The E3/E4 ubiquitin conjugation factor UBE4B interacts with and ubiquitinates the HTLV-1 Tax oncoprotein to promote NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), and the neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein persistently activates the NF-κB pathway to enhance the proliferation and survival of HTLV-1 infected T cells. Lysine 63 (K63)-linked polyubiquitination of Tax provides an important regulatory mechanism that promotes Tax-mediated interaction with the IKK complex and activation of NF-κB; however, the host proteins regulating Tax ubiquitination are largely unknown. To identify new Tax interacting proteins that may regulate its ubiquitination we conducted a yeast two-hybrid screen using Tax as bait. This screen yielded the E3/E4 ubiquitin conjugation factor UBE4B as a novel binding partner for Tax. Here, we confirmed the interaction between Tax and UBE4B in mammalian cells by co-immunoprecipitation assays and demonstrated colocalization by proximity ligation assay and confocal microscopy. Overexpression of UBE4B specifically enhanced Tax-induced NF-κB activation, whereas knockdown of UBE4B impaired Tax-induced NF-κB activation and the induction of NF-κB target genes in T cells and ATLL cell lines. Furthermore, depletion of UBE4B with shRNA resulted in apoptotic cell death and diminished the proliferation of ATLL cell lines. Finally, overexpression of UBE4B enhanced Tax polyubiquitination, and knockdown or CRISPR/Cas9-mediated knockout of UBE4B attenuated both K48- and K63-linked polyubiquitination of Tax. Collectively, these results implicate UBE4B in HTLV-1 Tax polyubiquitination and downstream NF-κB activation.     

UHRF1 epigenetically down-regulates UbcH8 to inhibit apoptosis in cervical cancer cells

UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is an important epigenetic regulator that plays a part in DNA methylation, protein methylation and ubiquitination. It is also frequently overexpressed in many types of cancers, including cervical cancer, which is caused by human papillomavirus (HPV). In this study, we showed that UHRF1 was up-regulated in HPV oncogene E7 expressing cells and HPV-positive cervical cancer cells. We demonstrated that UHRF1 down-regulated the expression of UBE2L6 gene that encodes the ISG15-conjugating enzyme UbcH8. Overexpression of UHRF1 reduced UBE2L6 while knockdown UHRF1 elevated the expression of UBE2L6. We showed that UHRF1 regulated UBE2L6 gene by promoter hypermethylation in cervical cancer cells. Consistent with the functions of UHRF1, restored expression of UbcH8 induced apoptosis. These findings establish UBE2L6 as a novel target of UHRF1 that regulates the apoptosis function of UHRF1. Our studies suggest that UHRF1/ UbcH8 can be manipulated for therapy in cervical cancer.     

ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A

Bladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.Subject terms: Cancer models, Bladder cancer     

Mitsugumin 53 (MG53) Ligase Ubiquitinates Focal Adhesion Kinase during Skeletal Myogenesis

The striated muscle-specific mitsugumin 53 (MG53) is a novel E3 ligase that induces the ubiquitination of insulin receptor substrate 1 (IRS-1) during skeletal myogenesis, negatively regulating insulin-like growth factor and insulin signaling. Here we show that focal adhesion kinase (FAK) is the second target of MG53 during skeletal myogenesis. The FAK protein level gradually decreased, whereas its mRNA level was constant during myogenesis in C2C12 cells and MyoD-overexpressing mouse embryonic fibroblasts. The FAK protein was associated with the E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation experiments. FAK ubiquitination and degradation was induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes. Because RING-disrupted MG53 mutants (C14A and ΔR) did not induce FAK ubiquitination and degradation, the RING domain was determined to be required for MG53-induced FAK ubiquitination. Taken together, these data indicate that MG53 induces FAK ubiquitination with the aid of UBE2H during skeletal myogenesis.     

DEPDC1 up‐regulates RAS expression to inhibit autophagy in lung adenocarcinoma cells

DEP domain containing 1(DEPDC1) is involved in the tumorigenesis of a variety of cancers. But its role in tumorigenesis of lung adenocarcinoma (LUAD) is not fully understood. Here, we investigated the role and the underlying mechanisms of DEPDC1 in the development of LUAD. The expression and prognostic values of DEPDC1 in LUAD were analysed by using the data from public databases. Gene enrichment in TCGA LUAD was analysed using GSEA software with the pre‐defined gene sets. Cell proliferation, migration and invasion of A549 cells were examined with colony formation, Transwell and wound healing assays. The function of DEPDC1 in autophagy and RAS‐ERK1/2 signalling was determined with Western blot assay upon DEPDC1 knockdown and/or overexpression in A549, HCC827 and H1993 cells. The results demonstrated that DEPDC1 expression was up‐regulated in LUAD tissues, and its high expression was correlated with unfavourable prognosis. The data also showed that DEPDC1 knockdown impaired proliferation, migration and invasion of A549 cells. Most notably, the results showed that DEPDC1 up‐regulated RAS expression and thus enhanced ERK1/2 activity, through which DEPDC1 could inhibit autophagy. In conclusion, our study revealed that DEPDC1 is up‐regulated in LUAD tissues and plays an oncogenic role in LUAD, and that DEPDC1 inhibits autophagy through the RAS‐ERK1/2 signalling in A549, HCC827 and H1993 cells.     

UBE2E Ubiquitin-conjugating Enzymes and Ubiquitin Isopeptidase Y Regulate TDP-43 Protein Ubiquitination

Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3^C145S was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43^K263E ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43^K263E ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.     

UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53

The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors.     

Down-regulation of epidermal growth factor receptor by curcumin-induced UBE1L in human bronchial epithelial cells

UBE1L, ubiquitin-activating enzyme E1-like, is the activating enzyme of ISG15ylation (ISG15, interferon stimulated gene 15). Loss of UBE1L and activation of epidermal growth factor receptor (EGFR) signaling are common events in lung carcinogenesis. Curcumin, a well-studied chemopreventive agent, is known to down-regulate EGFR. The present study demonstrated that curcumin decreased EGFR expression in human bronchial epithelial (HBE) Beas-2B cells and lung cancer A549 cells. For the first time, UBE1L was found to be induced by curcumin in HBE cells. Interestingly, overexpression of UBE1L reduced EGFR at posttranslational level in HBE cells. UBE1L triggered EGFR membrane internalization and promoted complex formation between ISG15 and EGFR. Curcumin decreased EGFR downstream signaling pAKT and nuclear factor κB (NF-κB). Overexpression or knockdown of UBE1L also resulted in down-regulation or up-regulation of phosphoinositide 3-kinase/AKT/NF-κB correspondently. In human samples, there was an inverse relationship between UBE1L and EGFR/AKT/NF-κB in non-small cell lung cancer tissues and adjacent tissues. These results uncover a novel chemopreventive mechanism of curcumin in inducing UBE1L and down-regulating EGFR signaling in HBE cells.     

Inhibitors of apoptosis proteins (IAPs) as potential molecular targets for therapy of hematological malignancies

Apoptosis, a programmed cell death, plays a key role in the regulation of tissue homeostasis. However, impairment of its regulation may promote formation and progression of malignancy. An important part of the apoptotic machinery are the inhibitor of apoptosis protein (IAP) family, regulating caspase activity, cell division or cell survival pathways through binding to their baculovirus AIP repeat (BIR) domains and/or by their ubiquitin-ligase RING zinc finger (RZF) activity. The following IAPs have been described so far: NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8). Several studies suggested a potential contribution of IAPs to oncogenesis and resistance to anti-tumor treatment. Increased IAP expression was found in variety of human cancers, including hematological malignancies, such as leukemias and B-cell lymphomas. A correlation between the progression of those diseases and high levels of survivin or XIAP has been reported. Overexpression of XIAP in acute myeloid leukemia or survivin in acute lymphoblastic leukemia and diffuse large B-cell lymphoma have been indicated as an unfavorable prognostic factors. Elevated cellular levels of cIAP1, cIAP2, XIAP and survivin correlated with a progressive course of chronic lymphocytic leukemia. Thus, targeting IAPs with small-molecule inhibitors by their antisense approaches or natural IAP antagonist mimetics, may be an attractive strategy of anti-cancer treatment. Such agents can either directly induce apoptosis of tumor cells or sensitize them to other cytotoxic agents, hence overcoming drug-resistance. This review demonstrates the current knowledge on IAP molecular biology, as well as the mechanisms of action and the development of IAP-targeting agents for treatment of hematological malignancies.     

A Ubiquitin Shuttle DC-UbP/UBTD2 Reconciles Protein Ubiquitination and Deubiquitination via Linking UbE1 and USP5 Enzymes

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.     

CHPF promotes gastric cancer tumorigenesis through the activation of E2F1

Chondroitin polymerizing factor (CHPF) is an important glycosyltransferase involved in the biosynthesis of chondroitin sulfate. However, the relationship between CHPF and gastric cancer has not been fully investigated. CHPF expression in gastric cancer tissues was detected by immunohistochemistry and correlated with gastric cancer patient prognosis. Cultured gastric cancer cells and human gastric epithelial cell line GES1 were used to investigate the effects of shCHPF and shE2F1 on the development and progression of gastric cancer by MTT, western blotting, flow cytometry analysis of cell apoptosis, colony formation, transwell and gastric cancer xenograft mouse models, in vitro and in vivo. In gastric cancer tissues, CHPF was found to be significantly upregulated, and its expression correlated with tumor infiltration and advanced tumor stage and shorter patient survival in gastric cancer. CHPF may promote gastric cancer development by regulating cell proliferation, colony formation, cell apoptosis and cell migration, while knockdown induced the opposite effects. Moreover, the results from in vivo experiments demonstrated that tumor growth was suppressed by CHPF knockdown. Additionally, E2F1 was identified as a potential downstream target of CHPF in the regulation of gastric cancer, and its knockdown decreased the CHPF-induced promotion of gastric cancer. Mechanistic study revealed that CHPF may regulate E2F1 through affecting UBE2T-mediated E2F1 ubiquitination. This study showed, for the first time, that CHPF is a potential prognostic indicator and tumor promoter in gastric cancer whose function is likely carried out through the regulation of E2F1.Subject terms: Gastric cancer, Cell biology     

UBE2T-mediated Akt ubiquitination and Akt/β-catenin activation promotes hepatocellular carcinoma development by increasing pyrimidine metabolism

The oncogene protein ubiquitin-conjugating enzyme E2T (UBE2T) is reported to be upregulated in hepatocellular carcinoma (HCC) and correlated with poor clinical outcomes of HCC patients. However, the underlying mechanism by which UBE2T exerts its oncogenic function in HCC remains largely unexplored. In this study, in vitro and in vivo experiments suggested that UBE2T promoted HCC development including proliferation and metastasis. GSEA analysis indicated that UBE2T was positively correlated with pyrimidine metabolism, and LC/MS-MS metabolomics profiling revealed that the key products of pyrimidine metabolism were significantly increased in UBE2T-overexpressing cells. UBE2T overexpression led to the upregulation of several key enzymes catalyzing de novo pyrimidine synthesis, including CAD, DHODH, and UMPS. Moreover, the utilization of leflunomide, a clinically approved DHODH inhibitor, blocked the effect of UBE2T in promoting HCC progression. Mechanistically, UBE2T increased Akt K63-mediated ubiquitination and Akt/β-catenin signaling pathway activation. The disruption of UBE2T-mediated ubiquitination on Akt, including E2-enzyme-deficient mutation (C86A) of UBE2T and ubiquitination-site-deficient mutation (K8/14 R) of Akt impaired UBE2T’s effect in upregulating CAD, DHODH, and UMPS. Importantly, we demonstrated that UBE2T was positively correlated with p-Akt, β-catenin, CAD, DHODH, and UMPS in HCC tumor tissues. In summary, our study indicates that UBE2T increases pyrimidine metabolism by promoting Akt K63-linked ubiquitination, thus contributing to HCC development. This work provides a novel insight into HCC development and a potential therapeutic strategy for HCC patients.Subject terms: Liver cancer, Ubiquitylation     

Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce -/- background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites.     

UBE2T基因对结直肠细胞增殖和凋亡的影响

目的:探讨人类泛素结合酶E2T(Ubiquitin-conjugating enzyme E2T,UBE2T)基因对结肠细胞增殖和凋亡的影响。方法:体外培养人正常结直肠粘膜细胞FHC,采用将UBE2T基因慢病毒质粒转染至FHC细胞48 h后,通过MTT法检测细胞增殖情况,western blotting检测细胞中增殖相关蛋白UBE2T蛋白、Ki67、促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达,流式细胞术检测细胞凋亡率。结果:与转染空质粒的FHC细胞相比,UBE2T基因慢病毒质粒转染FHC细胞48 h后,细胞增殖能力显著上调(P0.05),UBE2T蛋白明显增加,Ki67的表达明显增加(P0.05),细胞凋亡率显著降低(P0.05),且Bax的表达明显下调而Bcl-2的表达上调(P0.05)。结论:UBE2T基因能够促进正常结肠粘膜细胞的增殖,并抑制其凋亡。     

Increased UBE2L6 regulated by type 1 interferon as potential marker in TB

The aim of this study is to identify potential biomarker of tuberculosis (TB) and determine its function. Differentially expressed mRNAs(DEGs) were selected from a blood database GSE101805, and then, 30 key genes were screened using STING, Cytoscape and further functionally enriched. We then found that only 6 of 13 genes related to ubiquitination (the first in the functional enrichment) were increased significantly. ROC analysis showed that UBE2L6, among 6 genes, had the highest diagnostic value, and then, we found that it also had mild value in differential diagnosis. Moreover, our analysis showed that UBE2L6 may be upregulated by type I interferon, which was further confirmed by us. In addition, we also found that UBE2L6 inhibits the apoptosis of Mycobacterium tuberculosis(Mtb)infected macrophages. Subsequently, we discovered that miR-146a-5p, which may target UBE2L6, is reduced in peripheral blood mononuclear cells (PBMC) and plasma of TB, and it also had certain diagnostic efficiency(AUC=0.791). In brief, we demonstrated that UBE2L6 as well as miR-146a-5p is a potential biomarker for TB and UBE2L6,which may also plays important role in TB by, at least, modulating Mtb-infected macrophage apoptosis.     

沉默DEPDC1基因表达对鼻咽癌细胞系HNE-1生长和细胞周期的影响

为探讨沉默DEPDC,基因表达对鼻咽癌细胞系HNE．1生长和细胞周期的影响,该实验设计合成靶向DEPDCl的小分子干扰RNA（smallinterferingRNA,siRNA）转染人鼻咽癌HNE-1细胞。转染后,采用荧光定量PCR、免疫印迹、MTT及流式细胞术方法检测细胞内DEPDCl的表达量以及细胞周期、生长增殖、凋亡的变化及其可能机制。结果显示,转染DEPDClsiRNA后,DEPDC1基因在mRNA及蛋白水平的表达量明显降低;大量细胞被阻滞于G2／M期,生长增殖减慢,凋亡增加。荧光定量PCR结果表明,抑制NF—KB激活的A20基因表达量明显上调,受NF-κB调控的肿瘤相关靶基因的表达量下降,包括C-MYC、MMP9、ICAM-1、BCL-2基因。由此说日月,沉默DEPDC1基因可以影响HNE-1细胞的周期,抑制其生长增殖,促进凋亡,其机制可能与抑制NF-κB通路有关。     

The effect of lentivirus-mediated SIRT1 gene knockdown in the ATDC5 cell line via inhibition of the Wnt signaling pathway

SIRT1 is a highly conserved type III acetyltransferase gene located on chromosome 10 in mammals that belong to the Sirtuins family. In order to explore the effects of the SIRT1 gene in the ATDC5 cell line, an RNAi SIRT1 target sequence was designed and synthesized, aimed to knockdown the expression of SIRT1 in ATDC5 by a lentivirus. Gene chip, qrt-PCR, and WES analyses were used to detect the expression of SIRT1 and changes to the Wnt signaling pathway, while detecting any changes in proliferation and differentiation factors. The results showed that the expressions of the SIRT1 gene, mRNA, and protein were lower after transfection of the RNAi SIRT1sequence into ATDC5 cells. The Wnt signaling pathway, especially the classical pathway, was inhibited by the knockdown of SIRT1. The cartilaginous proliferation and differentiation of ATDC5 cells were simultaneously inhibited, and apoptosis was accelerated. In summary, knocking down SIRT1 gene increased the degeneration of ATDC5 cells via inhibiting the Wnt signaling pathway. We also found some novel factors related to the Wnt signaling pathway after SIRT1 gene knockdown (BIRC3, IL1RAP, PPP3CA, PPP2R2A, PPP2R5E, GSN, PPP2R1B, etc), which might provide new clues in disease research related to chondrocyte degeneration.     

敲低去泛素化酶USP13抑制K562细胞的增殖*

目的:研究去泛素化酶USP13对人慢性髓系白血病细胞系K562增殖和凋亡的影响,并进行初步的机制探究。方法:构建pLKO.1-shUSP13-GFP慢病毒干涉载体,慢病毒包装后感染并建立稳定敲低USP13的K562细胞株。免疫印迹检测K562细胞中USP13蛋白的敲低效率。流式细胞术分析敲低USP13对K562细胞增殖和凋亡的影响。免疫共沉淀和蛋白质泛素化实验探究USP13调控K562细胞的分子机制。结果:成功构建pLKO.1-shUSP13-GFP慢病毒干涉载体,同时利用慢病毒体系获得稳定敲低USP13的K562细胞株。流式细胞术结果显示,敲低USP13促进K562细胞凋亡、抑制细胞增殖。分子机制研究发现,敲低USP13通过增强c-Myc泛素化进而导致其蛋白质水平降低。结论:初步揭示了USP13调控K562细胞增殖和凋亡的分子机制,为治疗慢性髓系白血病提供了潜在的靶点。