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Transcription factor SNAI2 plays key roles during development and has also been known to promote metastasis by inducing invasive phenotype and tumor-initiating activity of cancer cells. However, the post-translational regulation of SNAI2 is less well studied. We performed a dual-luciferase-based, genome-wide E3 ligase siRNA library screen and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells promoted cell migration and decreased F-actin polymerization, while overexpression of ASB13 suppressed lung metastasis. Furthermore, ASB13 knockout decreased YAP expression, and such regulation is dependent on an increased protein level of SNAI2, which in turn represses YAP transcription. YAP suppresses tumor progression in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo. Clinical data analysis reveals that ASB13 expression is positively correlated with improved overall survival in breast cancer patients. These findings establish ASB13 as a suppressor of breast cancer metastasis by promoting degradation of SNAI2 and relieving its transcriptional repression of YAP., (© 2020 Fan et al.; Published by Cold Spring Harbor Laboratory Press.)

ERα positive breast cancer accounts for 70% of breast malignancies. Compared with ERα negative types, ERα positive breast cancer could be effective controlled by endocrine therapy. However, more than half of the patients will develop endocrine resistance, making it an important clinical issue for breast cancer therapy. Endocrine resistance might be caused by multiple alternations, including the components of ERα signaling, during tumor progression. Thus, it is urgent and necessary to uncover the molecular mechanisms that controls ERα expression and stability to improve breast cancer therapeutics. In our current study, we identifies that the ubiquitin ligase RNF181 stabilizes ERα and facilitates breast cancer progression. The expression of RNF181 is correlated with ERα level in human breast tumors and relates to poor survival in endocrine-treated patients. RNF181 depletion inhibits breast cancer progression in vivo and in vitro, reduces ERα protein level and its target gene expression, such as PS2 and GREB1. Unbiased RNA sequencing analysis indicates RNF181 is necessary for ERα signature gene expression in whole genomic level. Immuno-precipitation assays indicate that RNF181 associates with ERα and promotes its stability possibly via inducing ERα K63-linked poly-ubiquitination. In conclusion, our data implicate a non-genomic mechanism by RNF181 via stabilizing ERα protein controls ERα target gene expression linked to breast cancer progression.

Epigenetic regulation is important for establishing lineage-specific gene expression during early development. Although signaling pathways have been well-studied for regulation of trophectoderm reprogramming, epigenetic regulation of trophectodermal genes with histone modification dynamics have been poorly understood. Here, we identify that plant homeodomain finger protein 6 (PHF6) is a key epigenetic regulator for activation of trophectodermal genes using RNA-sequencing and ChIP assays. PHF6 acts as an E3 ubiquitin ligase for ubiquitination of H2BK120 (H2BK120ub) via its extended plant homeodomain 1 (PHD1), while the extended PHD2 of PHF6 recognizes acetylation of H2BK12 (H2BK12Ac). Intriguingly, the recognition of H2BK12Ac by PHF6 is important for exerting its E3 ubiquitin ligase activity for H2BK120ub. Together, our data provide evidence that PHF6 is crucial for epigenetic regulation of trophectodermal gene expression by linking H2BK12Ac to H2BK120ub modification., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eukaryotic proteomes are enormously sophisticated through versatile post-translational modifications (PTMs) of proteins. A large variety of code generated via PTMs of proteins by ubiquitin (ubiquitination) and ubiquitin-like proteins (Ubls), such as interferon (IFN)-stimulated gene 15 (ISG15), small ubiquitin-related modifier (SUMO) and neural precursor cell expressed, developmentally downregulated 8 (NEDD8), not only provides distinct signals but also orchestrates a plethora of biological processes, thereby underscoring the necessity for sophisticated and fine-tuned mechanisms of code regulation. Deubiquitinases (DUBs) play a pivotal role in the disassembly of the complex code and removal of the signal. Ubiquitin-specific protease 18 (USP18), originally referred to as UBP43, is a major DUB that reverses the PTM of target proteins by ISG15 (ISGylation). Intriguingly, USP18 is a multifaceted protein that not only removes ISG15 or ubiquitin from conjugated proteins in a deconjugating activity-dependent manner but also acts as a negative modulator of type I IFN signaling, irrespective of its catalytic activity. The function of USP18 has become gradually clear, but not yet been completely addressed. In this review, we summarize recent advances in our understanding of the multifaceted roles of USP18. We also highlight new insights into how USP18 is implicated not only in physiology but also in pathogenesis of various human diseases, involving infectious diseases, neurological disorders, and cancers. Eventually, we integrate a discussion of the potential of therapeutic interventions for targeting USP18 for disease treatment.

Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1 -/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCF Fbxw7α -dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The BH3-only protein NOXA is a regulator of mitochondrial apoptosis by specifically antagonizing the anti-apoptotic protein MCL-1. Here we show that the E3 ubiquitin ligase CHIP controls NOXA stability after DNA damage. Our findings reveal that CHIP and MCL-1 are binding partners of NOXA and differentially define the fate of NOXA. Whereas NOXA is initially targeted to mitochondria upon MCL-1-binding, CHIP mediates ubiquitylation of cytosolic NOXA and promotes lysosomal degradation of NOXA, which is not bound by MCL-1. Our data indicate that MCL-1 defines NOXA abundance and its pro-apoptotic activity. Increased NOXA levels beyond this threshold are effectively removed by lysosomal protein degradation triggered via CHIP-mediated ubiquitylation. Together, these results shed new light on regulatory circuits controlling DNA damage response and identified the E3 ligase CHIP as a new molecular guardian, which restricts the cytosolic accumulation of NOXA upon genotoxic stress.

There is existing evidence that elevated homocysteine (Hcy) levels are risk factors for some neurodegenerative disorders. The pathogenesis of neurological diseases could be contributed to excessive cell dysfunction and death caused by defective DNA damage response (DDR) and accumulated DNA damage. Hcy is a neurotoxic amino acid and acts as a DNA damage inducer. However, it is not clear whether Hcy participates in the DDR. To investigate the effects of Hcy on DNA damage and the DDR, we employed mitomycin C (MMC) to cause DNA damage in NE4C murine neural stem cells (NSCs). Compared to treatment with MMC alone, we found that co-treatment with MMC and Hcy worsened DNA damage and increased death in NE4C cells. Intriguingly, in this DNA damage model mimicked by MMC, immunoblotting results showed that the monoubiquitination levels of Fanconi anemia complementation group I (Fanci) and Fanconi anemia complementation group D2 (Fancd2) were decreased to about 60.3% and 55.7% by supplementing cell culture medium with Hcy, indicating Hcy inactivates the function of Fanci and Fancd2 in DNA damage conditions. Given Breast Cancer 1 (BRCA1) is an important downstream of FANCD2, we next detected the interaction between Fancd2 and Brca1 in NE4C cells. Compared to treatment with MMC alone, the Fancd2-Brca1 interaction and the amount of Brca1 on chromatin were decreased when cells were co-exposed to MMC and Hcy, suggesting Hcy could impair the Fanconi anemia (FA)/Brca1 pathway. Taken together, our study demonstrates that Hcy may enhance cell death, which contributes to the accumulation of DNA damage and promotion of hypersensitivity to cytotoxicity by impairing the FA/Brca1 pathway in murine NSCs in the presence of DNA damage., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G 1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator., Competing Interests: Conflict of interest:—Authors declare no competing interests., (© 2020 Manohar et al.)

Maresin1 is a potent lipid mediator exhibiting potential anti-inflammatory activity in a variety of inflammatory diseases, however, the underlying mechanisms remain poorly understood. Excessive activation of NLRP3 inflammasome has been established in multiple inflammatory diseases. Here, we show that Maresin1 dose-dependently inhibited the NLRP3 inflammasome activation and subsequent caspase-1 activation and IL-1β secretion. This inhibitory effect could be reversed by KH7 and H89, the inhibitors of the cAMP-PKA signaling pathway. Activation of PKA kinase induced by Maresin1 led to the K63-linked ubiquitination of NLRP3 in macrophages. Maresin1 attenuated serum IL-1β secretion through inhibition of NLRP3 inflammasome in vivo using Nlrp3-deficient mouse models of lipopolysaccharide (LPS)-induced sepsis. Maresin1 also repressed MSU-induced peritonitis. This study suggests that Maresin1 is an inhibitor of NLRP3 inflammasome activation and can be used clinically in the treatment of NLRP3 inflammasome-driven inflammatory diseases., (© 2020 Federation of American Societies for Experimental Biology.)

Objective: LncRNA Fendrr plays an important role in cardiac development, but its role in myocardial ischaemia-reperfusion (I/R) injury remains unclear. P53 has been shown to be an important regulator of apoptosis and is involved in myocardial I/R-induced apoptosis. This study aims at investigating whether Fendrr affects hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis through p53., Methods: The left anterior descending coronary artery of the rat was ligated for 30 min and then reperfusion for 120 min by releasing the suture. Neonatal rat ventricular myocytes (NRVM) and rat cardiac cell line H9c2 were cultured for 6 h in hypoxia (95% N 2 and 5% CO 2 ), followed by reoxygenation (95% air and 5% CO 2 ) for 6 h. Transfection were performed in cells. Apoptosis was detected by flow cytometry. Moreover, RNA pull-down, RNA immunoprecipitation, ubiquitination assay, GST pull-down assay and co-immunoprecipitation were used to detect the regulation of Fendrr on p53 protein., Key Findings: Fendrr was decreased in I/R-induced myocardium and H/R-induced cardiomyocyte, and overexpression of Fendrr inhibited H/R-induced NRVM or H9c2 cells apoptosis. Further research found that the 1381-2100 nt of Fendrr bound to p53 protein and Fendrr promoted t direct binding of p53 to Cop1. The inhibition of Fendrr reduced the binding of E3 ubiquitin-protein ligase constitutive photomorphogenesis protein 1 (COP1) to p53 and reduced the ubiquitination of p53. Furthermore, the inhibition of Fendrr on H/R-induced NRVM or H9c2 cells apoptosis could be reversed by overexpression of p53., Conclusions: Fendrr can inhibit H/R-induced cardiomyocyte apoptosis, which is partly through promoting the ubiquitination and degradation of p53 by increasing the binding of Cop1 and p53., (© 2020 Royal Pharmaceutical Society.)

Dysregulation of the ubiquitin-proteasomal system (UPS) enables pathogenic accumulation of disease-driving proteins in neurons across a host of neurological disorders. However, whether and how the UPS contributes to oligodendrocyte dysfunction and repair after white matter injury (WMI) remains undefined. Here we show that the E3 ligase VHL interacts with Daam2 and their mutual antagonism regulates oligodendrocyte differentiation during development. Using proteomic analysis of the Daam2-VHL complex coupled with conditional genetic knockout mouse models, we further discovered that the E3 ubiquitin ligase Nedd4 is required for developmental myelination through stabilization of VHL via K63-linked ubiquitination. Furthermore, studies in mouse demyelination models and white matter lesions from patients with multiple sclerosis corroborate the function of this pathway during remyelination after WMI. Overall, these studies provide evidence that a signaling axis involving key UPS components contributes to oligodendrocyte development and repair and reveal a new role for Nedd4 in glial biology., (© 2020 Ding et al.; Published by Cold Spring Harbor Laboratory Press.)

Epigenetic plasticity is a pivotal factor that drives metastasis. Here, we show that the promoter of the gene that encodes the ubiquitin ligase subunit FBXL7 is hypermethylated in advanced prostate and pancreatic cancers, correlating with decreased FBXL7 mRNA and protein levels. Low FBXL7 mRNA levels are predictive of poor survival in patients with pancreatic and prostatic cancers. FBXL7 mediates the ubiquitylation and proteasomal degradation of active c-SRC after its phosphorylation at Ser 104. The DNA-demethylating agent decitabine recovers FBXL7 expression and limits epithelial-to-mesenchymal transition and cell invasion in a c-SRC-dependent manner. In vivo, FBXL7-depleted cancer cells form tumours with a high metastatic burden. Silencing of c-SRC or treatment with the c-SRC inhibitor dasatinib together with FBXL7 depletion prevents metastases. Furthermore, decitabine reduces metastases derived from prostate and pancreatic cancer cells in a FBXL7-dependent manner. Collectively, this research implicates FBXL7 as a metastasis-suppressor gene and suggests therapeutic strategies to counteract metastatic dissemination of pancreatic and prostatic cancer cells.

Background: N-end rule ubiquitination pathway is known to be disrupted in many diseases, including cancer. UBR5, an E3 ubiquitin ligase, is mutated and/or overexpressed in human lung cancer cells suggesting its pathological role in cancer., Methods: We determined expression of UBR5 protein in multiple lung cancer cell lines and human patient samples. Using immunoprecipitation followed by mass spectrometry we determined the UBR5 interacting proteins. The impact of loss of UBR5 for lung adenocarcinoma cell lines was analyzed using cell viability, clonogenic assays and in vivo xenograft models in nude mice. Additional Western blot analysis was performed to assess the loss of UBR5 on downstream signaling. Statistical analysis was done by one-way ANOVA for in vitro studies and Wilcoxon paired t-test for in vivo tumor volumes., Results: We show variability of UBR5 expression levels in lung adenocarcinoma cell lines and in primary human patient samples. To gain better insight into the role that UBR5 may play in lung cancer progression we performed unbiased interactome analyses for UBR5. Data indicate that UBR5 has a wide range of interacting protein partners that are known to be involved in critical cellular processes such as DNA damage, proliferation and cell cycle regulation. We have demonstrated that shRNA-mediated loss of UBR5 decreases cell viability and clonogenic potential of lung adenocarcinoma cell lines. In addition, we found decreased levels of activated AKT signaling after the loss of UBR5 in lung adenocarcinoma cell lines using multiple means of UBR5 knockdown/knockout. Furthermore, we demonstrated that loss of UBR5 in lung adenocarcinoma cells results in significant reduction of tumor volume in nude mice., Conclusions: These findings demonstrate that deregulation of the N-end rule ubiquitination pathway plays a crucial role in the etiology of some human cancers, and blocking this pathway via UBR5-specific inhibitors, may represent a unique therapeutic target for human cancers.

While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.

It is universally accepted that aberrant metabolism facilitates tumor growth. However, how cancer cells coordinate glucose metabolism and tumor proliferation is largely unknown. Sine oculis homeobox homolog 1 (SIX1) is a transcription factor that belongs to the SIX family and is believed to play an important role in the regulation of the Warburg effect in tumors. However, whether the role of SIX1 and the molecular mechanisms that regulate its activity are similar in hepatocellular carcinoma (HCC) still needs further investigation. Methods: Western blotting was performed to determine the levels of SIX1 and O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) in HCC tissues. Cell Counting Kit 8 (CCK8), colony formation and mouse tumor model assays were used to establish the role of SIX1 and O-GlcNAcylation in HCC processes. Mass spectrometry, immunoprecipitation and site-directed mutagenesis were performed to confirm the O-GlcNAcylation of SIX1. Results: Here, we demonstrated that SIX1, the key transcription factor regulating the Warburg effect in cancer, promotes HCC growth in vitro and in vivo . Furthermore, we revealed that SIX1 could also enhance the levels of a posttranslational modification called O-GlcNAcylation. Importantly, we found that SIX1 was also highly modified by O-GlcNAcylation and that O-GlcNAcylation inhibited the ubiquitination degradation of SIX1. In addition, site-directed mutagenesis at position 276 (T276A) decreased the O-GlcNAcylation level and reversed the protumor effect of SIX1. Conclusions: We conclude that O-GlcNAcylation of SIX1 enhances its stability and promotes HCC proliferation. Our findings illustrate a novel feedback loop of SIX1 and O-GlcNAcylation and show that O-GlcNAcylation of SIX1 is an important way to coordinate glucose metabolism and tumor progression., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Autophagy and the ubiquitin-proteasome system are the major degradation processes for intracellular components in eukaryotes. Although ubiquitination acts as a signal inducing organelle-targeting autophagy, the interaction between ubiquitination and autophagy in chloroplast turnover has not been addressed. In this study, we found that two chloroplast-associated E3 enzymes, SUPPRESSOR OF PPI1 LOCUS1 and PLANT U-BOX4 (PUB4), are not necessary for the induction of either piecemeal autophagy of chloroplast stroma or chlorophagy of whole damaged chloroplasts in Arabidopsis ( Arabidopsis thaliana ). Double mutations of an autophagy gene and PUB4 caused synergistic phenotypes relative to single mutations. The double mutants developed accelerated leaf chlorosis linked to the overaccumulation of reactive oxygen species during senescence and had reduced seed production. Biochemical detection of ubiquitinated proteins indicated that both autophagy and PUB4-associated ubiquitination contributed to protein degradation in the senescing leaves. Furthermore, the double mutants had enhanced susceptibility to carbon or nitrogen starvation relative to single mutants. Together, these results indicate that autophagy and chloroplast-associated E3s cooperate for protein turnover, management of reactive oxygen species accumulation, and adaptation to starvation., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Protein ubiquitination is critical for various biological processes in eukaryotes. A ubiquitin (Ub) chain can be linked through one of the seven lysine (K) residues or the N-terminus methionine of the Ub, and the Ub-conjugating enzymes called E2s play a critical role in determining the linkage specificity of Ub chains. Further, while K48-linked polyubiquitin chain is important for protein degradation, much less is known about the functions of other types of polyubiquitin chains in plants. We showed previously that UBC22 is unique in its ability to catalyze K11-dependent Ub dimer formation in vitro and ubc22 knockout mutants had defects in megasporogenesis. In this study, further analyses of the Arabidopsis ubc22 mutants revealed four subtypes of plants based on the phenotypic changes in vegetative growth. These four subtypes appeared consistently in the plants of three independent ubc22 mutants. Transcriptomic analysis showed that transcript levels of genes related to several pathways were altered differently in different subtypes of mutant plants. In one subtype, the mutant plants had increased expression of genes related to plant defenses and showed enhanced resistance to a necrotrophic plant pathogen. These results suggest multiple functions of UBC22 during plant development and stress response., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Changes in the cellular environment modulate protein energy landscapes to drive important biology, with consequences for signaling, allostery and other vital processes. The effects of ubiquitination are particularly important because of their potential influence on degradation by the 26S proteasome. Moreover, proteasomal engagement requires unstructured initiation regions that many known proteasome substrates lack. To assess the energetic effects of ubiquitination and how these manifest at the proteasome, we developed a generalizable strategy to produce isopeptide-linked ubiquitin within structured regions of a protein. The effects on the energy landscape vary from negligible to dramatic, depending on the protein and site of ubiquitination. Ubiquitination at sensitive sites destabilizes the native structure and increases the rate of proteasomal degradation. In well-folded proteins, ubiquitination can even induce the requisite unstructured regions needed for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation.

This study was conducted to define the underlying molecular mechanism of tripartite motif (TRIM) 59-induced invasion of ectopic endometrial stromal cells in endometriosis. Primary endometriosis ectopic endometrial stromal cells and normal endometrial cells were isolated and purified. Western blot was used to detect the expression of TRIM59, protein phosphatase Mg 2+ /Mn 2+ -dependent 1A (PPM1A), smad2/3, and phosphorylated (p)-smad2/3. Lentiviral vector-mediated TRIM59 interference and overexpression were established. Cell Counting Kit-8 assay was used to detect cell proliferation, and the Transwell migration assay was used to detect cell invasion. Matrix metalloproteinase (MMP-2), MMP9, smad2/3, and p-smad2/3 expressions were also detected using Western blot analysis; degradation of PPM1A was verified to be through ubiquitination. We found that TRIM59 expression levels in the endometriosis group was significantly higher compared with the normal group ( P < 0.05), whereas the expression levels of PPM1A in the endometriosis group were significantly lower ( P < 0.05). Endometriosis did not alter smad2/3 ( P > 0.05) expression. However, after activating smad2/3 by phosphorylation, the expression of p-smad2/3 in the endometriosis group was significantly higher compared with the normal group ( P < 0.05). The content of PPM1A in the TRIM59 overexpression group was significantly lower than that in the control group ( P < 0.001), whereas the content of PPM1A in the siTRIM59 group was significantly higher than that in the control group ( P < 0.001). In addition, there were no significant differences in the mRNA levels of PPM1A among the five groups, indicating that TRIM59 affects the expression of PPM1A at the posttranslational level ( P < 0.05). Overexpression of TRIM59 significantly promoted the ubiquitination of PPM1A. We conclude that TRIM59 inhibits PPM1A through ubiquitination and activates the transforming growth factor-β/Smad pathway to promote the invasion of ectopic endometrial stromal cells in endometriosis.

Lung cancer is a devastating cancer with high morbidity and mortality. Ubiquitin‑specific protease (USP) is a type of deubiquitinating enzyme (DUB) that has been implicated in numerous cancers, including colorectal, myeloma and breast. In the present study, the expression of USP51 was determined in the lung cancer cell line A549 and cisplatin (also known as DDP)‑resistant lung cancer strain A549/DDP. The expression of zinc‑finger E‑box binding homeobox 1 (ZEB1), a transcriptional repressor, was also examined. The effects of USP51 knockdown or overexpression on proliferation and apoptosis, as well as the impact of ZEB1 overexpression and USP51 interference on apoptosis and ubiquitination were then assessed. Notably, increased expression of USP51 and ZEB1 in A549/DDP cells was observed, and treatment with DDP significantly inhibited proliferation in A549/DDP cells. In addition, knockdown of USP51 in A549/DDP cells significantly induced apoptosis, decreased ZEB1 expression and increased cleaved poly ADP‑ribose polymerase 1 (PARP1) and cleaved caspase‑3 levels. Consistently, USP51 overexpression in A549 cells displayed the opposite effects and potently attenuated DDP‑induced apoptosis. Notably, overexpression of ZEB1 in A549/DDP cells potently attenuated the effects of USP51 knockdown on apoptosis, and co‑IP experiments further demonstrated interaction between USP51 and ZEB. Lastly, knockdown of USP51 promoted ZEB1 ubiquitination, leading to ZEB1 degradation. Collectively, the present findings demonstrated that USP51 inhibition attenuated DDP resistance in A549/DDP cells via ubiquitin‑mediated degradation of ZEB1. Hence, targeting USP51 may serve as a novel therapeutic target for DDP resistance in lung cancer.

Purpose: For patients with refractory/relapsed Hodgkin lymphoma (roughly 20% of total cases), few effective therapeutic options exist. Currently, brentuximab vedotin (BV), a drug-conjugated anti-CD30 antibody, is one of the most effective approved therapy agents for these patients. However, many patients do not achieve complete remission and ultimately develop BV-resistant disease, necessitating a more detailed understanding of the molecular circuitry that drives BV sensitivity and the mechanism of BV resistance., Experimental Design: Here, we established a ubiquitin regulator-focused CRISPR library screening platform in Hodgkin lymphoma and carried out a drug sensitization screen against BV to identify genes regulating BV treatment sensitivity., Results: Our CRISPR library screens revealed the ubiquitin-editing enzymes A20 and RBX1 as key molecule effectors that regulate BV sensitivity in Hodgkin lymphoma line L428. A20 negatively regulates NF-κB activity which is required to prevent BV cytotoxicity. In line with these results, the RNA-seq analysis of the BV-resistant single-cell clones demonstrated a consistent upregulation of NF-κB signature genes, as well as the ABC transporter gene ABCB1. Mechanically, NF-κB regulates BV treatment sensitivity through mediating ABCB1 expression. Targeting NF-κB activity synergized well with BV in killing Hodgkin lymphoma cell lines, augmented BV sensitivity, and overcame BV resistance in vitro and in Hodgkin lymphoma xenograft mouse models., Conclusions: Our identification of this previously unrecognized mechanism provides novel knowledge of possible BV responsiveness and resistance mechanisms in Hodgkin lymphoma, as well as leads to promising hypotheses for the development of therapeutic strategies to overcome BV resistance in this disease., (©2020 American Association for Cancer Research.)

Objectives: Ubiquitination has a role as a host defense mechanism against pathogens. To channelize autophagic mycobacteria to destruction, ubiquitin ligase like, Makorin Ring Finger Protein 1 (MKRN1) was speculated to play a role in ubiquitinating M. tuberculosis. We have developed a flow cytometry based in vitro ubiquitin ligase assay to understand the role of MKRN1 in ubiquitinating mycobacteria and confirmed the results by western blotting., Results: MKRN1 was cloned and expressed in E. coli BL21 (DE3) strain. The recombinant MKRN1 protein was solubilised, purified and refolded to restore the activity. In addition, through autoubiquitination assay, the activity of protein was confirmed. The corresponding E1 and E2 enzymes for MKRN1, UBE1 and UBE2D3 respectively, were selected using BioGrid tool. Surprisingly, flow cytometric assay revealed that at a concentration of 300 nM of MKRN1, 38% of M. tuberculosis was found to be ubiquitinated in vitro with 3.5% of the cells having bound MKRN1. Immunoblot results also substantiates the ubiquitination of M. tuberculosis. MKRN1 did not ubiquitinate B. Subtilis and therefore, we speculate that the E3 Ub ligase activity might be specific to M. tuberculosis., Conclusion: This clearly demonstrates that recombinant MKRN1 ubiquitinates M. tuberculosis which opens up a novel, potential role of MKRN1 against mycobacteria which has to be unfolded.

The genomes of all living cells are under endogenous and exogenous attacks every day, causing diverse genomic lesions. Most of the lesions can be timely repaired by multiple DNA repair pathways. However, some may persist during S-phase, block DNA replication, and challenge genome integrity. Eukaryotic cells have evolved DNA damage tolerance (DDT) to mitigate the lethal effects of arrested DNA replication without prior removal of the offending DNA damage. As one important mode of DDT, translesion DNA synthesis (TLS) utilizes multiple low-fidelity DNA polymerases to incorporate nucleotides opposite DNA lesions to maintain genome integrity. Three different mechanisms have been proposed to regulate the polymerase switching between high-fidelity DNA polymerases in the replicative machinery and one or more specialized enzymes. Additionally, it is known that proliferating cell nuclear antigen (PCNA) mono-ubiquitination is essential for optimal TLS. Given its error-prone property, TLS is closely associated with spontaneous and drug-induced mutations in cells, which can potentially lead to tumorigenesis and chemotherapy resistance. Therefore, TLS process must be tightly modulated to avoid unwanted mutagenesis. In this review, we will focus on polymerase switching and PCNA mono-ubiquitination, the two key events in TLS pathway in mammalian cells, and summarize current understandings of regulation of TLS process at the levels of protein-protein interactions, post-translational modifications as well as transcription and noncoding RNAs. Environ. Mol. Mutagen. 61:680-692, 2020. © 2020 Wiley Periodicals, Inc., (© 2020 Wiley Periodicals, Inc.)

Protein modifications with highly conserved small proteins, such as ubiquitin (Ub) and small ubiquitin-like modifier (SUMO), regulate various cellular processes; however, the contribution of these protein modifications to larval development in insects has not yet been elucidated. We investigated the regulation of genes for these protein modifications in the posterior silk gland (PSG) during larval development of the silkworm Bombyx mori. We found that several genes encoding enzymes (E1, E2, and E3) for ubiquitination and SUMO-specific protease were upregulated by 20-hydroxyecdysone (20E), and, consistently, increases in ubiquitinated proteins were observed during the fourth molting stage. An injection of 20E into larvae at the fourth feeding stage induced higher expression levels of these E1, E2, and E3 genes and ecdysis approximately one day earlier than in mock-treated larvae. The expression of the fibroin heavy-chain gene (fibH) was simultaneously suppressed approximately one day earlier in 20E-injected larvae. The treatment of cultured PSG with 20E also induced these genes, which could be categorized into at least two types: those induced by a high dose of 20E, or by a pulse of 20E. In contrast to the 20E treatment, the administration of PR-619, an inhibitor of Ub- and SUMO-specific proteases in larvae, delayed ecdysis and prolonged the expression of fibH. These results suggest that the regulation of genes for ubiquitination and SUMO-specific protease is involved in the larval development of B. mori., (© 2020 Japanese Society of Developmental Biologists.)

Deubiquitinating enzymes (DUBs) are important regulators of the posttranslational protein ubiquitination system. Mammalian genomes encode about 100 different DUBs, which can be grouped into seven different classes. Members of other DUB classes are found in pathogenic bacteria, which use them to target the host defense. By combining bioinformatical and experimental approaches, we address the question if the known DUB families have a common evolutionary ancestry and share conserved features that set them apart from other proteases. By systematically comparing family-specific hidden Markov models, we uncovered distant relationships between established DUBs and other cysteine protease families. Most DUB families share a conserved aromatic residue linked to the active site, which restricts the cleavage of substrates with side chains at the S2 position, corresponding to Gly-75 in ubiquitin. By applying these criteria to Legionella pneumophila ORFs, we identified lpg1621 and lpg1148 as deubiquitinases, characterized their cleavage specificities, and confirmed the importance of the aromatic gatekeeper motif for substrate selection., (© 2020 Hermanns et al.)

Objective: Esophageal squamous cell carcinoma (ESCC) is one of the most commonly diagnosed cancer types in China. Recent genomic sequencing analysis indicated the over-activation of Hippo/YAP signaling might play important roles for the carcinogenic process and progression for ESCC patients. However, little is known about the molecular mechanisms that controls Hippo signaling activity in ESCC. Our previous studies indicated that PLCE1-an important risk factor for ESCC-linked to ESCC progression through snail signaling, during this period, we found PARK2 was an important downstream target of PLCE1-snail axis. PARK2 was decreased in ESCC human samples, and correlated with good prognosis in ESCC patients. Further research showed that PARK2 could inhibit YAP, which functions as key downstream effectors of the Hippo pathway. Here, we aim to reveal the molecular mechanisms of PARK2 modulated Hippo pathway in ESCC. Methods: To evaluate the function of PARK2 in ESCC, we used a tissue microarray (TMA) of 223 human ESCC patients and immunohistochemistry to analyze the correlation between PARK2 expression and clinicopathologic variables. Depletion of endogenous PARK2 and YAP from ESCC cells using CRISPR/Cas9 technologies. Flow cytometry and EdU cell proliferation assay were used to detect proliferation of ESCC cells. Nude mice subcutaneous injection and Ki-67 staining were used to evaluate tumor growth in vivo . Migration and invasion assays were performed. In addition, lung metastasis models in mice were used to validate the function of PARK2 in vivo . Identification of PARK2 involved in hippo pathway was achieved by expression microarray screening, double immunofluorescence staining and co-immunoprecipitation assays. The RNA-seq analysis results were validated through quantitative real-time PCR (qRT-PCR) analysis. The protein half-life of YAP was analyzed by Cycloheximide assay, and the TEAD activity was detected by Luciferase reporter assays. Results: Clinical sample of ESCC revealed that low PARK2 expression correlated with late tumor stage (P < 0.001), poor differentiation (P < 0.04), lymph node (P < 0.001) and distant metastasis (P = 0.0087). Multivariate Cox proportional regression analysis further revealed that PARK2 expression (P = 0.032) is an independent prognostic factor for the overall survival of ESCC patients. Besides, the immunohistochemistry results showed that PARK2 negatively correlated with YAP protein level (P < 0.001). PARK2 depletion promotes ESCC progression both through Hippo/YAP axis, while PARK2 overexpression suppresses ESCC tumor progression by Hippo signaling. Co-IP and ubiquitination assays revealed that PARK2 could interact with YAP in the cytosol and promotes YAP K48-linked ubiquitination at K90 sites. Conclusion: Clinical sample analysis and mechanistic study have validated PARK2 as a tumor suppressor for ESCC. Multivariate Cox proportional regression analysis further revealed that PARK2 is an independent prognostic factor for the overall survival of ESCC patients. Cellular and molecular mechanisms in this study showed that PARK2 associated with YAP protein in the cytosol, promoted YAP ubiquitination and proteasome-dependent degradation in ESCC cells. Therefore, as a novel modulator for Hippo signaling, modulation of PARK2 activity or gene expression level could be an appealing strategy to treat esophageal., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle., Competing Interests: The authors have declared that no competing interests exist.

The molecular mechanism for worsening left ventricular (LV) function after mitral valve (MV) repair for chronic mitral regurgitation remains unknown. We wished to assess the LV transcriptome and identify determinants associated with worsening LV function post-MV repair. A total of 13 patients who underwent MV repair for chronic primary mitral regurgitation were divided into two groups, preserved LV function (N = 8) and worsening LV function (N = 5), for the study. Specimens of LV from the patients taken during surgery were used for the gene microarray study. Cardiomyocyte cell line HL-1 cells were transfected with gene-containing plasmids and further evaluated for mRNA and protein expression, apoptosis, and contractile protein degradation. Of 67,258 expressed sequence tags, microarrays identified 718 genes to be differentially expressed between preserved-LVF and worsening-LVF, including genes related to the protein ubiquitination pathway, bone morphogenetic protein (BMP) receptors, and regulation of eIF4 and p70S6K signaling. In addition, worsening-LVF was associated with altered expressions of genes pathologically relevant to heart failure, such asdownregulated apelin receptors and upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). HL-1 cardiomyocyte cells transfected with ubiquitination-related genes demonstrated activation of the protein ubiquitination pathwaywith an increase in the ubiquitin activating enzyme E1 (UAE-E1). It also led to increased apoptosis, downregulated and ubiquitinated X-linked inhibitor of apoptosis protein (XIAP), and reduced cell viability. Overexpression of ubiquitination-related genes also resulted in degradation and increased ubiquitination of α-smooth muscle actin (SMA). In conclusion, worsening-LVF presented differential gene expression profiles from preserved-LVF after MV repair. Upregulation of protein ubiquitination-related genes associated with worsening-LVF after MV repair may exert adverse effects on LV through increased apoptosis and contractile protein degradation.

The RIG-I-like receptors (RLRs) retinoic acid-inducible gene I protein (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) are cytosolic pattern recognition receptors that recognize specific viral RNA products and initiate antiviral innate immunity. Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic member of the Bunyavirales RIG-I, but not MDA5, has been suggested to sense some bunyavirus infections; however, the roles of RLRs in anti-SFTSV immune responses remain unclear. Here, we show that SFTSV infection induces an antiviral response accompanied by significant induction of antiviral and inflammatory cytokines and that RIG-I plays a main role in this induction by recognizing viral 5'-triphosphorylated RNAs and by signaling via the adaptor mitochondrial antiviral signaling protein. Moreover, MDA5 may also sense SFTSV infection and contribute to IFN induction, but to a lesser extent. We further demonstrate that the RLR-mediated anti-SFTSV signaling can be antagonized by SFTSV nonstructural protein (NSs) at the level of RIG-I activation. Protein interaction and MS-based analyses revealed that NSs interacts with the host protein tripartite motif-containing 25 (TRIM25), a critical RIG-I-activating ubiquitin E3 ligase, but not with RIG-I or Riplet, another E3 ligase required for RIG-I ubiquitination. NSs specifically trapped TRIM25 into viral inclusion bodies and inhibited TRIM25-mediated RIG-I-Lys-63-linked ubiquitination/activation, contributing to suppression of RLR-mediated antiviral signaling at its initial stage. These results provide insights into immune responses to SFTSV infection and clarify a mechanism of the viral immune evasion, which may help inform the development of antiviral therapeutics., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Min et al.)

One important question in aging research is how differences in genomics and transcriptomics determine the maximum lifespan in various species. Despite recent progress, much is still unclear on the topic, partly due to the lack of samples in nonmodel organisms and due to challenges in direct comparisons of transcriptomes from different species. The novel ranking-based method that we employ here is used to analyze gene expression in the gray whale and compare its de novo assembled transcriptome with that of other long- and short-lived mammals. Gray whales are among the top 1% longest-lived mammals. Despite the extreme environment, or maybe due to a remarkable adaptation to its habitat (intermittent hypoxia, Arctic water, and high pressure), gray whales reach at least the age of 77 years. In this work, we show that long-lived mammals share common gene expression patterns between themselves, including high expression of DNA maintenance and repair, ubiquitination, apoptosis, and immune responses. Additionally, the level of expression for gray whale orthologs of pro- and anti-longevity genes found in model organisms is in support of their alleged role and direction in lifespan determination. Remarkably, among highly expressed pro-longevity genes many are stress-related, reflecting an adaptation to extreme environmental conditions. The conducted analysis suggests that the gray whale potentially possesses high resistance to cancer and stress, at least in part ensuring its longevity. This new transcriptome assembly also provides important resources to support the efforts of maintaining the endangered population of gray whales., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster . In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted.

Long intergenic non-coding RNAs (lincRNAs) play important roles in human cancer development, including cell differentiation, apoptosis, and tumor progression. However, their underlying mechanisms of action are largely unknown at present. In this study, we focused on a novel suppressor lincRNA that has the potential to inhibit progression of human hepatocellular carcinoma (HCC). Our experiments disclosed long intergenic non-protein coding RNA 1488 (LINC01488) as a key negative regulator of HCC. Clinically, patients with high LINC01488 expression displayed greater survival rates and better prognosis. In vitro and in vivo functional assays showed that LINC01488 overexpression leads to significant suppression of cell proliferation and metastasis in HCC. Furthermore, LINC01488 bound to cyclin E to induce its ubiquitination and reduced expression of vimentin mediated by both miR-124-3p/miR-138-5p. Our results collectively indicate that LINC01488 acts as a tumor suppressor that inhibits metastasis and tumorigenesis in HCC via the miR-124-3p/miR-138-5p/vimentin axis. Furthermore, LINC01488 interacts with and degrades cyclin E, which contributes to its anti-tumorigenic activity. In view of these findings, we propose that enhancement of LINC01488 expression could be effective as a potential therapeutic strategy for HCC.

Prostate cancer is the second leading cause of cancer-related death in the United States. As a first line treatment for hormone-refractory prostate cancer, docetaxel (DTX) treatment leads to suboptimal effect since almost all patients eventually develop DTX resistance. In this study, we investigated whether and how TGF-β affects DTX resistance of prostate cancer. Methods: Cytotoxicity of DTX in DU 145 and PC-3 cells was measured by CCK-8 and Matrigel colony formation assays. Resistance to DTX in DU 145 cells was examined in a xenograft tumorigenesis model. A luciferase reporter system was used to determine transcriptional activities. Gene expression was analyzed by RT-qPCR and Western blotting. Results: We found that KLF5 is indispensable in TGF-β-induced DTX resistance. Moreover, KLF5 acetylation at lysine 369 mediates DTX resistance in vitro and in vivo . We showed that the TGF-β/acetylated KLF5 signaling axis activates Bcl-2 expression transcriptionally. Furthermore, DTX-induced Bcl-2 degradation depends on a proteasome pathway, and TGF-β inhibits DTX-induced Bcl-2 ubiquitination. Conclusion: Our study demonstrated that the TGF-β-acetylated KLF5-Bcl-2 signaling axis mediates DTX resistance in prostate cancer and blockade of this pathway could provide clinical insights into chemoresistance of prostate cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

RNF12 is a widely expressed ubiquitin E3 ligase that is required for X-chromosome inactivation, regulation of LIM-domain containing transcription factors, and TGF-β signaling. A RING domain at the C terminus of RNF12 is important for its E3 ligase activity, and mutations in the RING domain are associated with X-linked intellectual disability. Here we have characterized ubiquitin transfer by RNF12, and show that the RING domain can bind to, and is active with, ubiquitin conjugating enzymes (E2s) that produce degradative ubiquitin chains. We report the crystal structures of RNF12 in complex with two of these E2 enzymes, as well as with an E2~Ub conjugate in a closed conformation. These structures form a basis for understanding the deleterious effect of a number of disease causing mutations. Comparison of the RNF12 structure with other monomeric RINGs suggests that a loop prior to the core RING domain has a conserved and essential role in stabilization of the active conformation of the bound E2~Ub conjugate. Together these findings provide a framework for better understanding substrate ubiquitylation by RNF12 and the impact of disease causing mutations., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple ( Malus domestica ) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA - A , which encodes a vacuolar H + -ATPase, and MdVHP1 , which encodes a vacuolar H + -pyrophosphatase, as well as MdALMT9 , which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells., (© 2020 Duan et al.)

The maintenance of ROS homeostasis, membrane biogenesis and recycling of molecules are common stress responses involving specific and complex regulatory network. Ubiquitination is an important and common mechanism which facilitates environmental adaptation in eukaryotes. In the present study we have cloned the AlRabring7, an E3-Ub-ligase, previously identified as AlRab7 interacting partner. The role of AlRabring7 for ubiquitinating AlRab7 and facilitating stress tolerance is analysed. The AlRabring7, with an open-reading frame of 702 bp encodes a protein of 233 amino acids, with RING-HC domain of 40 amino acids. In silico analysis shows that AlRabring7 is a C3HC4-type RING E3 Ub ligase. The protein - protein docking show interaction dynamics between AlRab7-AlRabring7-Ubiquitin proteins. The AlRab7 and AlRabring7 transcript showed up-regulation in response to different salts i.e: NaCl, KCl, CaCl 2 , NaCl + KCl, NaCl + CaCl 2 , imposing ionic as well as hyperosmotic stress, and also with oxidative stress by H 2 O 2 treatment. Interestingly, the AlRabring7 showed early transcript expression with maximum expression in shoots on combinatorial stresses. The AlRab7 showed delayed and maximum expression with NaCl + CaCl 2 stress treatment. The AlRab7 complements yeast ypt7Δ mutants and restored the fragmented vacuole. The in vitro ubiquitination assay revealed that AlRabring7 function as E3 ubiquitin ligase and mediates AlRab7 ubiquitination. Overexpression of AlRab7 and AlRabring7 independently and when co-transformed enhanced the growth of yeast cells during stress conditions. Further, the bimolecular fluorescence complementation assay shows the in planta interaction of the two proteins. Our results suggest that AlRab7 and AlRabring7 confers enhanced stress tolerance in yeast., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Circular RNAs (circRNAs), highly expressed in the central nervous system, are involved in various regulatory processes and implicated in some pathophysiology. However, the potential role of circRNAs in psychiatric diseases, particularly major depressive disorder (MDD), remains largely unknown. Here, we demonstrated that circular RNA DYM (circDYM) levels were significantly decreased both in the peripheral blood of patients with MDD and in the two depressive-like mouse models: the chronic unpredictable stress (CUS) and lipopolysaccharide (LPS) models. Restoration of circDYM expression significantly attenuated depressive-like behavior and inhibited microglial activation induced by CUS or LPS treatment. Further examination indicated that circDYM functions as an endogenous microRNA-9 (miR-9) sponge to inhibit miR-9 activity, which results in a downstream increase of target-HECT domain E3 ubiquitin protein ligase 1 (HECTD1) expression, an increase of HSP90 ubiquitination, and a consequent decrease of microglial activation. Taken together, the results of our study demonstrate the involvement of circDYM and its coupling mechanism in depression, providing translational evidence that circDYM may be a novel therapeutic target for depression.

Ubiquitin-activating enzyme E1, UBA1, functions at the apex of the enzymatic ubiquitylation cascade, catalysing ubiquitin activation. UBA1 is thus of fundamental importance to the modulation of ubiquitin homeostasis and to all downstream ubiquitylation-dependent cellular processes, including proteolysis through the ubiquitin-proteasome system and selective autophagy. The proteasome-dependent and -independent functions of UBA1 contribute significantly to a range of processes crucial to neuronal health. The significance of UBA1 activity to neuronal health is clear in light of accumulating evidence implicating impaired UBA1 activity in a range of neurodegenerative conditions, including Parkinson's disease, Alzheimer's disease, Huntington's disease and spinal muscular atrophy. Moreover, ubiquitylation-independent functions of UBA1 of importance to neuronal functioning have been proposed. Here, we summarise findings supporting the significant role of UBA1 in regulating neuronal functioning, and discuss the detrimental consequences of UBA1 impairment that contribute to neuronal dysfunction and degeneration., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Protein degradation is tightly regulated inside cells because of its utmost importance for protein homeostasis (proteostasis). The two major intracellular proteolytic pathways are the ubiquitin-proteasome and the autophagy-lysosome systems which ensure the fate of proteins when modified by various members of the ubiquitin family. These pathways are tightly interconnected by receptors and cofactors that recognize distinct chain architectures to connect with either the proteasome or autophagy under distinct physiologic and pathologic situations. The degradation of proteasome by autophagy, known as proteaphagy, plays an important role in this crosstalk since it favours the activity of autophagy in the absence of fully active proteasomes. Recently described in several biological models, proteaphagy appears to help the cell to survive when proteostasis is broken by the absence of nutrients or the excess of proteins accumulated under various stress conditions. Emerging evidence indicates that proteaphagy could be permanently activated in some types of cancer or when chemoresistance is observed in patients.

The study explores the role of neddylation in early trophoblast development and its alteration during the pathogenesis of recurrent spontaneous abortion (RSA). Immunofluorescence and western blot were conducted to evaluate the expression pattern of NEDD8 protein in the first-trimester placentas of healthy control and RSA patients. Neddylated-cullins, especially neddylated-cullin1, were downregulated and their substrate, p21, was accumulated in RSA samples. NEDD8 cytoplasmic recruitment was observed in extravillous trophoblast (EVT) progenitors of RSA placentas. Consistent with the results of clinical samples, neddylation inhibition using MLN4924 in trophoblast cell lines caused obvious p21 accumulation and free NEDD8 cytoplasmic recruitment. Further in vitro study demonstrated neddylation inhibition attenuated proliferation of Jeg-3 cells via p21 accumulation. Moreover, when trophoblast stem (TS) cells derived from first-trimester placentas were cultured for differentiation analyses. MLN4924 impaired the differentiation of TS cells towards EVTs by downregulating HLA-G and GATA3. p21 knockdown could partly rescue MLN4924-suppressed HLA-G and GATA3 expression. In conclusion, cullin1 neddylation-mediated p21 degradation is required for trophoblast proliferation and can affect trophoblast plasticity by affecting HLA-G and GATA3 expression. The results provide insights into the pathological mechanism of RSA and the biological regulation of trophoblast development., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.

Vaccinia virus (VACV) strain Western Reserve gene A49L encodes a small intracellular protein with a Bcl-2 fold that is expressed early during infection and has multiple functions. A49 co-precipitates with the E3 ubiquitin ligase β-TrCP and thereby prevents ubiquitylation and proteasomal degradation of IκBα, and consequently blocks activation of NF-κB. In a similar way, A49 stabilizes β-catenin, leading to activation of the wnt signalling pathway. However, a VACV strain expressing a mutant A49 that neither co-precipitates with β-TrCP nor inhibits NF-κB activation, is more virulent than a virus lacking A49, indicating that A49 has another function that also contributes to virulence. Here we demonstrate that gene A49L encodes a second, smaller polypeptide that is expressed via leaky scanning translation from methionine 20 and is unable to block NF-κB activation. Viruses engineered to express either only the large protein or only the small A49 protein both have lower virulence than wild-type virus and greater virulence than an A49L deletion mutant. This demonstrates that the small protein contributes to virulence by an unknown mechanism that is independent of NF-κB inhibition. Despite having a large genome with about 200 genes, this study illustrates how VACV makes efficient use of its coding potential and from gene A49L expresses a protein with multiple functions and multiple proteins with different functions.

Intellectual disability (ID) and autism spectrum disorder (ASD) are two of the most common neurodevelopmental disorders. Both disorders are extremely heterogenous, and only ~ 40% of reported cases have so far been attributed to genetic mutations. Of the many cellular processes that are affected, the ubiquitin system (UbS) is of particular relevance in that it can rapidly regulate multiple signaling cascades simultaneously. The UbS is a post-translational modification process that revolves around the covalent attachment of a ubiquitin moiety to a substrate, thereby influencing different elements of protein biology, including trafficking, signal transduction, and degradation. Importantly, the UbS has been implicated in regulating multiple pathophysiological pathways related to ASD and ID. This review will discuss how the UbS acts as major signaling hub in the pathogenesis of ASD and ID, raising the prospect of treating broader patient cohorts by targeting the UbS as a common point of convergence of various mutations.

Tumor necrosis factor alpha-induced protein 1 (TNFAIP1) modulates a plethora of important biological processes, including tumorigenesis and cancer cell migration. However, the regulatory mechanism of TNFAIP1 degradation remains largely elusive. In the present study, with a label-free quantitative proteomic approach, TNFAIP1 was identified as a novel ubiquitin target of the Cullin-RING E3 ubiquitin ligase (CRL) complex. More importantly, Cul3-ROC1 (CRL3), a subfamily of CRLs, was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation. Mechanistically, BTBD9, a specific adaptor component of CRL3 complex, was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells. As such, downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1, whereas TNFAIP1 deletion abrogated this effect. Finally, bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer, which was associated with poor overall survival of patients. Taken together, these findings reveal a previously unrecognized mechanism by which the CRL3 BTBD9 ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration.

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.

Protein ubiquitination is one of the most prevalent post-translational modifications, controlling virtually every process in eukaryotic cells. Recently, the Legionella effector MavC was found to mediate a unique ubiquitination through transglutamination, linking ubiquitin (Ub) to UBE2N through Ub Gln40 in a process that can be inhibited by another Legionella effector, Lpg2149. Here, we report the structures of MavC/UBE2N/Ub ternary complex, MavC/UBE2N-Ub (product) binary complex, and MavC/Lpg2149 binary complex. During the ubiquitination, the loop containing the modification site K92 of UBE2N undergoes marked conformational change, and Lpg2149 inhibits this ubiquitination through competing with Ub to bind MavC. Moreover, we found that MavC itself also exhibits weak deubiquitinase activity towards this non-canonical ubiquitination. Together, our study not only provides insights into the mechanism and inhibition of this transglutaminase-induced ubiquitination by MavC, but also sheds light on the future studies into UBE2N inhibition by this modification and deubiquitinases of this unique ubiquitination.

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin. Mutation of the LIR motif on Cx37, Cx43, Cx46 and Cx50 impairs interaction with LC3B and GABARAP without compromising protein ubiquitination. Through in vitro protein-protein interaction assays, we demonstrate that this LIR motif is required for the binding of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins.