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Esophageal cancer is one of the leading malignancies worldwide, while around sixty percent of newly diagnosed cases are in China. In recent years, genome-wide sequencing studies and cancer biology studies show that Hippo signaling functions a critical role in esophageal squamous cell carcinoma (ESCC) progression, which could be a promising therapeutic targets in ESCC treatment. However, the detailed mechanisms of Hippo signaling dys-regulation in ESCC remain not clear. Here we identify SHARPIN protein as an endogenous inhibitor for YAP protein. SHARPIN depletion significantly decreases cell migration and invasion capacity in ESCC, which effects could be rescued by further YAP depletion. Depletion SHARPIN increases YAP protein level and YAP/TEAD target genes, such as CTGF and CYR61 in ESCC. Immuno-precipitation assay shows that SHARPIN associates with YAP, promoting YAP degradation possibly via inducing YAP K48-dependent poly-ubiquitination. Our study reveals a novel post-translational mechanism in modulating Hippo signaling in ESCC. Overexpression or activation of SHARPIN could be a promising strategy to target Hippo signaling for ESCC patients., 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 © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Maternal immune activation (MIA) is an independent risk factor for psychiatric disorders including depression spectrum in the offsprings, but the molecular mechanism is unclear. Recent studies show that interferon-stimulated gene-15 (ISG15) is involved in inflammation and neuronal dendrite development; here we studied the role of ISG15 in MIA-induced depression and the underlying mechanisms., Methods: By vena caudalis injecting polyinosinic: polycytidylic acid (poly I:C) into the pregnant rats to mimic MIA, we used AAV or lentivirus to introduce or silence ISG15 expression. Synaptic plasticity was detected by confocal microscope and Golgi staining. Cognitive performances of the offspring were measured by Open field, Forced swimming and Sucrose preference test., Results: We found that MIA induced depression-like behaviors with dendrite impairments in the offspring with ISG15 level increased in the offsprings' brain. Overexpressing ISG15 in the prefrontal cortex of neonatal cubs (P0) could mimic dendritic pathology and depressive like behaviors, while downregulating ISG15 rescued these abnormalities in the offsprings. Further studies demonstrated that MIA-induced upregulation of inflammatory cytokines promoted ISG15 expression in the offspring' brain which suppressed Rap2A ubiquitination via NEDD4 and thus induced Rap2A accumulation, while upregulating NEDD4 abolished ISG15-induced dendrite impairments., Conclusions: These data reveal that MIA impedes offsprings' dendrite development and causes depressive like behaviors by upregulating ISG15 and suppressing NEDD4/Rap2A signaling. The current findings suggest that inhibiting ISG15 may be a promising intervention of MIA-induced psychiatric disorders in the offsprings., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is an oncoviral protein that plays a pivotal role in EBV-induced oncogenic transformation. The function of LMP1 in EBV-induced oncogenesis has been well studied. However, the molecular mechanisms underlying LMP1 protein stability remain poorly understood. In this study, we found that ribosomal protein s27a (RPS27a) regulates LMP1 stability by a tandem affinity purification analysis. RPS27a interacts directly with LMP1 in vitro and in vivo. Furthermore, overexpression of RPS27a increases the half-life of LMP1 in 293T cells, whereas downregulation of RPS27a using lentiviral shRNA technology accelerates the decrease in LMP1 protein level in EBV-transformed B cells. We show that LMP1 ubiquitination via the proteasome is completely inhibited by overexpression of RPS27a. RPS27a also enhances LMP1-mediated proliferation and invasion, suggesting that RPS27a interacts with LMP1 and stabilizes it by suppressing proteasome-mediated ubiquitination. These results suggest that RSP27a could be a potential target in EBV-infected LMP1-positive cancer cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL., (© 2016 by The American Society of Hematology.)

Hepatocellular carcinoma is an aggressive and rapidly fatal malignancy representing the common cancer worldwide. The specific cellular gene involved in carcinogenesis has not been fully characterized. The ubiquitin-like modifier FAT10, recently reported to be overexpressed in 90% of hepatocellular carcinoma carcinomas, was attributed to transcriptional upregulation upon the loss of p53 and induced chromosome instability in long-term in vitro culture. However, the exact function of FAT10 in hepatocellular carcinoma is not clear. In the present study, we utilized adenovirus-mediated RNA interference to knock down FAT10 expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. The results demonstrated that interference of FAT10 could inhibit cell proliferation by inhibiting the cell cycle S-phase entry and inducing cell apoptosis. In addition, in vivo experiments showed that adenovirus Ad-siRNA/FAT10 significantly suppressed tumor growth and prolonged the lifespan of tumor-bearing mice. These results suggest that knockdown of FAT10 by adenovirus-delivered siRNA may be a promising therapeutical strategy for treatment of hepatocellular carcinoma., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In previous studies we identified the interferon stimulated gene 15 (ISG15) as a pro-viral host factor in the pathogenesis of hepatitis C virus (HCV) infection. However, the functional link between ISG15 and the HCV replication cycle is not well understood. Aim of the present study was to functionally analyze the role of ISG15 and to identify possible HCV promoting effector molecules. Isg15 suppression was investigated in the murine subgenomic HCV replicon (MH1) transfected with Isg15-specific siRNA and in C57BL/6 mice intravenously injected with lipid nanoparticles (LNP)-formulated siRNA. Interestingly, the LNP-formulated siRNA led to hepatocyte-specific knockdown of Isg15 in vivo, which mediated a hypo-responsiveness to endogenous and exogenous interferon. A label free proteome analysis accompanied by western blot and quantitative RT-PCR techniques led to identification of five candidate proteins (Heterogeneous nuclear ribonucleoprotein A3 (HnrnpA3), Heterogeneous nuclear ribonucleoprotein K (HnrnpK), Hydroxymethylglutaryl-CoA synthase (Hmgcs1), Isocitrate dehydrogenase cytoplasmic (Idh1) and Thioredoxin domain-containing protein 5 (Txndc5)) that are either involved in lipid metabolism or belong to the family of Heterogeneous nuclear ribonucleoprotein (Hnrnp). All candidate proteins are likely to be associated with the HCV replication complex. Furthermore treatment with HnrnpK-specific siRNA directly suppressed HCV replication in vitro. Taken together these data suggest that targeting Isg15 may represent an attractive novel therapeutic option for the treatment of chronic HCV infection.

CDC6 is an important component of the pre-replication complex and plays an essential role in the regulation of DNA replication in eukaryotic cells. Deregulation of CDC6 protein levels results in rereplication and genomic instability. CDC6 expression is tightly regulated during the cell cycle. One major mechanism of cell cycle dependent regulation of CDC6 is APC(Cdh1) mediated protein ubiquitination and degradation during G1 phase. In addition to APC(Cdh1) dependent degradation, alternative, Cullin RING E3 ubiquitin ligase dependent degradation pathways have been characterized in yeast. Here we studied whether Cullin RING E3 ligases also play a role in the turnover of CDC6 protein in mammalian cells. To this end, we used the Nedd8 E1 inhibitor MLN4924, which blocks the activity of all Cullin E3 ligases. We observed that treatment with MLN4924 increased CDC6 protein expression. However, this effect was due to a delay in cell cycle progression from G1 to S phase, resulting in accumulation of cells with high CDC6 protein levels. Therefore, our results indicate that Cullin E3 ligases are not involved in the basal turnover of CDC6 in mammalian cells. Interestingly, we also found that the DNA cross-linker mitomycin C induces marked CDC6 protein degradation. Mitomycin C induced CDC6 degradation is not mediated by APC(Cdh1), Cullin or HUWE1 E3 ubiquitin ligases. Notably, mitomycin C mediated CDC6 degradation requires the neddylation pathway. Our results provide evidence for a novel, cullin independent mechanism of CDC6 posttranslational regulation upon DNA damage that involves the neddylation pathway., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.

The conjugation of proteins with the ubiquitin-like protein Nedd8 is an essential cellular process and an important anti-cancer therapeutic target. The major known role of Nedd8 is the attachment to and activation of Cullin RING E3 ubiquitin ligases (CRL). The attachment of Nedd8 to its substrates occurs via a process analogous to ubiquitin transfer, involving a Nedd8 E1 activating enzyme and a Nedd8 E2 conjugating enzyme, Ubc12, which transfers Nedd8 onto lysine residues of target proteins. In this study, we utilize dominant-negative Ubc12 (dnUbc12) and the Nedd8 E1 inhibitor MLN4924 to inhibit cellular neddylation. We demonstrate that dnUbc12 functions by depleting cellular Nedd8 concentrations. Inhibition of cellular neddylation leads to rapid accumulation of CRL substrates and an enlarged and flattened morphology in HEK293 cells. Inhibiting Nedd8 conjugation also causes abnormalities in the actin cytoskeleton. This is likely at least partially mediated via accumulation of the small GTPase RhoA, a recently identified CRL substrate. We indeed found that siRNA mediated knockdown of RhoA can reverse the morphological changes observed upon inhibition of cellular neddylation. In conclusion, the Nedd8 pathway plays an important role in regulating the actin cytoskeleton and cellular morphology. Dysfunction of the actin cytoskeleton may contribute to the anti-cancer effect of Nedd8 inhibition., (Copyright 2010 Elsevier Inc. All rights reserved.)

A common cause for pharmacogenetic alteration in drug response is genetic variation in encoded amino acid sequence. We have used the catecholamine and drug-metabolizing enzyme sulfotransferase (SULT)1A3 to create an artificial model system to study mechanisms-especially possible aggresome formation-by which genetic alteration in amino acid sequence might influence function. Specifically, we created a double variant SULT1A3 allozyme that included the naturally occurring Asn234 polymorphism plus an additional Trp172Arg mutation. Analysis of the SULT1A3 X-ray crystal structure had indicated that the Trp172Arg mutation might destabilize the protein's structure. Expression of SULT1A3 Arg172,Asn234 in COS-1 cells resulted in undetectable enzyme activity and a virtual lack of enzyme protein. Rabbit reticulocyte lysate degradation studies showed that the double variant allozyme was degraded much more rapidly than was wild type SULT1A3 by a ubiquitin-proteasome-dependent process. In addition, after expression in COS-1 cells, the double variant allozyme localized to aggresomes, a process not previously described or studied in pharmacogenetics. Therefore, the alteration of only one or two amino acids can lead to decreased levels of protein as a result of both aggresome formation and accelerated degradation. The possible role of aggresome formation in pharmacogenetics should be evaluated in naturally occurring systems with inherited alteration in encoded amino acid sequence.

Cytoplasmic poly(A) elongation is widely utilized during the early development of many organisms as a mechanism for translational activation. Targeting of mRNAs for this mechanism requires the presence of a U-rich element, the cytoplasmic polyadenylation element (CPE), and its binding protein, CPEB. Blocking cytoplasmic polyadenylation by interfering with the CPE or CPEB prevents the translational activation of mRNAs that are crucial for oocyte maturation. The CPE sequence and CPEB are also important for translational repression of mRNAs stored in the Xenopus oocyte during oogenesis. To understand the contribution of protein metabolism to these two roles for CPEB, we have examined the mechanisms influencing the expression of CPEB during oogenesis and oocyte maturation. Through a comparison of CPEB mRNA levels, protein synthesis, and accumulation, we find that CPEB is synthesized during oogenesis and stockpiled in the oocyte. Minimal synthesis of CPEB, <3.6%, occurs during oocyte maturation. In late oocyte maturation, 75% of CPEB is degraded coincident with germinal vesicle breakdown. Using proteasome and ubiquitination inhibitors, we demonstrate that CPEB degradation occurs via the proteasome pathway, most likely through ubiquitin-conjugated intermediates. In addition, we demonstrate that degradation requires a 14 amino acid PEST domain., (Copyright 2001 Academic Press.)

The proteasome is not simply a 'garbage disposal unit' but also has functions in the control of the cell cycle and immune responses. The structure of an archaebacterial proteasome has recently been determined to high resolution, and provides insight into the unusual mechanism of proteolytic cleavage by the proteasome.