LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation.

LNX is a RING finger and PDZ domain containing protein that interacts with the cell fate determinant Numb. To investigate the function of LNX, we tested its RING finger domain for ubiquitin ligase activity. The isolated RING finger domain was able to function as an E2-dependent, E3 ubiquitin ligase in vitro and mutation of a conserved cysteine residue within the RING domain abolished its activity, indicating that LNX is the first described PDZ domain-containing member of the E3 ubiquitin ligase family. We have identified Numb as a substrate of LNX E3 activity in vitro and in vivo. In addition to the RING finger, a region of LNX, including the Numb PTB domain-binding site and the first PDZ domain, is required for Numb ubiquitylation. Expression of wild-type but not mutant LNX causes proteasome-dependent degradation of Numb and can enhance Notch signalling. These results suggest that the levels of mammalian Numb protein and therefore, by extension, the processes of asymmetric cell division and cell fate determination may be regulated by ubiquitin-dependent proteolysis.     

Characterization of human LNX, a novel ligand of Numb protein X that is downregulated in human gliomas

Gliomas are major tumors of the central nervous system with a wide spectrum of different tumor types. Ligand of Numb protein X (LNX) is PDZ domain containing protein that interacts with cell fate determinant Numb. cDNA microarray analysis was used to determine the expression of 13,939 genes in a set of 18 gliomas. It showed that human LNX was downregulated in 100% of gliomas including low- and high-grade ones, which was confirmed by Northern blot. In situ hybridization analysis revealed that LNX was lowly expressed in cytoplasm of glioma cells. Thus, LNX might act as a diagnostic marker and a potential therapeutic target for glioma. Two-hybrid screen in yeast was used to identify human LNX interacting proteins important for LNX function. It showed that human LNX interacted with Ski interacting protein (SKIP) via PDZ domains. The co-immunoprecipitation results suggested that LNX interacted with SKIP in HEK293 cells. LNX could affect the subcellular localization of Numb, which indicated that LNX might function as a molecular anchor that localized Numb to the subcellular site of its interaction with Notch. The presence of multiple protein binding domains involved in signal transduction and interaction with Numb and SKIP suggested an important role for LNX in tumorogenesis.     

The active zone protein CAST directly associates with Ligand-of-Numb protein X

The presynaptic active zone (AZ) is a specialized site where neurotransmitter release occurs in a precisely regulated manner. The cytomatrix at the AZ (CAZ)-associated protein CAST and its family member ELKS form a large molecular complex at the AZ and regulate neurotransmitter release by binding other AZ proteins including Bassoon, Piccolo, Munc13-1, and RIM1. Here, yeast two-hybrid screening was used to identify Ligand-of-Numb Protein X (LNX) as a potential binding partner for CAST. LNX is an interactor of Numb and has four PDZ domains. CAST bound LNX both in vivo and in vitro. This binding required the COOH-terminus of CAST and the second PDZ domain of LNX. CAST and LNX were further colocalized with each other in a heterologous expression system, in which LNX was recruited to a Triton X-insoluble structure. Moreover, exogenously expressed LNX was partially colocalized with endogenous CAST in the axonal varicosities of cultured rat hippocampal neurons. These results suggest that CAST and LNX might form a protein complex in neurons.     

Biochemical and computational analysis of LNX1 interacting proteins

PDZ (Post-synaptic density, 95 kDa, Discs large, Zona Occludens-1) domains are protein interaction domains that bind to the carboxy-terminal amino acids of binding partners, heterodimerize with other PDZ domains, and also bind phosphoinositides. PDZ domain containing proteins are frequently involved in the assembly of multi-protein complexes and clustering of transmembrane proteins. LNX1 (Ligand of Numb, protein X 1) is a RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligase that also includes four PDZ domains suggesting it functions as a scaffold for a multi-protein complex. Here we use a human protein array to identify direct LNX1 PDZ domain binding partners. Screening of 8,000 human proteins with isolated PDZ domains identified 53 potential LNX1 binding partners. We combined this set with LNX1 interacting proteins identified by other methods to assemble a list of 220 LNX1 interacting proteins. Bioinformatic analysis of this protein list was used to select interactions of interest for future studies. Using this approach we identify and confirm six novel LNX1 binding partners: KCNA4, PAK6, PLEKHG5, PKC-alpha1, TYK2 and PBK, and suggest that LNX1 functions as a signalling scaffold.     

Ectopic expression of Ligand-of-Numb protein X promoted TGF-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells

Ligand-of-Numb protein X (LNX) was initially characterized as a RING finger type E3 ubiquitin ligase that targeted the intrinsic cell fate determinant Numb for ubiquitination dependent degradation. However, the physiological function of LNX remains largely unknown. In the present study, we demonstrate that ectopic expression of LNX in human proximal tubular epithelial cells (HK-2 cells) significantly enhanced TGF-β1 induced epithelial to mesenchymal transition (EMT). The EMT-promoting effect of LNX manifested as strong inhibition of E-cadherin expression, enhanced expression of vimentin, fibronectin or PAI-1, and increased cell migration. This function of LNX was shown to be independent of its ligase activity because ectopic expression of a mutant form of LNX (C48ALNX) that lacks E3 ligase activity had the similar effect as the wild-type LNX. Overexpression of E-cadherin could inhibit LNX augmented EMT. This study suggests a potential role for LNX in promoting EMT in human proximal tubular epithelial cells.     

A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb

LNX was originally cloned as a Numb PTB-binding molecule, and it was subsequently found to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of mNumb. Numb is a PTB domain-containing protein that functions as an intrinsic determinant of cell fate in asymmetric cell division. In mammals, four protein isoforms arise from alternative mRNA splicing. Here we report that while all four protein isoforms bind to LNX, only p72 and p66 Numb isoforms are ubiquitylated and degraded. The p72 and p66 Numb proteins differ from the other two isoforms by the presence of an 11-amino acid sequence insert in the PTB domain (PTBi). We demonstrate that the isoform-specific ubiquitylation of mNumb is due to a novel interaction between the first PDZ domain (PDZ1) of LNX and the PTBi variant. Deletion of LNX PDZ1 domain resulted in loss of ubiquitylation and subsequent degradation of the PTBi form of Numb. Interestingly efficient PTBi ubiquitylation not only depends on association with the LNX PDZ1 domain but also requires binding to the canonical PTB-binding motif NPAY in LNX. Thus two distinct modes of PTBi-mediated interaction with LNX work in concert to allow the effective and specific degradation of the p72 and p66 isoforms of mNumb.     

A novel lncRNA PLK4 up‐regulated by talazoparib represses hepatocellular carcinoma progression by promoting YAP‐mediated cell senescence

A growing number of studies recognize that long non‐coding RNAs (lncRNAs) are essential to mediate multiple tumorigenic processes, including hepatic tumorigenesis. However, the pathological mechanism of lncRNA‐regulated liver cancer cell growth remains poorly understood. In this study, we identified a novel function lncRNA, named polo‐like kinase 4 associated lncRNA (lncRNA PLK4, GenBank Accession No. RP11‐50D9.3), whose expression was dramatically down‐regulated in hepatocellular carcinoma (HCC) tissues and cells. Interestingly, talazoparib, a novel and highly potent poly‐ADP‐ribose polymerase 1/2 (PARP1/2) inhibitor, could increase lncRNA PLK4 expression in HepG2 cells. Importantly, we showed that talazoparib‐induced lncRNA PLK4 could function as a tumour suppressor gene by Yes‐associated protein (YAP) inactivation and induction of cellular senescence to inhibit liver cancer cell viability and growth. In summary, our findings reveal the molecular mechanism of talazoparib‐induced anti‐tumor effect, and suggest a potential clinical use of talazoparib‐targeted lncRNA PLK4/YAP‐dependent cellular senescence for the treatment of HCC.     

泛素连接酶LNX对上皮细胞间连接的影响

为阐明泛素连接酶LNX在维持上皮细胞之间连接的作用,构建了四环素诱导表达LNX的MDCK细胞株;以免疫荧光法观察细胞连接蛋白E-钙黏素和ZO-1在细胞内的分布,发现LNX的表达使E-钙黏素和ZO-1在细胞内的分布发生明显改变,大量E-钙黏素和ZO-1聚集在胞浆中,而在细胞膜上的分布则明显减少;透射电镜观察上皮细胞间连接的超微结构显示,LNX的表达导致紧密连接和黏附连接的正常结构消失;用钙离子转换实验检测黏附连接的形成发现,表达LNX的MDCK细胞间的黏附连接形成的速度明显滞后于正常细胞.上述结果表明,LNX的表达影响了E-钙黏素在细胞膜上的正常分布,从而延迟黏附连接复合体的形成,导致上皮细胞间连接结构的异常.     

EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

The ligands of Numb proteins X1 and X2 are specific markers for chronic Q fever

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is spontaneously resolutive and is characterized by an efficient immune response. In contrast, chronic Q fever is characterized by dysregulated immune response, as demonstrated by the failure of C. burnetii to induce lymphoproliferation and the lack of granulomas. Recently, it has been demonstrated that when co-expressed in heterologous mammalian cell lines, the ligands of Numb proteins X1 and X2 (LNX1 and LNX2) regulate the level of the T-cell co-receptor CD8, which plays an essential role in T-cell-mediated immune response. We decided to investigate the expression of LNX1 and LNX2 genes in patients with acute or chronic Q fever. Interestingly, we found a high level of LNX1 and LNX2 mRNAs in endocarditis, the principal manifestation of chronic Q fever, but not in acute Q fever. Our data suggest that LNXs may be used as complementary biomarkers to follow the prognosis of chronic Q fever.     

Tight,cell type-specific control of LNX expression in the nervous system,at the level of transcription,translation and protein stability

LNX1 and LNX2 are E3 ubiquitin ligases that can interact with Numb — a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, largely because LNX proteins are present at very low levels in vivo. Here, we demonstrate expression of both LNX1 and LNX2 proteins in the brain for the first time. We clarify the cell-type specific expression of LNX isoforms in both the CNS and PNS, and identify a novel LNX1 isoform. Using luciferase reporter assays, we show that the 5′ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, we find some evidence that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. These observations provide plausible explanations for the low levels of LNX1 proteins detected in vivo.     

Progress in the research of p53 tumour suppressor activity controlled by Numb in triple‐negative breast cancer

Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.     

LncRNA HOXA11‐AS promotes hepatocellular carcinoma progression by repressing miR‐214‐3p

Accumulating studies supported that lncRNAs played important roles in tumorigenesis. LncRNA HOXA11‐AS was a novel lncRNA that has been proved to involved in several tumours. However, the role of HOXA11‐AS in the development of hepatocellular carcinoma (HCC) remains to be explained. In our study, we showed that HOXA11‐AS expression was up‐regulated in the HCC tissues, and the higher expression of HOXA11‐AS was associated with the advanced stage in the HCC samples. In addition, we indicated that the expression of HOXA11‐AS was up‐regulated in HCC cell lines (Hep3B, SMMC‐7721, MHCC97‐H and BEL‐7402) compared with normal liver cell lines (HL‐7702). Overexpression of HOXA11‐AS promoted HCC proliferation and invasion and induced the epithelial‐mesenchymal transition (EMT) and knockdown of HOXA11‐AS suppressed the HCC cell proliferation and invasion. However, we showed that miR‐214‐3p expression was down‐regulated in the HCC tissues and cell lines. Ectopic expression of miR‐214‐3p suppressed HCC cell proliferation and invasion. Furthermore, we indicated that overexpression of HOXA11‐AS decreased the miR‐214‐3p expression and the expression of miR‐214‐3p was negatively related with the HOXA11‐AS expression in HCC samples. Ectopic expression of HOXA11‐AS increased HCC proliferation and invasion and induced EMT through inhibiting miR‐214‐3p expression. These data suggested that HOXA11‐AS/miR‐214‐3p axis was responsible for development of HCC.     

microRNA‐1914, which is regulated by lncRNA DUXAP10, inhibits cell proliferation by targeting the GPR39‐mediated PI3K/AKT/mTOR pathway in HCC

Increasing studies have confirmed that abnormally expressed microRNAs (miRNAs) take part in the carcinogenesis as well as the aggravation of hepatocellular carcinoma (HCC). However, little information is currently available about miR‐1914 in HCC. Here, we first confirmed that miR‐1914 inhibition in HCC cell lines and tumour specimens correlates with tumour size and histological grade. In a series of functional experiments, miR‐1914 inhibited tumour proliferation and colony formation, resulting in cell cycle arrest and increased apoptosis. Moreover, miR‐1914 mediated its functional effects by directly targeting GPR39 in HCC cells, leading to PI3K/AKT/mTOR repression. Restoring GPR39 expression incompletely counteracted the physiological roles of miR‐1914 in HCC cells. In addition, down‐regulation of AKT phosphorylation inhibited the effects of miR‐1914 in HCC. Furthermore, the overexpression of lncRNA DUXAP10 negatively correlated with the expression of miR‐1914 in HCC; thus, lncRNA DUXAP10 regulated miR‐1914 expression and modulated the GPR39/PI3K/AKT‐mediated cellular behaviours. In summary, the present study demonstrated for the first time that lncRNA DUXAP10–regulated miR‐1914 plays a functional role in inhibiting HCC progression by targeting GPR39‐mediated PI3K/AKT/mTOR pathway, and this miRNA represents a novel therapeutic target for patients with HCC.     

泛素连接酶LNX1的表达、活性测定和功能初探

目的:在大肠杆菌中表达重组人泛素连接酶LNX1,并研究其对钾离子通道蛋白Kv1.4的泛素化作用。方法:构建人LNX1重组蛋白原核表达载体pGEX-LNX1,在大肠杆菌中通过IPTG诱导表达重组蛋白,表达产物经GSTrap FF纯化,并通过体外泛素化(in vitroubiquitination)方法测定其泛素连接酶活性,同样用体外泛素化方法研究其对含有Kv1.4的C端的人工底物的泛素化。结果:获得了纯化的有泛素连接酶活性的重组人LNX1蛋白,重组LNX1可以在体外泛素化体系中泛素化含有Kv1.4的C端的人工底物。结论:重组人LNX1原核表达成功,具有泛素连接酶活性,并催化Kv1.4的泛素化。