RING Domain Research Articles

Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings.

CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.

Liposomal UHRF1 siRNA shows lung fibrosis treatment potential through regulation of fibroblast activation.

Pulmonary fibrosis is a chronic and progressive interstitial lung disease associated with the decay of pulmonary function, which leads to a fatal outcome. As an essential epigenetic regulator of DNA methylation, the involvement of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) in fibroblast activation remains largely undefined in pulmonary fibrosis. In the present study, we found that TGF-β1-mediated upregulation of UHRF1 repressed beclin 1 via methylated induction of its promoter, which finally resulted in fibroblast activation and lung fibrosis both in vitro and in vivo. Moreover, knockdown of UHRF1 significantly arrested fibroblast proliferation and reactivated beclin 1 in lung fibroblasts. Thus, intravenous administration of UHRF1 siRNA-loaded liposomes significantly protected mice against experimental pulmonary fibrosis. Accordingly, our data suggest that UHRF1 might be a novel potential therapeutic target in the pathogenesis of pulmonary fibrosis.

Structural and functional asymmetry of RING trimerization controls priming and extension events in TRIM5α autoubiquitylation

TRIM5α is an E3 ubiquitin ligase of the TRIM family that binds to the capsids of primate immunodeficiency viruses and blocks viral replication after cell entry. Here we investigate how synthesis of K63-linked polyubiquitin is upregulated by transient proximity of three RING domains in honeycomb-like assemblies formed by TRIM5α on the surface of the retroviral capsid. Proximity of three RINGs creates an asymmetric arrangement, in which two RINGs form a catalytic dimer that activates E2-ubiquitin conjugates and the disordered N-terminus of the third RING acts as the substrate for N-terminal autoubiquitylation. RING dimerization is required for activation of the E2s that contribute to the antiviral function of TRIM5α, UBE2W and heterodimeric UBE2N/V2, whereas the proximity of the third RING enhances the rate of each of the two distinct steps in the autoubiquitylation process: the initial N-terminal monoubiquitylation (priming) of TRIM5α by UBE2W and the subsequent extension of the K63-linked polyubiquitin chain by UBE2N/V2. The mechanism we describe explains how recognition of infection-associated epitope patterns by TRIM proteins initiates polyubiquitin-mediated downstream events in innate immunity.

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

TRIM13 Is a Novel Cell Cycle Hub Regulated By CHAF1B to Promote Acute Myeloid Leukemogenesis

CHAF1B (Chromatin Assembly Factor 1B) functions as a histone chaperone within the DNA replication machinery. It is almost universally overexpressed in cancers, and we have previously shown that this upregulation is necessary to enforce the differentiation block found in acute myeloid leukemia (AML) through CHAF1B's chromatin binding function. In this study, we found that CHAF1B creates a pro-leukemic state in hematopoietic cells by repressing the expression of the E3 ubiquitin ligase TRIM13. Our data suggest that TRIM13 is a strong promoter of entry into the cell cycle functioning through novel nuclear localization and activating ubiquitination of key cell cycle genes. We propose that repression of TRIM13 by CHAF1B is a critical event for leukemogenesis because unchecked entry into cell cycle leads to loss of self-renewal and eventual exhaustion of the tumor. First, we measured the expression levels of CHAF1B and TRIM13 in several bone marrow aspirates from primary pediatric AML patients using immunohistochemistry. TRIM13 was expressed in healthy marrow and almost undetectable upon leukemic progression. CHAF1B, on the other hand, had low to moderate expression in healthy tissues and substantially higher expression upon leukemic progression and relapse. We found restoration of healthy CHAF1B and TRIM13 expression levels upon resolution of the tumor in matched patient aspirates upon remission. To determine if CHAF1B is capable of directly repressing TRIM13 expression, first we observed CHAF1B occupancy at the TRIM13 promoter by ChIPseq in multiple AML cell lines. Then we found loss of CHAF1B by shRNA resulted in increased TRIM13 transcription, and overexpression of CHAF1B by dox-inducible lentivirus drove TRIM13 transcriptional repression. Overexpression of a CHAF1B chromatin binding deficient mutant (CHAF1BRRAA) was unable to repress TRIM13, suggesting that CHAF1B chromatin binding is necessary for TRIM13 repression. This led us to hypothesize that TRIM13 may be a novel tumor suppressor in AML. To test this hypothesis, we overexpressed TRIM13 in AML cell lines and several patient-derived xenografts (PDXs) spanning many subtypes of AML and observed consistent repression of colony formation, increase in apoptosis, and reduction of leukemic progression in vivo. Conversely, loss of TRIM13 expression by shRNA led to an increase in colony formation in vitro and enhanced leukemogenicity of PDXs and AML cell lines in vivo. To better understand the mechanistic contribution of TRIM13 to myeloid leukemogenesis, we started by identifying potential catalytic targets of TRIM13. Since TRIM13 is a RING-type E3 ubiquitin ligase, we used CRISPR to generate in-frame RING domain deleted alleles of TRIM13 (T13RINGdel) and performed ubiquitin proteomics comparing our T13RINGdel and TRIM13 WT clones. We expected that loss of the catalytic function would lead to stabilization of pro-leukemic proteins as has been shown in solid tumor models. We did not find that, but we did observe a profound reduction in ubiquitination of proteins associated with the cell cycle, including CCNA1, CCNA2, CCNB2, and CDK1. Additionally, the total levels of these cell cycle-promoting proteins was reduced in T13RINGdel cells suggesting the ubiquitination event leads to their stability and activity. We confirmed that our T13RINGdel clones of AML cell lines had reduced entry and progression through cell cycle. Using imaging cytometry, we found a novel nuclear localization of TRIM13 and an expression pattern characterized by increased TRIM13 expression as cell cycle progressed. We confirmed the stabilization/activation relationship between TRIM13 and CCNA1 by restoring CCNA1 expression and confirming loss of the colony formation benefit in T13RINGdel AML cell lines. This study is the first to link CHAF1B, a cell cycle gene, with transcriptional repression of genes that promote cell cycle progression. We believe this relationship is critical for AML cells to support high levels of CHAF1B expression, thereby promoting an immature leukemic state throughout replication, without premature entry into multiple damaging rounds of proliferation.

The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy

The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.

MEX3A Mediates p53 Degradation to Suppress Ferroptosis and Facilitate Ovarian Cancer Tumorigenesis.

Degradation of p53 mediated by MEX3A drives ovarian cancer growth by circumventing p53 tumor suppressive functions, suggesting targeting MEX3A as a potential strategy for treating of ovarian cancer expressing WT p53.

Dual Targeting of MDM4 and FTH1 by MMRi71 for Induced Protein Degradation and p53-Independent Apoptosis in Leukemia Cells.

MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.

Inter-Fighting between Influenza A Virus NS1 and β-TrCP: A Novel Mechanism of Anti-Influenza Virus.

Influenza A virus (IAV) prevents innate immune signaling during infection. In our previous study, the production of pro-inflammatory cytokines was associated with Cullin-1 RING ligase (CRL1), which was related to NF-κB activation. However, the underlying mechanism is unclear. Here, an E3 ligase, β-transducin repeat-containing protein (β-TrCP), was significantly downregulated during IAV infection. Co-IP analysis revealed that non-structural 1 protein (NS1) interacts with β-TrCP. With co-transfection, an increase in NS1 expression led to a reduction in β-TrCP expression, affecting the level of IκBα and then resulting in repression of the activation of the NF-κB pathway during IAV infection. In addition, β-TrCP targets the viral NS1 protein and significantly reduces the replication level of influenza virus. Our results provide a novel mechanism for influenza to modulate its immune response during infection, and β-TrCP may be a novel target for influenza virus antagonism.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein.

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215-300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cβ chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.

Apigenin suppresses mycoplasma-induced alveolar macrophages necroptosis via enhancing the methylation of TNF-α promoter by PPARγ-Uhrf1 axis

BackgroundMycoplasma-associated pneumonia is characterized by severe lung inflammation and immunological dysfunction. However, current anti-mycoplasma agents used in clinical practice do not prevent dysfunction of alveolar macrophages caused by the high level of the cytokine tumor necrosis factor-α (TNF-α) after mycoplasma infection. Apigenin inhibits the production of TNF-α in variet inflammation associated disease. PurposeThis study aimed to investigate apigenin's effect on mycoplasma-induced alveolar immune cell injury and the mechanism by which it inhibits TNF-α transcription. MethodsIn this study, we performed a mouse model of Mycoplasma hyopneumoniae infection to evaluate the effect of apigenin on reducing mycoplasma-induced alveolar immune cell injury. Furthermore, we carried out transcriptome analysis, RNA interference assay, methylated DNA bisulfite sequencing assay, and chromatin immunoprecipitation assay to explore the mechanism of action for apigenin in reducing TNF-α. ResultsWe discovered that M. hyopneumoniae infection-induced necroptosis in alveolar macrophages MH-S cells and primary mouse alveolar macrophages, which was activated by TNF-α autocrine. Apigenin inhibited M. hyopneumoniae-induced elevation of TNF-α and necroptosis in alveolar macrophages. Apigenin inhibited TNF-a mRNA production via increasing ubiquitin-like with PHD and RING finger domains 1 (Uhrf1)-dependent DNA methylation of the TNF-a promotor. Finally, we demonstrated that apigenin regulated Uhrf1 transcription via peroxisome proliferator activated receptor gamma (PPARγ) activation, which acts as a transcription factor binding to the Uhrf1 promoter and protected infected mice's lungs, and promoted alveolar macrophage survival. ConclutsionThis study identified a novel mechanism of action for apigenin in reducing alveolar macrophage necroptosis via the PPARγ/ Uhrf1/TNF-α pathway, which may have implications for the treatment of Mycoplasma pneumonia.

Ubiquitin Proteasome Gene Signatures in Ependymoma Molecular Subtypes.

The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients.

A novel inhibitor of apoptosis protein (IAPs) from Crassostrea gigas is involved in anti-apoptosis and innate defense role under Vibrio alginolyticus stimulation

Apoptosis is an essential biological process in vertebrates and invertebrates for the development and maintenance of homeostasis. Inhibition of apoptosis can help some pathogens survive in host cells and escape from immune surveillance. Importantly, it also plays an important role in immune signaling regulation from insects to mammals. In this study, we identified a new inhibitor of the apoptosis protein gene (IAP), CgIAP3 from the Pacific oyster C. gigas, which contained one BIR domain and one RING domain. Blast analysis showed that CgIAP3 share the highest sequence identity of 65.68% with C. virginica IAP8, and clustered with other shellfish IAP in the phylogenetic tree, indicating that CgIAP3 belonged to a member of the IAPs family of shellfish. Homology comparison showed that the BIR domain of CgIAP3 was highly conserved with the BIR3 domain of human IAP. The tissue distribution of CgIAP3 in healthy C. gigas showed that CgIAP3 is ubiquitously expressed in all analyzed tissues, with the highest level of expression in the gills and digestive gland than the adductor muscle and was rapidly up-regulated after V. alginolyticus stimulation. Moreover, cell experiments showed CgIAP3 could inhibit apoptosis by maintaining cell morphology and cell viability. The result of subcellular localization showed that CgIAP3 is widely distributed in cytoplasm and nucleus and only expressed in A549 cells with damaged nucleus. Taken together, we speculated that CgIAP3 might be involved in the innate immune process of the Pacific oyster against V. alginolyticus through maintaining host cell morphology and viability.

Complete deletion of Ectromelia virus p28 impairs virus genome replication in a mouse strain, cell type, and multiplicity of infection-dependent manner

p28 is a poxvirus-encoded E3 ubiquitin ligase that possesses an N-terminal KilA-N domain and a C-terminal RING domain. In Ectromelia virus (ECTV), disruption of the p28 RING domain severely attenuated virulence in A strain mice, which normally succumb to ECTV infection. Moreover, this mutant virus exhibited dramatically reduced genome replication and impaired factory formation in A strain mice peritoneal macrophages (PMs) infected at high multiplicity of infection (MOI) These defects were not observed in PMs isolated from C57BL/6 mice which survive ECTV infection, demonstrating that p28 functions in a context-specific manner. To further investigate p28 function, we completely deleted the p28 gene from ECTV (ECTV-Δp28). In contrast to previous findings, we found that the ECTV-Δp28 virus exhibited severely compromised virus production and genome replication in PMs isolated from A strain mice only when infected at low MOI. This defect was minimal in bone marrow-derived macrophages and two cell lines derived from A strain mice. Furthermore, this low MOI defect in virus production was also observed in PMs isolated from the susceptible BALB/c mouse strain, but not PMs isolated from C57BL/6 mice. Taken together, our data demonstrate that the requirement for ECTV p28 to establish a productive infection depends on the MOI, the cell type, as well as the mouse strain.

Eight TRIM32 isoforms from oriental river prawn Macrobrachium nipponense are involved in innate immunity during white spot syndrome virus infection

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. In this study, full-length MnTRIM32 cDNA was obtained from oriental river prawn Macrobrachium nipponense, and eight MnTRIM32 isoforms generated by alternative splicing were identified. The open reading frames of the eight MnTRIM32 isoforms were predicted to be separately composed of 402, 346, 347, 346, 414, 358, 359, and 358 amino acid residues. Protein structural analysis revealed that all MnTRIM32 isoforms contained a RING domain and a coiled coil region. MnTRIM32 was ubiquitously expressed in all tissues tested, with the highest expression in the hepatopancreas. The mRNA levels of MnTRIM32 in the gills, stomach, and intestine of prawns were found to undergo time-dependent enhancement following white spot syndrome virus (WSSV) stimulation. Double-stranded RNA interference studies revealed that MnTRIM32 silencing significantly downregulated the expression levels of interferon (IFN) regulatory factor MnIRF, IFN-like factor MnVago4, and tumor necrosis factor MnTNF. Furthermore, knockdown of MnTRIM32 in WSSV-challenged prawns increased the expression of VP28 and the number of WSSV copies, suggesting that MnTRIM32 plays a positive role in limiting WSSV infection. These findings provided strong evidence for the important role of MnTRIM32 in the antiviral innate immunity of M. nipponense.

Identification of prognostic miRNA-mRNA regulatory network in the progression of HCV-associated cirrhosis to hepatocellular carcinoma.

Long-term hepatitis C virus (HCV) infection is strongly associated with hepatocellular carcinoma (HCC), yet the mechanisms of the progression process remain unclear. The research is aiming to establish a crucial prognostic model that indicates the risk of HCV-associated cirrhosis evolving into HCC. Differentially expressed microRNAs (DE-miRNAs) and differentially expressed genes (DEGs) between HCV-associated cirrhosis and HCC were screened from the GSE40744 and GSE6764 datasets, respectively. Downstream target genes of DE-miRNAs were predicted by the miRNet tool and then overlapped with the DEGs to select intersection genes. The GSE15654 was downloaded to establish a prognostic model. Expression levels of risk genes and their corresponding miRNAs were measured in liver tissues of clinical patients. HCC cell lines with UHRF1 knockdown or overexpression were assayed for cell proliferation and migration. Thirty-nine DE-miRNAs and 796 DEGs are identified between HCV-associated cirrhosis and HCC. Main intersection genes and their corresponding miRNAs constitute a miRNA-mRNA regulatory network. PABPC1 (Polyadenylate-binding protein 1), SLC2A9 (solute carrier gene family 2, member 9), and UHRF1 (ubiquitin-like with PHD and ring finger domains 1) form a prognostic model indicating the risk of HCC development among HCV-associated cirrhosis. The genetic mutations of PABPC1, SLC2A9, and UHRF1 in HCC patients are 9%, 0.8%, and 0.6%, respectively. Compared to that in HCV-associated cirrhosis, the expression levels of PABPC1 and UHRF1 are higher while the expression level of SLC2A9 is lower in clinical HCV-associated HCC samples. UHRF1 enhances the proliferation and migration ability of HCC cells. PABPC1, SLC2A9, and UHRF1 and their corresponding miRNAs are involved in the evolution process of HCV-associated cirrhosis into malignant HCC. UHRF1 serves as an oncogene that promotes the proliferation and migration of HCC cells.

TRIM50 promotes NLRP3 inflammasome activation by directly inducing NLRP3 oligomerization.

Tripartite motif protein (TRIM) 50 is a new member of the tripartite motif family, and its biological function and the molecular mechanism it is involved in remain largely unknown. The NOD-like receptor family protein (NLRP)3 inflammasome is actively involved in a wide array of biological processes while mechanisms of its regulation remain to be fully clarified. Here, we demonstrate the role of TRIM50 in NLRP3 inflammasome activation. In contrast to the conventional E3 ligase functions of TRIM proteins, TRIM50 mediates direct oligomerization of NLRP3, thereby suppressing its ubiquitination and promoting inflammasome activation. Mechanistically, TRIM50 directly interacts with NLRP3 through its RING domain and induces NLRP3 oligomerization via its coiled-coil domain. Finally, we show that TRIM50 promotes NLRP3 inflammasome-mediated diseases in mice. We thus reveal a novel regulatory mechanism of NLRP3 via TRIM50 and suggest that modulating TRIM50 might represent a therapeutic strategy for NLRP3-dependent pathologies.

Peli1 contributes to myocardial ischemia/reperfusion injury by impairing autophagy flux via its E3 ligase mediated ubiquitination of P62

Autophagy flux is impaired during myocardial ischemia/reperfusion (M-I/R) via the accumulation of autophagosome and insufficient clearance, which exacerbates cardiomyocyte death. Peli1 (Pellion1) is a RING finger domain-containing ubiquitin E3 ligase that could catalyze the polyubiquitination of substrate proteins. Peli1 has been demonstrated to play an important role in ischemic cardiac diseases. However, little is known about whether Peli1 is involved in the regulation of autophagy flux during M-I/R. The present study investigated whether M-I/R induced impaired autophagy flux could be mediated through Peli1 dependent mechanisms. We induced M-I/R injury in wild type (WT) and Peli1 knockout mice and observed that M-I/R significantly decreased cardiac function that was associated with increased cardiac Peli1 expression and upregulated autophagy-associated protein LC3II and P62. In contrast, Peli1 knockout mice exhibited significant improvement of M-I/R induced cardiac dysfunction and decreased LC3II and P62 expression. Besides, inhibitors of autophagy also increased the infarct size in Peli1 knockout mice after 24 h of reperfusion. Mechanistic studies demonstrated that in vivo I/R or in vitro hypoxia/reoxygenation (H/R) markedly increased the Peli1 E3 ligase activity which directly promoted the ubiquitination of P62 at lysine(K)7 via K63-linkage to inhibit its dimerization and autophagic degradation. Co-immunoprecipitation and GST-pull down assay indicated that Peli1 interacted with P62 via the Ring domain. In addition, Peli1 deficiency also decreased cardiomyocyte apoptosis. Together, our work demonstrated a critical link between increased expression and activity of Peli1 and autophagy flux blockage in M-I/R injury, providing insight into a promising strategy for treating myocardium M-I/R injury.

TRAF5 splicing variants associate with TRAF3 and RIP1 in NF-κB and type I IFN signaling in large yellow croaker Larimichthys crocea

As a member of the tumor necrosis factor receptor-associated factor (TRAF) family, TRAF5 acts as a crucial adaptor molecule and plays important roles in the host innate immune responses. In the present study, the typical form and a splicing variant of TRAF5, termed Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-TRAF5_tv1 protein is constituted of 577 aa, contains a RING finger domain, two zinc finger domains, a coiled-coil domain, and a MATH domain, whereas Lc-TRAF5_tv2 protein is constituted of 236 aa and only contains a RING finger domain due to a premature stop resulted from the intron retention. Subcellular localization analysis revealed that both of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were localized in the cytoplasm, with Lc-TRAF5_tv2 found to aggregate around the nucleus. It was revealed that Lc-TRAF5_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-TRAF5_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulations in vivo. Interestingly, overexpression of Lc-TRAF5_tv1 and Lc-TRAF5_tv2 could significantly induce NF-κB but not IFN1 activation, whereas co-expression of them remarkably induced IFN1 activation but impaired NF-κB activation. In addition, both Lc-TRAF5_tv1 and Lc-TRAF5_tv2 were associated with TRAF3 and RIP1 in IFN1 activation, whereas only Lc-TRAF5_tv1 cooperated with TRAF3 and RIP1 in NF-κB activation. These results collectively indicated that the splicing variant together with the typical form of TRAF5 function importantly in the regulation of host immune signaling in teleosts.

Unveiling the Essential Role of Arkadia's Non-RING Elements in the Ubiquitination Process.

Arkadia is a positive regulator of the TGFβ-SMAD2/3 pathway, acting through its C-terminal RING-H2 domain and targeting for degradation of its negative regulators. Here we explore the role of regions outside the RING domain (non-RING elements) of Arkadia on the E2-E3 interaction. The contribution of the non-RING elements was addressed using Arkadia RING 68 aa and Arkadia 119 aa polypeptides. The highly conserved NRGA (asparagine-arginine-glycine-alanine) and TIER (threonine-isoleucine-glutamine-arginine) motifs within the 119 aa Arkadia polypeptide, have been shown to be required for pSMAD2/3 substrate recognition and ubiquitination in vivo. However, the role of the NRGA and TIER motifs in the enzymatic activity of Arkadia has not been addressed. Here, nuclear magnetic resonance interaction studies with the E2 enzyme, UBCH5B, C85S UBCH5B-Ub oxyester hydrolysis, and auto-ubiquitination assays were used to address the role of the non-RING elements in E2-E3 interaction and in the enzymatic activity of the RING. The results support that the non-RING elements including the NRGA and TIER motifs are required for E2-E3 recognition and interaction and for efficient auto-ubiquitination. Furthermore, while Arkadia isoform-2 and its close homologue Arkadia 2C are known to interact with free ubiquitin, the results here showed that Arkadia isoform-1 does not interact with free ubiquitin.