short hairpin RNA (shRNA) against VPS72. This study reveals the molecular mechanism of ACTL6A/VPS72/MYC in HCC, providing a theoretical basis and therapeutic target for this malignancy.

CLEC-1 is a death sensor that limits antigen cross-presentation by dendritic cells and represents a target for cancer immunotherapy.

Tumors exploit numerous immune checkpoints, including those deployed by myeloid cells to curtail antitumor immunity. Here, we show that the C-type lectin receptor CLEC-1 expressed by myeloid cells senses dead cells killed by programmed necrosis. Moreover, we identified Tripartite Motif Containing 21 (TRIM21) as an endogenous ligand overexpressed in various cancers. We observed that the combination of CLEC-1 blockade with chemotherapy prolonged mouse survival in tumor models. Loss of CLEC-1 reduced the accumulation of immunosuppressive myeloid cells in tumors and invigorated the activation state of dendritic cells (DCs), thereby increasing T cell responses. Mechanistically, we found that the absence of CLEC-1 increased the cross-presentation of dead cell-associated antigens by conventional type-1 DCs. We identified antihuman CLEC-1 antagonist antibodies able to enhance antitumor immunity in CLEC-1 humanized mice. Together, our results demonstrate that CLEC-1 acts as an immune checkpoint in myeloid cells and support CLEC-1 as a novel target for cancer immunotherapy.

TRIM21‑a potential biomarker for the prognosis of thyroid cancer.

Thyroid cancer (THCA) is one of the commonest malignancies associated with increased recurrence. Therefore, identifying the putative molecular markers and therapeutic targets to improve the treatment of THCA is essential. The present study analyzed the potential role of tripartite motif-containing 21 (TRIM21), a member of the TRIM family belonging to the subfamily of E3 ubiquitin ligases, in the progression of THCA. Using bioinformatics analysis and immunohistochemistry of THCA tissues, it was observed that TRIM21 is overexpressed in THCA tissues. The present study also found that TRIM21 is associated with lymph node metastasis and high-risk recurrence of THCA. Furthermore, it identified a promotional role of TRIM21 in THCA cell migration and invasion. In addition, the present study analyzed TRIM21-enriched pathways and co-expressed genes in THCA. The present study suggested that TRIM21 may serve as a potential biomarker for THCA prognosis.

Tumor Cell-Intrinsic E3 Ligase TRIM21 Restrains Radiation-Induced Antitumor Immunity by Decreasing Mitochondrial DNA Release from VDAC2 Oligomers

<h3>Purpose/Objective(s)</h3> Radiotherapy has been proved to activate antitumor immunity but how aberrantly expressed genes in cancer regulated this process remains elusive. <h3>Materials/Methods</h3> We identified TRIM21, an E3 ligase gene, by bioinformatics analysis based on gross sequencing and single-cell sequencing data of nasopharyngeal carcinoma (NPC). The impact of TRIM21on radiation-induced antitumor immune response was identified in vitro and in vivo by TRIM21 knockout. We also determined the prognosis value of TRIM21 in NPC tissues with immunohistochemical analysis. <h3>Results</h3> Tumor cell-intrinsic E3 ligase tripartite motif-containing 21(TRIM21) is inversely associated with CD8+ T cell antitumor immunity and radiation response in NPC. Knockout of TRIM21 promoted radiation-induced antigen presentation, chemokine expression, dendritic cells, and CD8+ T cells activation in vitro, and led to enhanced radiation efficacy and immune response in MC38 murine colon adenocarcinoma, and transplanted NPC tumor in humanized mice in vivo. In mechanism, knockout of TRIM21 reversed its degradation of mitochondrial voltage-dependent anion-selective channel protein 2 (VDAC2), which resulted in sufficient VDAC2 oligomerization, mitochondrial DNA release, and thus strengthened activation of STING and type I interferon signaling during radiation. Clinically, high expression of TRIM21 in NPC indicated poor prognosis and tumor relapse after radiation. <h3>Conclusion</h3> Tumor cell-intrinsic TRIM21 suppressed radiation-induced antitumor immunity and predicted poor survival in NPC patients.

The emerging roles of TRIM21 in coordinating cancer metabolism, immunity and cancer treatment.

Tripartite motif containing-21 (TRIM21), an E3 ubiquitin ligase, was initially found to be involved in antiviral responses and autoimmune diseases. Recently studies have reported that TRIM21 plays a dual role in cancer promoting and suppressing in the occurrence and development of various cancers. Despite the fact that TRIM21 has effects on multiple metabolic processes, inflammatory responses and the efficacy of tumor therapy, there has been no systematic review of these topics. Herein, we discuss the emerging role and function of TRIM21 in cancer metabolism, immunity, especially the immune response to inflammation associated with tumorigenesis, and also the cancer treatment, hoping to shine a light on the great potential of targeting TRIM21 as a therapeutic target.

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

Trim-21 promotes proteasomal degradation of CED-1 for apoptotic cell clearance in C. elegans.

The phagocytic receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans. Whether appropriate levels of CED-1 are maintained for executing the engulfment function remains unknown. Here, we identified the C. elegans E3 ubiquitin ligase tripartite motif containing-21 (TRIM-21) as a component of the CED-1 pathway for apoptotic cell clearance. When the NPXY motif of CED-1 was bound to the adaptor protein CED-6 or the YXXL motif of CED-1 was phosphorylated by tyrosine kinase SRC-1 and subsequently bound to the adaptor protein NCK-1 containing the SH2 domain, TRIM-21 functioned in conjunction with UBC-21 to catalyze K48-linked poly-ubiquitination on CED-1, targeting it for proteasomal degradation. In the absence of TRIM-21, CED-1 accumulated post-translationally and drove cell corpse degradation defects, as evidenced by direct binding to VHA-10. These findings reveal a unique mechanism for the maintenance of appropriate levels of CED-1 to regulate apoptotic cell clearance.

Direct knockdown of phospho-PTM targets mediated by TRIM21 can improve personalized treatment in breast cancer.

In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'. To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7-8days. This significantly inhibited BC survival (p < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity (p < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.

TRIM21 deficiency promotes cell proliferation and tumorigenesis via regulating p21 expression in ovarian cancer

ABSTRACT Tripartite motif-containing 21 (TRIM21) has been reported to have a cancer-promoting or anticancer effect in various tumors; however, its role in ovarian cancer (OC) remains to be elucidated. In this study, we explored the biological function of TRIM21 in OC progression and investigated the potential mechanisms. We found that TRIM21 was remarkably decreased in OC tissues and cell lines compared with adjacent-cancerous tissues and normal ovarian epithelium cell. Decreased expression of TRIM21 in OC patients was significantly correlated with shorter overall and disease-specific survival by The Cancer Genome Atlas database (TCGA) analysis. Functional assays revealed that TRIM21 inhibited the migration and invasion of OC cells; and that TRIM21 also obviously impaired cell proliferation by inhibiting cell cycle progression in vitro and in vivo. Taken together, our results suggest that TRIM21 may be a promising biomarker and target for OC diagnosis and treatment.