E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

The FBXW7-binding sites on FAM83D are potential targets for cancer therapy.

Increasing evidence shows the oncogenic function of FAM83D in human cancer, but how FAM83D exerts its oncogenic function remains largely unclear. Here, we investigated the importance of FAM83D/FBXW7 interaction in breast cancer (BC). We systematically mapped the FBXW7-binding sites on FAM83D through a comprehensive mutational analysis together with co-immunoprecipitation assay. Mutations at the FBXW7-binding sites on FAM83D led to that FAM83D lost its capability to promote the ubiquitination and proteasomal degradation of FBXW7; cell proliferation, migration, and invasion in vitro; and tumor growth and metastasis in vivo, indicating that the FBXW7-binding sites on FAM83D are essential for its oncogenic functions. A meta-evaluation of FAM83D revealed that the prognostic impact of FAM83D was independent on molecular subtypes. The higher expression of FAM83D has poorer prognosis. Moreover, high expression of FAM83D confers resistance to chemotherapy in BCs, which is experimentally validated in vitro. We conclude that identification of FBXW7-binding sites on FAM83D not only reveals the importance for FAM83D oncogenic function, but also provides valuable insights for drug target.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

IMPORTANCE: Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon ß. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

Promotion of colorectal cancer progression by immune-related lnc-SOX9-4 via suppression of YBX1 poly-ubiquitination and degradation.

BACKGROUND: Recent research has highlighted the versatile functions of long non-coding RNAs (lncRNAs) in the onset and progression of various malignancies. Still, insufficient knowledge is available on how lnc-SOX9-4 functions in colorectal cancer (CRC) progression. METHODS: Bioinformatics analysis was used to identify a novel lncRNA (lnc-SOX9-4), and the expression pattern of the RNA in CRC was verified using qRT-PCR. Gene ontology (GO) term analysis and Gene set enrichment analysis (GSEA) were implemented for the identification of the related mechanisms and roles of lnc-SOX9-4. Immune infiltration analysis was conducted for assessment of how lnc-SOX9-4 is linked to tumor immune cell infiltration level. Both in vitro and in vivo phenotype analyses were conducted for scrutinizing how lnc-SOX9-4 impacts the proliferation and metastasis of CRC. RNA pulldown, mass spectrometry analysis, fluorescent in situ hybridization (FISH), western blotting, and RIP assay aided in verifying lnc-SOX9-4 mechanisms linked to CRC progression. RESULTS: An upregulation of lnc-SOX9-4 was observed in the sample CRC cells and tissues. Elevated lnc-SOX9-4 levels showed a positive association with poor clinical prognosis. Lnc-SOX9-4 was closely correlated to several types of immune infiltrating cells. Functionally, the knockdown of lnc-SOX9-4 significantly inhibited CRC cell proliferation, migration, and invasion abilities. Mechanistically, YBX1 was identified as lnc-SOX9-4, specifically interacting protein in the nucleus. Lnc-SOX9-4 could stabilize YBX1 protein levels by inhibiting poly-ubiquitination and degradation of YBX1. Furthermore, phenotype rescue experiments reveal that lnc-SOX9-4 enhanced the CRC cellular potential to proliferate and metastasize by regulating YBX1 levels. CONCLUSIONS: Lnc-SOX9-4 promoted CRC progression by suppressing cytoplasmic translocation and promoting protein levels of YBX1 can serve as novel treatment targets for diagnosing and treating CRC.

Tumor-suppressive E3 ubiquitin ligase CHIP inhibits the PBK/ERK axis to repress stem cell properties and radioresistance in non-small cell lung cancer.

Recently, radioresistant cancer cells surviving radiotherapy have been suggested to show more aggressive phenotypes than parental cells, and the underlying mechanisms may be associated with cancer stem cells. This study provided novel mechanistic insights for E3 ubiquitin ligase CHIP in stem cell properties and radioresistance of non-small cell lung cancer (NSCLC). After bioinformatic prediction for key genes involved, NSCLC tissues and cells were collected to measure the expression of CHIP and PBK. E3 ubiquitin ligase CHIP was poorly expressed, while PBK was highly expressed in NSCLC tissues and cells. CHIP reduced the protein stability of PBK through the ubiquitin-protease pathway to repress the activation of ERK pathway. Based on the gain- or loss-of-function experiments, it was noted that restoration of CHIP curtailed stem cell properties and radioresistance in NSCLC, as manifested by inhibited sphere formation and cell proliferation, decreased number of CD133+CD44+ cells and expression of OCT4, SOX2, and NANOG, as well as facilitated apoptosis of NSCLC cells. Besides, in vivo animal experiments further confirmed that CHIP restrained tumorigenic ability and improved radiosensitivity of NSCLC cells by inhibiting PBK/ERK axis. Collectively, CHIP suppressed stem cell properties and radioresistance of NSCLC cells by inhibiting PBK/ERK axis, therefore offering a potential therapeutic target for enhancing efficacy of radiotherapy.

E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway.

BACKGROUND: Recent studies have indicated that some members of the tripartite motif (TRIM) proteins function as important regulators for non-small cell lung cancer (NSCLC), However, the regulatory mechanism underpinning aberrant expression of TRIM in NSCLC remains unclear. Here we report that TRIM15 plays important roles in NSCLC progression through modulating Keap1-Nrf2 signaling pathway. METHODS: TRIM15 expression was evaluated by western blot analysis, tissue microarray-based immunohistochemistry analysis. The interactions between TRIM15 and Keap1 were analyzed by co-immunoprecipitation (Co-IP) and immunofluorescence co-localization assay. The correlation between TRIM15 and Keap1 was measured by Co-IP and ubiquitination analysis in vitro. Gain- and lost-of-function experiments were used to detect TRIM15 promotes proliferation and invasion of NSCLC cells both in vitro and vivo. RESULTS: Here, we revealed that TRIM15 was frequently upregulated in NSCLC samples and associated with poor prognosis. Functionally, TRIM15 knockdown resulted in decreased cancer cell proliferation and metastasis, whereas ectopic TRIM15 expression facilitated tumor cancer cell proliferation and metastasis in vitro and in vivo. Moreover, TRIM15 promoted cell proliferation and metastasis depends on its E3 ubiquitin ligase. Mechanistically, TRIM15 directly targeted Keap1 by ubiquitination and degradation, the principal regulator of Nrf2 degradation, leading to Nrf2 escaping from Keap1-mediated degradation, subsequently promoting antioxidant response and tumor progression. CONCLUSIONS: Therefore, our study characterizes the pivotal roles of TRIM15 promotes NSCLC progression via Nrf2 stability mediated by promoting Keap1 ubiquitination and degradation and could be a valuable prognostic biomarker and a potential therapeutic target in NSCLC. Video Abstract.

miR-221-5p-Mediated Downregulation of JNK2 Aggravates Acute Lung Injury.

Sepsis and acute lung injury (ALI) are linked to mitochondrial dysfunction; however, the underlying mechanism remains elusive. We previously reported that c-Jun N-terminal protein kinase 2 (JNK2) promotes stress-induced mitophagy by targeting small mitochondrial alternative reading frame (smARF) for ubiquitin-mediated proteasomal degradation, thereby preventing mitochondrial dysfunction and restraining inflammasome activation. Here we report that loss of JNK2 exacerbates lung inflammation and injury during sepsis and ALI in mice. JNK2 is downregulated in mice with endotoxic shock or ALI, concomitantly correlated inversely with disease severity. Small RNA sequencing revealed that miR-221-5p, which contains seed sequence matching to JNK2 mRNA 3' untranslated region (3'UTR), is upregulated in response to lipopolysaccharide, with dynamically inverse correlation with JNK2 mRNA levels. miR-221-5p targets the 3'UTR of JNK2 mRNA leading to its downregulation. Accordingly, miR-221-5p exacerbates lung inflammation and injury during sepsis in mice by targeting JNK2. Importantly, in patients with pneumonia in medical intensive care unit, JNK2 mRNA levels in alveolar macrophages flow sorted from non-bronchoscopic broncholaveolar lavage (BAL) fluid were inversely correlated strongly and significantly with the percentage of neutrophils, neutrophil and white blood cell counts in BAL fluid. Our data suggest that miR-221-5p targets JNK2 and thereby aggravates lung inflammation and injury during sepsis.

The Anti-Apoptotic Role of EBV-LMP1 in Lymphoma Cells.

BACKGROUND: Epstein-Barr virus (EBV) has been indicated in the development of some tumors, including lymphoma. However, the potential role of latent membrane protein 1 (LMP1) encoded by EBV in the tumorigenesis of lymphoma remains debated. Herein, we examined the function of LMP1 in lymphoma. METHODS: The expression of LMP1 was downregulated or upregulated in EBV negative cell line SNT-8 and positive cell line KHYG-1, respectively. Subsequently, the cell viability, apoptosis, as well as the expression patterns of p53, mouse double minute 2 (MDM2), B-cell CLL/lymphoma 2 (Bcl-2) and NF-κB were evaluated. Next, the binding relationship between MDM2 and p53 along with p53 ubiquitination in cells was tested by Western blot and co-immunoprecipitation. Finally, the effects of LMP1 on lymphoma cell growth through p53, Bcl-2 and NF-κB pathways were verified by functional rescue experiments. RESULTS: Overexpression of LMP1 promoted KHYG-1 cell growth and inhibited cell apoptosis. Moreover, LMP1 upregulation significantly enhanced the activation of NF-κB pathway, thus increasing MDM2 binding to p53, leading to p53 ubiquitination and degradation as well as Bcl-2 expression enhancement. Further inhibition of the NF-κB pathway or Bcl-2 expression significantly weakened the promotive role of LMP1 in the growth of KHYG-1 cells. CONCLUSION: EBV-LMP1 promoted the p53 ubiquitination and degradation by activating NF-κB signaling pathway and the following binding of MDM2 and p53 in cells to enhance Bcl-2 expression, thus promoting the growth of lymphoma cells and inhibiting cell apoptosis.

GBP5 Is an Interferon-Induced Inhibitor of Respiratory Syncytial Virus.

Guanylate binding protein 5 (GBP5) belongs to the GTPase subfamily, which is mainly induced by interferon gamma (IFN-γ) and is involved in many important cellular processes, including inflammasome activation and innate immunity against a wide variety of microbial pathogens. However, it is unknown whether GBP5 inhibits respiratory syncytial virus (RSV) infection. In this study, we identified GBP5 as an effector of the anti-RSV activity of IFN-γ and found that in children, the weaker immune response, especially the weaker IFN-γ response and the decreased GBP5 expression, leads to RSV susceptibility. Furthermore, we revealed that GBP5 reduced the cell-associated levels of the RSV small hydrophobic (SH) protein, which was identified as a viroporin. In contrast, overexpression of the SH protein rescued RSV replication in the presence of GBP5. The GBP5-induced decrease in intracellular SH protein levels is because GBP5 promotes the release of the SH protein into the cell culture. Moreover, the GBP5 C583A mutants with changes at the C terminus or the GBP5 ΔC mutant lacking the C-terminal region, which impairs GBP5 localization in the Golgi, could not inhibit RSV infection, whereas the GTPase-defective GBP5 maintained RSV inhibition, suggesting that Golgi localization but not the GTPase activity of GBP5 is required for RSV inhibition. Interestingly, we found that RSV infection or RSV G protein downregulates GBP5 expression by upregulating DZIP3, an E3 ligase, which induces GBP5 degradation through the K48 ubiquitination and proteasomal pathways. Thus, this study reveals a complicated interplay between host restrictive factor GBP5 and RSV infection and provides important information for understanding the pathogenesis of RSV.IMPORTANCE RSV is a highly contagious virus that causes multiple infections in infants within their first year of life. It can also easily cause infection in elderly or immunocompromised individuals, suggesting that individual differences in immunity play an important role in RSV infection. Therefore, exploring the pathogenic mechanisms of RSV and identifying essential genes which inhibit RSV infection are necessary to develop an effective strategy to control RSV infection. Here, we report that the IFN-inducible gene GBP5 potently inhibits RSV replication by reducing the cell-associated levels of the RSV small hydrophobic (SH) protein, which is a viroporin. In contrast, the RSV G protein was shown to upregulate the expression of the DZIP3 protein, an E3 ligase that degrades GBP5 through the proteasomal pathway. Our study provides important information for the understanding of the pathogenic mechanisms of RSV and host immunity as well as the complicated interplay between the virus and host.

The Ubiquitin Code of NODs Signaling Pathways in Health and Disease.

NOD1 and NOD2 belong to the family of intracellular Nod-like receptors (NLRs) that are involved in the maintenance of tissue homeostasis and host defense against bacteria and some viruses. When sensing such microbes, those NLRs act as hitherto scaffolding proteins for activating multiple downstream inflammatory signaling pathways to promote the production of cytokines and chemokines that are ultimately important for pathogen clearance. In recent years, substantial advances have been made on our understanding of a contextual series of intracellular processes that regulate such group of innate immune molecules, including phosphorylation and ubiquitination. Specifically, we will herein discuss those recently described posttranslational modifications of either NOD1 or NOD2 that fundamentally contribute to the robustness of protective responses within specific tissues through either internal domain association or external interactions with various proteins. From a public health perspective, it is then anticipated that a better understanding how genetic mutations and deregulation of these activating and repressing mechanisms might break down in diseases would open up new therapeutic avenues for humanity.

Long noncoding RNA GAS5 inhibits progression of colorectal cancer by interacting with and triggering YAP phosphorylation and degradation and is negatively regulated by the m6A reader YTHDF3.

BACKGROUND: YAP activation is crucial for cancer development including colorectal cancer (CRC). Nevertheless, it remains unclear whether N6-Methyladenosine (m6A) modified transcripts of long noncoding RNAs (lncRNAs) can regulate YAP activation in cancer progression. We investigated the functional link between lncRNAs and the m6A modification in YAP signaling and CRC progression. METHODS: YAP interacting lncRNAs were screened by RIP-sequencing, RNA FISH and immunofluorescence co-staining assays. Interaction between YAP and lncRNA GAS5 was studied by biochemical methods. MeRIP-sequencing combined with lncRNA-sequencing were used to identify the m6A modified targets of YTHDF3 in CRC. Gain-of-function and Loss-of-function analysis were performed to measure the function of GAS5-YAP-YTHDF3 axis in CRC progression in vitro and in vivo. RESULTS: GAS5 directly interacts with WW domain of YAP to facilitate translocation of endogenous YAP from the nucleus to the cytoplasm and promotes phosphorylation and subsequently ubiquitin-mediated degradation of YAP to inhibit CRC progression in vitro and in vivo. Notably, we demonstrate the m6A reader YTHDF3 not only a novel target of YAP but also a key player in YAP signaling by facilitating m6A-modified lncRNA GAS5 degradation, which profile a new insight into CRC progression. Clinically, lncRNA GAS5 expressions is negatively correlated with YAP and YTHDF3 protein levels in tumors from CRC patients. CONCLUSIONS: Our study uncovers a negative functional loop of lncRNA GAS5-YAP-YTHDF3 axis, and identifies a new mechanism for m6A-induced decay of GAS5 on YAP signaling in progression of CRC which may offer a promising approach for CRC treatment.

A patent review of the ubiquitin ligase system: 2015-2018.

INTRODUCTION: Ubiquitin-proteasome system (UPS) has been validated as a novel anticancer drug target in the past 20 years. The UPS contains two distinct steps: ubiquitination of a substrate protein by ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3), and substrate degradation by the 26S proteasome complex. The E3 enzyme is the central player in the ubiquitination step and has a wide range of specific substrates in cancer cells, offering great opportunities for discovery and development of selective drugs. Areas covered: This review summarizes the recent advances in small molecule inhibitors of E1s, E2s, and E3s, with a focus on the latest patents (from 2015 to 2018) of E3 inhibitors and modulators. Expert opinion: One strategy to overcome limitations of current 20S proteasome inhibitors is to discover inhibitors of the upstream key components of the UPS, such as E3 enzymes. E3s play important roles in cancer development and determine the specificity of substrate ubiquitination, offering novel target opportunities. E3 modulators could be developed by rational design, natural compound or library screening, old drug repurposes, and application of other novel technologies. Further understanding of mechanisms of E3-substrate interaction will be essential for discovering and developing next-generation E3 inhibitors as effective anticancer drugs.

Nicotine increases apoptosis in HUVECs cultured in high glucose/high fat via Akt ubiquitination and degradation.

It is well-documented that nicotine, the main active ingredient in cigarettes, results in endothelial cell injury in numerous diseases. However, whether nicotine plays a crucial role in endothelial cell injury in diabetes and the exact molecular mechanism that mediates this process have not been fully elucidated. The current study aimed to investigate the effects of nicotine on endothelial cell injury in diabetes and the specific molecular mechanism by which it plays a role. Human umbilical vein endothelial cells (HUVECs) were incubated in HG/HF media and treated with nicotine, PYR-41 (a selective ubiquitin E1 inhibitor), Akt-overexpressing adenovirus, or TTC3 and MUL1 shRNA adenovirus. Cell viability was subsequently detected by the CCK8 assay, and apoptosis was examined by caspase-3 cleavage and activity analysis. Compared to the HG/HF incubated group, nicotine incubation significantly decreased cell survival and increased apoptosis. Moreover, nicotine induced Akt degradation via UPS, and Akt overexpression blocked nicotine-induced apoptosis in HUVECs cultured in HG/HF media. Furthermore, the TTC3 and MUL1 shRNA adenovirus dramatically decreased the Akt ubiquitination and apoptosis induced by nicotine. These results indicate that nicotine-induced Akt ubiquitination and degradation occurs through TTC3 and MUL1 and results in a dramatic increase in apoptosis in HUVECs cultured in HG/HF media.

Hepatitis C virus NS3 protein enhances hepatocellular carcinoma cell invasion by promoting PPM1A ubiquitination and degradation.

BACKGROUND: Growing evidence suggests that hepatitis C virus (HCV) contributes to hepatocellular carcinoma (HCC) by directly modulating oncogenic signaling pathways. Protein phosphatase magnesium-dependent 1A (PPM1A) has recently emerged as an important tumor suppressor as it can block a range of tumor-centric signaling pathways through protein dephosphorylation. However, the role and regulatory mechanisms of PPM1A in HCV-infected cells have not been reported. METHODS: Total, cytoplasmic, and nuclear PPM1A protein after HCV infection or overexpression of HCV nonstructural protein 3 (NS3) were detected by western blotting. The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry. The effects of HCV infection and NS3 expression on the PPM1A protein level were systematically analyzed, and the ubiquitination level of PPM1A was determined by precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody. Finally, the roles of NS3 and PPM1A in hepatoma cell migration and invasion were assessed by wound healing and transwell assays, respectively. RESULTS: HCV infection and replication decreased PPM1A abundance, mediated by NS3, in hepatoma cells. Compared to normal liver tissues, the expression of PPM1A was significantly decreased in the HCC tumor tissues and adjacent non-tumor tissues. NS3 directly interacted with PPM1A to promote PPM1A ubiquitination and degradation, which was dependent on its protease domain. Blockade of PPM1A through small interfering RNA significantly promoted HCC cell migration, invasion, and epithelial mesenchymal transition (EMT), which were further intensified by TGF-ß1 stimulation, in vitro. Furthermore, restoration of PPM1A abrogated the NS3-mediated promotion of HCC migration and invasion to a great extent, which was dependent on its protein phosphatase function. CONCLUSIONS: Our findings demonstrate that the HCV protein NS3 can downregulate PPM1A by promoting its ubiquitination and proteasomal degradation, which might contribute to the migration and invasion of hepatoma cells and may represent a new strategy of HCV in carcinogenesis.

Hsp70 and Hsp90 oppositely regulate TGF-ß signaling through CHIP/Stub1.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulation of a wide variety of cellular processes. Canonical TGF-ß signaling is mediated by Smads which were further regulated by several factors. We previously reported that E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein, also named Stub1) controlled the sensitivity of TGF-ß signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation. Here, we present evidence that Hsp70 and Hsp90 regulate the complex formation of Smad3/CHIP. Furthermore, we observed that over-expressed Hsp70 or inhibition of Hsp90 by geldanamycin (GA) leads to facilitated CHIP-induced ubiquitination and degradation of Smad3, which finally enhances TGF-ß signaling. In contrast, over-expressed Hsp90 antagonizes CHIP mediated Smad3 ubiquitination and degradation and desensitizes cells in response to TGF-ß signaling. Taken together, our data reveal an opposite role of Hsp70 and Hsp90 in regulating TGF-ß signaling by implicating CHIP-mediated Smad3 ubiquitination and degradation. This study provides a new insight into understanding the regulation of the TGF-ß signaling by chaperones.