Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

TRIM38 protects chondrocytes from IL-1ß-induced apoptosis and degeneration via negatively modulating nuclear factor (NF)-κB signaling.

Tripartite motif protein 38 (TRIM38) has been documented as a vital modulator of inflammation. However, the relevance of TRIM38 in osteoarthritis is not yet known. In this work, we aimed to explore any possible effects of TRIM38 on interleukin-1ß (IL-1ß)-stimulated chondrocytes, an in vitro cellular model of osteoarthritis. Analyzing our data showed significant decreases in the levels of TRIM38 in chondrocytes following IL-1ß stimulation. Gain-of-function studies revealed that overexpression of TRIM38 markedly increased the viability of IL-1ß-stimulated chondrocytes while decreasing their rate of apoptosis and degeneration. Conversely, depletion of TRIM38 enhanced the sensitivity of chondrocytes to IL-1ß-induced injury. Further research demonstrated that TRIM38 was capable of inhibiting IL-1ß-induced activation of nuclear factor (NF)-κB signaling. Reactivation of NF-κB markedly reversed TRIM38-overexpression-mediated effects, while inhibition of NF-κB significantly abolished TRIM38-depletion-induced effects in IL-1ß-stimulated chondrocytes. In summary, the findings of this work suggest that TRIM38 is capable of ameliorating IL-1ß-induced apoptosis and degeneration of chondrocytes via suppression of NF-κB signaling. Our work indicates a potential role of TRIM38 in osteoarthritis and proposes it as a new therapeutic target for osteoarthritis.

TRIM37 negatively regulates inflammatory responses induced by virus infection via controlling TRAF6 ubiquitination.

Virus-induced cytokine storm has been a devastating actuality in clinic. The abnormal production of type I interferon (IFN-1) and upregulation of multiple cytokines induced strong inflammation and thus lead to shock and organ failure. As an E3 ubiquitin ligase, tripartite motif-containing 37 (TRIM37) regulates the ubiquitination of multiple proteins including TRAFs. RNA sequencing was performed to investigated the alteration of transcriptional profile of H1N1-infected patients. qRT-PCR assay was performed to investigate the RNA levels of certain genes. The group of immune cells was examined by the Flow cytometry analysis. H&E staining was applied to evaluate lung inflammation of WT and TRIM37-KO mice. ELISA assay was performed to demonstrate the alteration of multiple cytokines. The protein levels in NF-kB signaling was estimated by western blotting and immunoprecipitation assays were applied to demonstrate the direct interaction between TRIM37 and TRAF-6. The RNA level of TRIM37 decreased in CD11b+ cells of Flu-infected patients. Knockout of TRIM37 inhibited the immune responses of H1N1-infected mice. TRIM37 deficiency reduced the levels of virous proinflammatory cytokines in bone marrow derived macrophages (BMDMs). Mechanically, TRIM37 promoted the K63-linked ubiquitination of TRAF6. TRIM37 negatively regulated inflammatory responses induced by virus infection via promoting TRAF6 ubiquitination at K63.

Knockdown of TRIM32 inhibits tumor growth and increases the therapeutic sensitivity to temozolomide in glioma in a p53-dependent and -independent manner.

Tripartite motif protein 32 (TRIM32), an E3 ubiquitin ligase, has been reported to participate in many human cancers. However, the underlying role of TRIM32 in glioma remains largely unknown. Here, we aimed to explore the function of TRIM32 in glioma cells and the clinical implications and found that TRIM32 was upregulated in glioma tissues. Consistently, overexpression of TRIM32 promoted glioma U87 and U251 cell proliferation and conferred cell resistance to temozolomide (TMZ). Conversely, knockdown of TRIM32 inhibited glioma cells proliferation in vitro and in vivo and sensitized glioma cells to the treatment of TMZ in a p53-dependent and -independent manner. Mechanistically, knockdown of TRIM32 induced apoptosis of U87 an U251 cells. In addition, TRIM32 interacted with the antiapoptotic proteins BCL-xL and BCL-w, which antagonized the inhibitory effect of TRIM32 knockdown in U87 cells. Together, our study uncovered the role of TRIM32 in glioma and TRIM32 may be a potential therapeutic target for gliomas.

Brain-specific deletion of TRIM13 promotes metabolic stress-triggered insulin resistance, glucose intolerance, and neuroinflammation.

Diabetes has been associated with metabolic disorder, insulin resistance and neuroinflammation. However, the pathogenesis for HFD-induced injury of central nervous system (CNS) is still unclear. Tripartite Motif Containing 13 (TRIM13), also known as RFP2, is a member of TRIM proteins, and is associated with multiple cellular processes, such as apoptosis, survival and inflammation. However, the effects of TRIM13 on brain injury, especially the HFD-induced CNS damage, have not been investigated. To address this issue, the TRIM13flox/flox (fl/fl) mice were produced and then crossed them with Nestin-Cre mice to delete TRIM13 specifically in the brain (cKO). Then, T2D mice with obesity were established by chronic feeding of HFD. We found that brain-specific deletion of TRIM13 accelerated HFD-induced metabolic disorder, insulin resistance and systematic inflammatory response. In addition, HFDcKO mice exhibited significantly higher pro-inflammatory cytokines, including interleukin (IL)-6, IL-1ß and tumor necrosis factor-α (TNF-α), in cortex, hippocampus and hypothalamus tissues, which were comparable to the HFDfl/fl mice. Consistently, the activation of nuclear factor-κB (NF-κB) induced by HFD was further aggravated in mice with brain-specific loss of TRIM13. Moreover, glial activation in CNS stimulated by HFD was further promoted by TRIM13 knockout in brain, as evidenced by the up-regulated expression of glial fibrillary acidic protein (GFAP) and Iba-1. In hypothalamus, HFD reduced proopiomelanocortin (POMC) and enhanced neuropeptide Y (NPY) expression, which were further promoted in mice with brain-specific deletion of TRIM13. Meanwhile, insulin signaling pathway was disrupted by HFD in hypothalamus of mice, and these effects were exacerbated in HFDcKO mice. The in vitro analysis confirmed that TRIM13 knockout in glial cells considerably promoted palmitate (PAL)-induced inflammatory response by accelerating NF-κB signal, contributing to the insulin resistance in the isolated primary neurons. Together, these findings demonstrated that TRIM13 was involved in HFD-induced CNS injury and insulin resistance through regulating neuroinflammatory response, contributing to the modulation of peripheral metabolic disorders.

Tripartite motif 10 regulates cardiac hypertrophy by targeting the PTEN/AKT pathway.

The pathogenesis of cardiac hypertrophy is tightly associated with activation of intracellular hypertrophic signalling pathways, which leads to the synthesis of various proteins. Tripartite motif 10 (TRIM10) is an E3 ligase with important functions in protein quality control. However, its role in cardiac hypertrophy was unclear. In this study, neonatal rat cardiomyocytes (NRCMs) and TRIM10-knockout mice were subjected to phenylephrine (PE) stimulation or transverse aortic constriction (TAC) to induce cardiac hypertrophy in vitro and in vivo, respectively. Trim10 expression was significantly increased in hypertrophied murine hearts and PE-stimulated NRCMs. Knockdown of TRIM10 in NRCMs alleviated PE-induced changes in the size of cardiomyocytes and hypertrophy gene expression, whereas TRIM10 overexpression aggravated these changes. These results were further verified in TRIM10-knockout mice. Mechanistically, we found that TRIM10 knockout or knockdown decreased AKT phosphorylation. Furthermore, we found that TRIM10 knockout or knockdown increased ubiquitination of phosphatase and tensin homolog (PTEN), which negatively regulated AKT activation. The results of this study reveal the involvement of TRIM10 in pathological cardiac hypertrophy, which may occur by prompting of PTEN ubiquitination and subsequent activation of AKT signalling. Therefore, TRIM10 may be a promising target for treatment of cardiac hypertrophy.

Skeletal muscle in MuRF1 null mice is not spared in low-gravity conditions, indicating atrophy proceeds by unique mechanisms in space.

Microgravity exposure is associated with loss of muscle mass and strength. The E3 ubiquitin ligase MuRF1 plays an integral role in degrading the contractile apparatus of skeletal muscle; MuRF1 null (KO) mice have shown protection in ground-based models of muscle atrophy. In contrast, MuRF1 KO mice subjected to 21 days of microgravity on the International Space Station (ISS) were not protected from muscle atrophy. In a time course experiment microgravity-induced muscle loss on the ISS showed MuRF1 gene expression was not upregulated. A comparison of the soleus transcriptome profiles between spaceflight and a publicly available data set for hindlimb suspension, a claimed surrogate model of microgravity, showed only marginal commonalities between the models. These findings demonstrate spaceflight induced atrophy is unique, and that understanding of effects of space requires study situated beyond the Earth's mesosphere.

Muscle-specific regulation of right ventricular transcriptional responses to chronic hypoxia-induced hypertrophy by the muscle ring finger-1 (MuRF1) ubiquitin ligase in mice.

BACKGROUND: We recently identified a role for the muscle-specific ubiquitin ligase MuRF1 in right-sided heart failure secondary to pulmonary hypertension induced by chronic hypoxia (CH). MuRF1-/- mice exposed to CH are resistant to right ventricular (RV) dysfunction whereas MuRF1 Tg + mice exhibit impaired function indicative of heart failure. The present study was undertaken to understand the underlying transcriptional alterations in the RV of MuRF1-/- and MuRF1 Tg + mice. METHODS: Microarray analysis was performed on RNA isolated from the RV of MuRF1-/-, MuRF1 Tg+, and wild-type control mice exposed to CH. RESULTS: MuRF1-/- RV differentially expressed 590 genes in response to CH. Analysis of the top 66 genes (> 2-fold or < - 2-fold) revealed significant associations with oxidoreductase, transcription regulation, and transmembrane component annotations. The significant genes had promoters enriched for HOXD12, HOXC13, and RREB-1 protein transcription factor binding sites. MuRF1 Tg + RV differentially expressed 150 genes in response to CH. Analysis of the top 45 genes (> 3-fold or < - 3-fold) revealed significant associations with oxidoreductase-metabolic, glycoprotein-transmembrane-integral proteins, and alternative splicing/splice variant annotations. The significant genes were enriched for promoters with ZIC1 protein transcription factor binding sites. CONCLUSIONS: The differentially expressed genes in MuRF1-/- and MuRF1 Tg + RV after CH have common functional annotations related to oxidoreductase (including antioxidant) and transmembrane component functions. Moreover, the functionally-enhanced MuRF1-/- hearts regulate genes related to transcription, homeobox proteins, and kinases/phosphorylation. These studies also reveal potential indirect effects of MuRF1 through regulating Rreb-1, and they reveal mechanisms by which MuRF1 may transcriptionally regulate anti-oxidant systems in the face of right heart failure.

TRIM22 knockdown suppresses chronic myeloid leukemia via inhibiting PI3K/Akt/mTOR signaling pathway.

Tripartite motif-containing 22 (TRIM22) is reported to participate in numerous cellular activities. Recent studies confirm that TRIM22 is a target gene for P53, and inhibits clonogenic growth of leukemic U-937 cells. The current study aims to discover the effect of TRIM22 in progression of human chronic myeloid leukemia (CML) and explore the related mechanism. TRIM22 was knocked down by siRNA transfection in CML cell K562. We observed that TRIM22 knockdown decreased proliferation and invasion in K562 cells. TRIM22 knockdown significantly induced cell cycle arrest by regulating the level of CDK4, Cyclin D1, P70S6K, and P53 in K562 cell. Moreover, loss of TRIM22 also promoted apoptosis through modulation of Bcl-2, Bax and active Caspase 3 in K562 cell. Furthermore, we demonstrated that TRIM22 knockdown inhibited the activation of PI3K/Akt/mTOR pathway by decreasing the level of the phosphorylated form p-Akt and p-mTOR in K562 cell. In conclusion, loss of TRIM22 suppresses the progression and invasion of CML through regulation of PI3K/Akt/mTOR pathway, suggesting that TRIM22 might be as a potential target for the treatment strategy of CML.

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling.

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-ß, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-ß (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

TRIM56 Suppresses Multiple Myeloma Progression by Activating TLR3/TRIF Signaling

PURPOSE: Tripartite-motif-containing protein 56 (TRIM56) has been found to exhibit a broad antiviral activity, depending upon E3 ligase activity. Here, we attempted to evaluate the function of TRIM56 in multiple myeloma (MM) and its underlying molecular basis. MATERIALS AND METHODS: TRIM56 expression at the mRNA and protein level was measured by qRT PCR and western blot analysis. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry analysis was performed to investigate the effect of TRIM56 on MM cell proliferation and apoptosis. The concentrations of interferon (IFN)-β, interleukin (IL)-6, and tumor necrosis factor-α in MM cell culture supernatants were detected with respective commercial ELISA kits. Western blot was employed to determine the effect of TRIM56 on toll-like receptor 3 (TLR3)/toll-IL-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) signaling pathway. RESULTS: TRIM56 expression was prominently decreased in MM cells. Poly (dA:dT)-induced TRIM56 overexpression in U266 cells suppressed proliferation, induced apoptosis, and enhanced inflammatory cytokine production, while TRIM56 knockdown improved growth, diminished apoptosis, and inhibited inflammatory cytokine secretion in RPMI8226 cells. Moreover, TRIM56 knockdown blocked TLR3 signaling pathway. Furthermore, poly (I:C), a TLR3 agonist, markedly abolished TRIM56 depletion-induced increase of proliferation, decrease of apoptosis, and reduction of inflammatory factor in MM cells. CONCLUSION: TRIM56 may act as a tumor suppressor in MM through activation of TLR3/TRIF signaling pathway, contributing to a better understanding of the molecular mechanism of TRIM56 involvement in MM pathogenesis and providing a promising therapy strategy for patients with MM.

An HIV-1 capsid binding protein TRIM11 accelerates viral uncoating.

BACKGROUND: Several members of the TRIM family have been implicated in antiviral defense. Our previous report showed that human TRIM11 potently inhibited HIV-1 transduction by reducing the viral reverse transcripts. These results prompted us to examine the effect of TRIM11 on HIV-1 uncoating, which is closely related to viral reverse transcription. RESULTS: Using a combination of in vitro binding and in situ proximity ligation assay, we showed that TRIM11 could interact with HIV-1 capsid. Overexpression of TRIM11 accelerates HIV-1 uncoating and reduces viral reverse transcription indicated by the fate-of-capsid assay and quantitative PCR respectively. Knockdown of TRIM11 enhanced HIV-1 capsid stability and increased viral reverse transcription. However, the replication of another retrovirus MLV is not affected by TRIM11. Moreover, the reverse transcription of HIV-1 mutant bearing capsid G89V showed insensitivity to restriction by TRIM11, indicating that the viral determinant of restriction by TRIM11 might reside on capsid. Using microtubule dynamics inhibitors, we revealed that microtubule dynamics contributes to TRIM11-mediated HIV-1 capsid premature disassembly and the reduction of reverse transcription levels. Finally, we demonstrated that TRIM11 inhibits HIV-1 transduction and accelerates viral uncoating in HIV-1 permissive THP-1-derived macrophages. CONCLUSIONS: We identify TRIM11 as a new HIV-1 capsid binding protein. Our data also reveal that TRIM11 restricts HIV-1 reverse transcription by accelerating viral uncoating, and microtubule dynamics is implicated in TRIM11-imposed block to early events of HIV-1 replication.

Fenofibrate unexpectedly induces cardiac hypertrophy in mice lacking MuRF1.

The muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) is critical in regulating both pathological and physiological cardiac hypertrophy in vivo. Previous work from our group has identified MuRF1's ability to inhibit serum response factor and insulin-like growth factor-1 signaling pathways (via targeted inhibition of cJun as underlying mechanisms). More recently, we have identified that MuRF1 inhibits fatty acid metabolism by targeting peroxisome proliferator-activated receptor alpha (PPARα) for nuclear export via mono-ubiquitination. Since MuRF1-/- mice have an estimated fivefold increase in PPARα activity, we sought to determine how challenge with the PPARα agonist fenofibrate, a PPARα ligand, would affect the heart physiologically. In as little as 3 weeks, feeding with fenofibrate/chow (0.05% wt/wt) induced unexpected pathological cardiac hypertrophy not present in age-matched sibling wild-type (MuRF1+/+) mice, identified by echocardiography, cardiomyocyte cross-sectional area, and increased beta-myosin heavy chain, brain natriuretic peptide, and skeletal muscle α-actin mRNA. In addition to pathological hypertrophy, MuRF1-/- mice had an unexpected differential expression in genes associated with the pleiotropic effects of fenofibrate involved in the extracellular matrix, protease inhibition, hemostasis, and the sarcomere. At both 3 and 8 weeks of fenofibrate treatment, the differentially expressed MuRF1-/- genes most commonly had SREBP-1 and E2F1/E2F promoter regions by TRANSFAC analysis (54 and 50 genes, respectively, of the 111 of the genes >4 and <-4 log fold change; P ≤ .0004). These studies identify MuRF1's unexpected regulation of fenofibrate's pleiotropic effects and bridges, for the first time, MuRF1's regulation of PPARα, cardiac hypertrophy, and hemostasis.