The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia.

BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.

HBV X Protein Induces Degradation of UBXN7, a Novel Negative Regulator of NF-κB Signaling, to Promote HBV Replication.

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using in vitro and in vivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase ß through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.

Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase.

The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

CircUBXN7 mitigates H/R-induced cell apoptosis and inflammatory response through the miR-622-MCL1 axis.

BACKGROUND: Hypoxia/reoxygenation (H/R)-mediated apoptosis and inflammation are major causes of tissue injury in acute myocardial infarction (AMI). Exploring the underlying mechanisms of cardiomyocyte injury induced by H/R is important for AMI treatment. Circular RNAs have been demonstrated to paly vital roles in the pathogenesis of AMI. Our study aimed to explore the function of circular RNA UBXN7 (circUBXN7) in regulating H/R-induced cardiomyocyte injury. METHODS: H/R-treated H9c2 cells and a mouse model of AMI were used to investigate the function of circUBXN7 in H/R damage and AMI. The expressions of circUNXN7, miR-622 and MCL1 were analyzed by RT-qPCR. CCK-8 was used for examining cell viability. Cell apoptosis was evaluated with caspase 3 activity and Annexin V/PI staining. MCL1, Bax, Bcl-2 and cleaved-caspase 3 were examined with western blot. ELISA was used to examine the secretion of IL-6, TNF-α and IL-1ß. RESULTS: CircUBXN7 was downregulated in patients and mice with AMI, as well as in H/R-treated cells. Overexpression of circUBXN7 mitigated H/R-mediated apoptosis and secretion of inflammatory factors including IL-6, TNF-α and IL-1ß. CircUBXN7 suppressed cell apoptosis and inflammatory reaction induced by H/R via targeting miR-622. MiR-622 targeted MCL1 to restrain its expression in H9c2 cells. Knockdown of MCL1 abrogated circUBXN7-mediated alleviation of apoptosis and inflammation after H/R treatment. CONCLUSION: CircUBXN7 mitigates cardiomyocyte apoptosis and inflammatory reaction in H/R injury by targeting miR-622 and maintaining MCL1 expression. Our study provides novel potential therapeutic targets for AMI treatment.

UBXN7 cofactor of CRL3KEAP1 and CRL2VHL ubiquitin ligase complexes mediates reciprocal regulation of NRF2 and HIF-1α proteins.

UBXN7 is a cofactor protein that provides a scaffold for both CRL3KEAP1 and CRL2VHL ubiquitin ligase complexes involved in the regulation of the NRF2 and HIF-1α protein levels respectively. NRF2 and HIF-1α are surveillance transcription factors that orchestrate the cellular response to oxidative stress (NRF2) or to hypoxia (HIF-1α). Since mitochondria are the main oxygen sensors as well as the principal producers of ROS, it can be presumed that they may be able to modulate the activity of CRL3KEAP1 and CRL2VHL complexes in response to stress. We have uncovered a new mechanism of such regulation that involves the UBXN7 cofactor protein and its regulation by mitochondrial MUL1 E3 ubiquitin ligase. High level of UBXN7 leads to HIF-1α accumulation, whereas low level of UBXN7 correlates with an increase in NRF2 protein. The reciprocal regulation of HIF-1α and NRF2 by UBXN7 is coordinated under conditions of oxidative stress or hypoxia. In addition, this molecular mechanism leads to different metabolic states; high level of UBXN7 and accumulation of HIF-1α support glycolysis, whereas inactivation of UBXN7 and activation of NRF2 confer increased OXPHOS. We describe a new mechanism by which MUL1 E3 ubiquitin ligase modulates the UBXN7 cofactor protein level and provides a reciprocal regulation of CRL3KEAP1 and CRL2VHL ubiquitin ligase complexes. Furthermore, we delineate how this regulation is reflected in NRF2 and HIF-1α accumulation and determines the metabolic state as well as the adaptive response to mitochondrial stress.

[Circular RNA-UBXN7 promotes proliferation, migration and suppresses apoptosis in hepatocellular cancer].

Objective: To investigate the effect and molecular mechanism of circular RNA-UBXN7 (circ_UBXN7) on the proliferation, migration and apoptosis of hepatocellular carcinoma cells. Methods: Circ_UBXN7 expression in the tissues and cells of hepatocellular cancer was detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the relationship between circ_UBXN7 expression and clinicopathological features, including age, gender, tumor volume, pathological classification, staging, and lymph node metastasis was analyzed. The full-length sequence of circ_UBXN7 with lentivirus carrying lenti circ_UBXN7 and lenti circ_UBXN7 shRNA was constructed to transfect hepatocellular cell lines (HepG2 and Huh-7), respectively. CCK-8 experiments were performed to detect the ability of up- or down-regulation of circ_UBXN7 on the proliferation of HEPG2 and HUH-7 cells. Annexin V / PI experiment was used to detect the changes in apoptosis of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression. JC-1 assay was used to detect the changes in mitochondrial potential energy of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression. Transwell was used to detect the migration ability of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression. Western blotting was used to detect the expressional change of TWIST, E-cadherin, N-cadherin and vimentin. Statistical analysis: The expression levels of circ_UBXN7 and clinicopathological features were measured by chi-square test. Two groups were compared by t-test and three groups and above were compared by single factor analysis of variance. LSD method was used for comparison between groups. Results: The expression of circ_UBXN7 in liver cancer tissues was significantly higher than adjacent tissues, and its expression level was significantly positively correlated with tumor volume, stage, and lymph node metastasis (P < 0.05). Lenti-circ_UBXN7 had up-regulated the expression of circ_UBXN7 in HEPG2 and HUH-7 cells and promoted cell proliferation. Lenti-circ_UBXN7-shRNA had down-regulated the expression of circ_UBXN7 and induced apoptosis. Lenti-circ_UBXN7-shRNA had reduced the mitochondrial membrane potential of cells. Lenti-circ_UBXN7 had promoted cell migration, while lenti-circ_UBXN7-shRNA had inhibited cell migration. Lenti-circ_UBXN7 had induced increased expression of Twist, N-cadherin, and Vimentin proteins, and reduced the expression of E-cadherin protein. Lenti-circ_UBXN7-shRNA had opposite effects on the expression levels of each protein. Starbase V2.0 software showed that miR-203a and circ_UBXN7 had potential binding sites, and miR-203a and circ_UBXN7 expression levels were negatively correlated in HEP ​​G2 and HUH-7 cells. Conclusion: circ_UBXN7 plays an important role in promoting the occurrence and development of liver cancer, and is expected to become a potential target for the treatment of liver cancer.