Ligand Of Numb protein-X Research Articles

Hypereosinophilic Syndrome with Endomyocarditis: Identification by Next-Generation Sequencing of the JAK2-V617F Mutation.

Hypereosinophilic syndrome requires a peripheral absolute eosinophil count of ≥1.5 × 109 /L with clinical manifestations attributable to peripheral or tissue hypereosinophilia. Clinical manifestations can vary greatly, with the majority of patients relatively asymptomatic and the eosinophilia detected incidentally. However, in a minority of hypereosinophilia cases, they may present with severe lifethreatening organ dysfunction affecting skin, lung, heart, gastrointestinal tract, and nervous system. A case of hypereosinophilia with potentially life-threatening cardiovascular involvement is discussed. Initial laboratory investigations showed an elevated white blood cell count with 60% eosinophils. An endomyocardial biopsy revealed eosinophilic endomyocarditis with granuloma, rare giant cells, and no vasculitis, microorganisms, or malignancy. Her presentation met the criteria for either hypereosinophilic syndrome or eosinophilic granulomatosis with polyangitis. Molecular genetic analysis was negative for myelodysplastic syndrome panel/ Platelet Derived Growth Factor Receptor Beta (PDGFRB) (5q32)/Fibroblast Growth Factor Receptor 1 (FGFR1) Fluorescence In Situ Hybridization (FISH), Feline McDonough Sarcoma-related Tyrosine Kinase 3 (FLT3) Internal Tandem Duplication (ITD) mutation, Calregulin (CALR) exon 9 mutation, and T-cell gene rearrangement/polymerase chain reaction. Bone marrow biopsy revealed a mildly hypocellular marrow with multilineage hematopoiesis,+ megakaryocyte dysplasia, and focal eosinophilia. No excess blasts, no monotypic B-cell population, and no discrete pan T-cell aberrancies were found. Bone marrow cytogenetic studies showed a normal signal pattern for myeloproliferative neoplasms panel/Sec1 Family Domain Containing 2 (SCFD2)-Ligand of Numb Protein-X (LNX)-Platelet-derived Growth Factor Receptor Alpha (PDGFRA) fluorescence in situ hybridization with a normal karyotype of 46 XX. Next-generation sequencing, however, was positive for the JAK2-V617F mutation, a rare molecular abnormality in hypereosinophilic syndrome. The prevalence ranges from approximately 0% to 4%. The JAK2 point mutation leads to aberrant tyrosine phosphorylation and increased cytokine activation. The case demonstrates the complexity and challenging nature of advanced diagnostic opportunities in hypereosinophilia and the potential use, in select subsets, of targeted treatments such as tyrosine kinase inhibitors.

PDZRN4 suppresses tumorigenesis and androgen therapy-resistance in prostate cancer.

Background: PDZRN4 (PDZ domain-containing RING finger 4), a member of the LNX (ligand of numb protein-X) family that regulates the levels of NUMB, plays a critical role in suppressing the proliferation and invasion of hormone-related malignant tumours. There are few studies on the role of PDZRN4 in the pathogenesis of prostate cancer (PCa). We aimed to examine whether PDZRN4 regulates the growth and development of PCa.Methods: Cell transduction and Western blotting were used to establish and confirm PDZRN4 knock down in PC cells. Using the MTT, wound healing, transwell migration, and animal experiments, we explored the biological function of PDZRN4 knockdown (PDZRN4-kd) cells. Via PCR and immunohistochemistry, the mRNA and protein expression of PDZRN4 was examined in PC cells and tissues.Results: Hormone-dependent (LNCap) and hormone-independent (DU145, PC3, and C4-2) PC lines were transfected with lentivirus carrying PDZRN4 shRNA. The Western blotting results showed that the expression of PDZRN4 was stably downregulated in PDZRN4 knockdown (PDZRN4-kd) cells. The proliferation, invasion and migration of PDZRN4-kd cells were dramatically increased in vivo. To explore the expression of PDZRN4 in prostate cancer samples, we analysed TCGA data and found that PDZRN4 was negatively correlated with the development of PC. PDZRN4 levels were downregulated by androgen deprivation in hormone-sensitive cells. Moreover, PDZRN4 failed to induce proliferation in DU145 cells with androgen deprivation.Conclusions: PDZRN4 is a functional suppressor of prostate cancer growth and development and is a potential target of biochemical therapy in hormone-resistant PC.

The Molecular and Pathophysiological Functions of Members of the LNX/PDZRN E3 Ubiquitin Ligase Family.

The ligand of Numb protein-X (LNX) family, also known as the PDZRN family, is composed of four discrete RING-type E3 ubiquitin ligases (LNX1, LNX2, LNX3, and LNX4), and LNX5 which may not act as an E3 ubiquitin ligase owing to the lack of the RING domain. As the name implies, LNX1 and LNX2 were initially studied for exerting E3 ubiquitin ligase activity on their substrate Numb protein, whose stability was negatively regulated by LNX1 and LNX2 via the ubiquitin-proteasome pathway. LNX proteins may have versatile molecular, cellular, and developmental functions, considering the fact that besides these proteins, none of the E3 ubiquitin ligases have multiple PDZ (PSD95, DLGA, ZO-1) domains, which are regarded as important protein-interacting modules. Thus far, various proteins have been isolated as LNX-interacting proteins. Evidence from studies performed over the last two decades have suggested that members of the LNX family play various pathophysiological roles primarily by modulating the function of substrate proteins involved in several different intracellular or intercellular signaling cascades. As the binding partners of RING-type E3s, a large number of substrates of LNX proteins undergo degradation through ubiquitin-proteasome system (UPS) dependent or lysosomal pathways, potentially altering key signaling pathways. In this review, we highlight recent and relevant findings on the molecular and cellular functions of the members of the LNX family and discuss the role of the erroneous regulation of these proteins in disease progression.

Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice.

NUMB is a key regulator of neurogenesis and neuronal differentiation that can be ubiquitinated and targeted for proteasomal degradation by ligand of numb protein-X (LNX) family E3 ubiquitin ligases. However, our understanding of LNX protein function in vivo is very limited. To examine the role of LNX proteins in regulating NUMB function in vivo, we generated mice lacking both LNX1 and LNX2 expression in the brain. Surprisingly, these mice are healthy, exhibit unaltered levels of NUMB protein and do not display any neuroanatomical defects indicative of aberrant NUMB function. Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. Given the apparent absence of major NUMB dysfunction in LNX null animals, we performed a proteomic analysis to identify neuronal LNX-interacting proteins other than NUMB that might contribute to the anxiolytic phenotype observed. We identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2 and LIPRIN-αs (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homologue) and SRGAP2. These and other novel LNX-interacting proteins identified are promising candidates to mediate LNX functions in the central nervous system, including their role in modulating anxiety-related behaviour.

Structural basis for the indispensable role of a unique zinc finger motif in LNX2 ubiquitination.

LNX (Ligand of Numb Protein-X) proteins, LNX1 and LNX2, are RING- and PDZ-based E3-ubiquitin ligases known to interact with Numb. Silencing of LNX2 has been reported to down-regulate WNT and NOTCH, two key signaling pathways in tumorigenesis. Here we report the identification of the domain boundary of LNX2 to confer its ubiquitination activity, its crystal structure along with functional studies. We show that the RING domain in LNX2 is flanked by two Zinc-binding motifs (Zn-RING-Zn), in which the N-terminal Zinc-binding motif adopts novel conformation. Although this motif follows the typical Cys2His2-type zinc finger configuration, it is devoid of any secondary structure and forms an open circle conformation, which has not been reported yet. This unique N-terminal Zn-finger motif is indispensable for the activity and stability of LNX2, as verified using mutational studies. The Zn-RING-Zn domain of LNX2 is a dimer and assumes a rigid elongated structure that undergoes autoubiquitination and undergoes N-terminal polyubiquitination. The ubiquitin chains consist of all seven possible isopeptide linkages. These results were validated using full-length LNX2. Moreover we have demonstrated the ubiquitination of cell fate determinant protein, Numb by LNX2. Our study provides a structural basis for the functional machinery of LNX2 and thus provides the opportunity to investigate suitable drug targets against LNX2.

PDZRN4 acts as a suppressor of cell proliferation in human liver cancer cell lines.

Recently, some reports show that Ligand of Numb Protein-X 1 (LNX1) could be a suppressor gene in gliomas, while our current research has firstly shown that PDZ domain containing ring finger 4 (PDZRN4), another member of LNX family, could also be a potential suppressor in hepatocellular carcinoma (HCC). PDZRN4, also named LNX4 (Ligand of Numb Protein-X 4), is a member of the LNX family. We recently found that PDZRN4, but not LNX1, was down-regulated in HCC samples, and the role of PDZRN4 in the progression of HCC had not been studied before. To address this question, firstly, we evaluated the expression of PDZRN4 in HCC samples and adjacent non-cancerous tissues. Semi-quantitative polymerase chain reaction showed that PDZRN4 was down-regulated in 24/36 (66.7%) HCC samples separately. In addition, our research shows that PDZRN4 is silenced in all of the 12 HCC cell lines tested. Subsequently, cell-based functional assay exhibited that ectopic expression of PDZRN4 inhibits the proliferation, plate colony formation and anchorage-independent colony formation of HCC cells. Collectively, our results showed that PDZRN4 might be a potential tumour suppressor gene and had anti-proliferative effect on HCC cell proliferation, which would be of great significance to the researches on HCC.

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain-containing E3 Ubiquitin Ligase

Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.

Molecular evolution of the LNX gene family

BackgroundLNX (Ligand of Numb Protein-X) proteins typically contain an amino-terminal RING domain adjacent to either two or four PDZ domains - a domain architecture that is unique to the LNX family. LNX proteins function as E3 ubiquitin ligases and their domain organisation suggests that their ubiquitin ligase activity may be targeted to specific substrates or subcellular locations by PDZ domain-mediated interactions. Indeed, numerous interaction partners for LNX proteins have been identified, but the in vivo functions of most family members remain largely unclear.ResultsTo gain insights into their function we examined the phylogenetic origins and evolution of the LNX gene family. We find that a LNX1/LNX2-like gene arose in an early metazoan lineage by gene duplication and fusion events that combined a RING domain with four PDZ domains. These PDZ domains are closely related to the four carboxy-terminal domains from multiple PDZ domain containing protein-1 (MUPP1). Duplication of the LNX1/LNX2-like gene and subsequent loss of PDZ domains appears to have generated a gene encoding a LNX3/LNX4-like protein, with just two PDZ domains. This protein has novel carboxy-terminal sequences that include a potential modular LNX3 homology domain. The two ancestral LNX genes are present in some, but not all, invertebrate lineages. They were, however, maintained in the vertebrate lineage, with further duplication events giving rise to five LNX family members in most mammals. In addition, we identify novel interactions of LNX1 and LNX2 with three known MUPP1 ligands using yeast two-hybrid asssays. This demonstrates conservation of binding specificity between LNX and MUPP1 PDZ domains.ConclusionsThe LNX gene family has an early metazoan origin with a LNX1/LNX2-like protein likely giving rise to a LNX3/LNX4-like protein through the loss of PDZ domains. The absence of LNX orthologs in some lineages indicates that LNX proteins are not essential in invertebrates. In contrast, the maintenance of both ancestral LNX genes in the vertebrate lineage suggests the acquisition of essential vertebrate specific functions. The revelation that the LNX PDZ domains are phylogenetically related to domains in MUPP1, and have common binding specificities, suggests that LNX and MUPP1 may have similarities in their cellular functions.

LNX (Ligand of Numb-protein X) interacts with RhoC, both of which regulate AP-1-mediated transcriptional activation

LNX (Ligand of Numb-protein X) was originally isolated as a binding partner to the cell-fate Determinant Numb during development, and then identified to act as a RING finger-type E3 ubiquitin ligase for the ubiquitylation and degradation of Numb. LNX contains 4 PDZ domains which are proved to play a central role in organizing diverse cell signaling assemblies. A yeast two-hybrid screening was used to identify LNX as a potential binding partner for RhoC. RhoC, a member of the Ras family of small GTPases, promotes reorganization of the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between LNX and RhoC in mammalian cells was identified by co-immunoprecipitation assays, and the efficient binding required the first PDZ domain of LNX. LNX and RhoC were further colocalized with each other in mammalian cells, in which RhoC changed its sublocalization from cytoplasm to nucleus when co-transferred with LNX. Furthermore, co-expression of RhoC reduced the transcriptional activity of AP-1, which was up-regulated by over-expression of LNX alone. These results suggest that LNX and RhoC might be part of a larger protein complex that would have important functions in signaling transduction about regulating the transcriptional activities of AP-1.

Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like

The organizer anchors the primary embryonic axis, and balance between dorsal (organizer) and ventral domains is fundamental to body patterning. Ligand of Numb protein-X (LNX) is a RING finger and four PDZ domain containing E3 ubiquitin ligase1,2. LNX serves as a binding platform and may have a role in cell fate determination, but its in vivo functions are unknown1–5. Here we show that Lnx-l (Lnx-like) acts as a critical regulator of dorso-ventral (D-V) axis formation in zebrafish. Depletion of Lnx-l using specific antisense morpholinos (MO), caused strong embryonic dorsalization. We identified Bozozok (Boz; also called Dharma or Nieuwkoid) as a binding partner and substrate of Lnx-l. Boz is a homeodomain-containing transcriptional repressor induced by canonical Wnt signaling that is critical for dorsal organizer formation6–12. Lnx-l induced K48-linked polyubiquitination of Boz, leading to its proteasomal degradation in human 293T cells and in zebrafish embryos. Dorsalization induced by Boz overexpression was suppressed by raising the level of Lnx-l, but Lnx-l failed to counteract dorsalization caused by mutant Boz lacking a critical motif for Lnx-l binding. Further, dorsalization induced by depletion of Lnx-l was alleviated by attenuation of Boz expression. We conclude that Lnx-l modulates Boz activity to prevent the invasion of ventral regions of the embryo by organizer tissue. These studies introduce a ubiquitin ligase, Lnx-l, as a balancing modulator of axial patterning in the zebrafish embryo.

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.

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.

Coxsackievirus and adenovirus receptor (CAR) is expressed in male germ cells and forms a complex with the differentiation factor JAM-C in mouse testis

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein important for viral binding to target cells. Using RT-PCR, Western analysis, GST pull-down assay and indirect immunofluorescence, it was shown that CAR is expressed in male germ cells from mice, rats, and humans. CAR was detected in round spermatids in the testis as well as in purified, mature spermatozoa. The two membrane-bound isoforms of CAR occupied different subcellular sites in the acrosomal region of the spermatozoa. CAR was exposed on the surface of acrosome-reacted, but not acrosome-intact cells. Two CAR-binding proteins belonging to the ligand-of-numb protein-X (LNX) family also occupied distinct regions in spermatozoa. Finally, co-immunoprecipitation experiments demonstrated an interaction between CAR and JAM-C, a protein required for spermatid differentiation. Together, these findings imply a function for CAR in male fertility. The results also suggest that CAR in spermatozoa is inaccessible to adenovirus-based gene therapy vectors, and that the risk of germ line infection therefore is low.

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.

Np9 protein of human endogenous retrovirus K interacts with ligand of numb protein X.

We have recently identified Np9 as a novel nuclear protein produced by the human endogenous retrovirus K and were able to document the exclusive presence of np9 transcript in tumors and transformed cells. With the aim of studying whether Np9 has a role in tumorigenesis, a systematic search for interacting proteins was performed. Here, we identify the RING-type E3 ubiquitin ligase LNX (ligand of Numb protein X) as an Np9-interacting partner. We furthermore show that the interaction involves N- and C-terminal domains of both proteins and can affect the subcellular localization of LNX. LNX has been reported to target the cell fate determinant and Notch antagonist Numb for proteasome-dependent degradation, thereby causing an increase in transactivational activity of Notch. We document that LNX-interacting Np9, like Numb, is unstable and degraded via the proteasome pathway and that ectopic Numb can stabilize recombinant Np9. Combined, these findings point to the possibility that Np9 affects tumorigenesis through the LNX/Numb/Notch pathway.

The Coxsackievirus and Adenovirus Receptor (CAR) Forms a Complex with the PDZ Domain-containing Protein Ligand-of-Numb Protein-X (LNX)

The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.