Ran-binding Protein Research Articles

RANBP9 as potential therapeutic target in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the Western world. Despite progress made with targeted therapies and immune checkpoint inhibitors, the vast majority of patients have to undergo chemotherapy with platinum-based drugs. To increase efficacy and reduce potential side effects, a more comprehensive understanding of the mechanisms of the DNA damage response (DDR) is required. We have shown that overexpressby live cell imaging (Incuyion of the scaffold protein RAN binding protein 9 (RANBP9) is pervasive in NSCLC. More importantly, patients with higher levels of RANBP9 exhibit a worse outcome from treatment with platinum-based drugs. Mechanistically, RANBP9 exists as a target and an enabler of the ataxia telangiectasia mutated (ATM) kinase signaling. Indeed, the depletion of RANBP9 in NSCLC cells abates ATM activation and its downstream targets such as pby live cell imaging (Incuy53 signaling. RANBP9 knockout cells are more sensitive than controls to the inhibition of the ataxia and telangiectasia-related (ATR) kinase but not to ATM inhibition. The absence of RANBP9 renders cells more sensitive to drugs inhibiting the Poly(ADP-ribose)-Polymerase (PARP) resulting in a “BRCAness-like” phenotype. In summary, as a result of increased sensitivity to DNA damaging drugs conferred by its ablation in vitro and in vivo, RANBP9 may be considered as a potential target for the treatment of NSCLC. This article aims to report the results from past and ongoing investigations focused on the role of RANBP9 in the response to DNA damage, particularly in the context of NSCLC. This review concludes with future directions and speculative remarks which will need to be addressed in the coming years.

Ran-binding protein 3 is associated with human spermatogenesis and male infertility.

Ran-binding protein 3 (RanBP3) is a Ran-interacting protein, which participates in the Ran GTPase system in cancer cell biology. However, the expression pattern and physiological role of RanBP3 remain largely unknown. In this study, we found that RanBP3 was expressed in human testes and localised to spermatogonium and spermatocyte of germ cells. In subcellular structure, its localisation is in the nucleus and cytoplasm. Interestingly, compared with normal groups, RanBP3 expression was lower in groups of patients with Maturation Arrest (MA) and Sertoli cell-only syndrome (SCO) when considered by the Johnson Score. RanBP3 expression in the MA group and SCO groups was dramatically lower than that in the normal control group. Studies have shown that RanBP3, which is one of the helper factors of Ran, is mainly participate in the nucleocytoplasmic transport of cells. RanBP3 helps Ran to achieve some functions such as nucleocytoplasmic transport, spindle assembly during mitosis and nuclear assembly after mitosis. Consequent changes in the expression of RanBP3 may associate with human spermatogenesis disorders and male infertility. The identification and characterisation of RanBP3 enhances our understanding of the molecular mechanisms underpinning its function in human spermatogenesis and male infertility.

Reduced RANBP9 expression is associated with poor prognosis in colorectal cancer patients.

BackgroundColorectal cancer (CRC) is the third most common malignancy worldwide. RANBP9 is a RAN-binding protein that has been reported to be a reliable predictor for prognosis in some human cancers. The mechanism of RANBP9 involvement in CRC carcinogenesis is unknown. This study measured RANBP9 expression levels in CRC to determine its association with clinicopathological parameters.MethodsThis study included 228 CRC patients who underwent radical resection. RANBP9 expression was determined using immunohistochemistry. Based on follow-up data, the correlation of RANBP9 expression levels with clinicopathological parameters, including disease free survival (DFS) and overall survival (OS) was evaluated.ResultsReduced RANBP9 expression was correlated with tumor location (P=0.014), vascular invasion (P=0.057) and normal serum carcinoembryonic antigen levels (P=0.001). Patients with reduced RANBP9 expression had a 5-year DFS rate of 63.0% compared to 78.9% for patients with high expression levels of RANBP9 (P=0.015). Subgroup analysis demonstrated that reduced RANBP9 expression was significantly correlated with a worse DFS rate (P=0.037) for patients with left-sided colon cancer. RANBP9 was found to be an independent predictive factor for estimating DFS rate (P=0.029, hazard ratio: 0.580, 95% confidence interval: 0.356–0.946) and OS.ConclusionsRANBP9 expression levels is a potential prognostic factor for estimating CRC survival rates in patients after surgery.

MoRBP9 Encoding a Ran-Binding Protein Microtubule-Organizing Center Is Required for Asexual Reproduction and Infection in the Rice Blast Pathogen Magnaporthe oryzae.

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis.

Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-γ (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome β5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.

Abstract 4824: Targeting CD30 as a novel treatment strategy in RANBP2-ALK-rearranged inflammatory myofibroblastic tumor

Abstract Rationale: Inflammatory myofibroblastic tumors (IMTs) are a particularly rare type of soft tissue sarcoma comprised of myofibroblastic spindle cells and an accompanying inflammatory infiltrate. There is an unmet clinical need for effective treatment regimens for patients diagnosed with IMT with anaplastic lymphoma kinase (ALK) rearrangement, who relapse following ALK inhibitor (ALKi) therapy or who present with aggressive disease. Fusion of RAN Binding Protein 2 (RANBP2) with ALK in IMT is associated with aggressive disease and has been correlated with tumor cell expression of CD30. This study investigated CD30 as a potential therapeutic target in IMT and the efficacy of the CD30-targeted antibody-monomethyl auristatin E conjugate, Brentuximab Vedotin (BV). Methods and Results: In a cohort of five recent IMT patients at the Sydney Children’s Hospital, RANBP2-ALK fusion was identified in three patients (IMT1, IMT2 and IMT3) by RNA capture sequencing, while patients IMT4 and IMT5 (who did not relapse) harbored CLTC-ALK or SEC31A-ALK fusions respectively. Expression of CD30 was confirmed two of three RANBP2-ALK fusion positive tumors by immunohistochemistry. We established cell cultures and xenografts from malignant ascites of IMT1, at diagnosis (IMT1A) and at relapse (IMT1D) after treatment with ALKi’s and low dose chemotherapy. CD30 expression was retained in the cell cultures and xenograft tumors, as demonstrated by flow cytometry and tumor histology. BV was investigated as a potential treatment for IMT with RANBP2-ALK fusion. BV reduced IMT1A and IMT1D cell viability in vitro in resazurin cell viability assays. IMT1A and IMT1D xenograft mice had a partial response to BV which significantly (p<0.0001) prolonged survival compared to untreated controls. However, tumors eventually recurred. Resistance to BV correlated with upregulation of P-glycoprotein and reduced CD30 antigen expression in cells from treated IMT xenograft tumors. The combination of the ALK inhibitor crizotinib with BV has also been investigated. In vivo, the combination of crizotinib and BV resulted in complete resolution of tumor and significantly (p<0.0001) improved survival compared to the individual agents. Conclusion: CD30 is a promising therapeutic target in RANBP2-ALK-rearranged IMT. BV successfully reduced IMT cell viability in vitro and prolonged survival in IMT xenografted mice, both as a single agent and when given in combination with crizotinib. Since BV is current clinical use for the treatment of Hodgkin lymphoma it may be possible to rapidly translate these findings into clinical practice for the treatment of IMT. Citation Format: Ashleigh M. Fordham, James Blackburn, Erin E. Heyer, Jinhan Xie, Emily V. Mould, Andrew J. Gifford, Lisa T. Morgan, Carol Wadham, Mitali Fadia, Jamie I. Fletcher, Karen L. MacKenzie, Toby N. Trahair. Targeting CD30 as a novel treatment strategy in RANBP2-ALK-rearranged inflammatory myofibroblastic tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4824.

Abstract 3514: RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage

Abstract Lung cancer is the leading cause of cancer-related deaths. Despite recent success with targeted and immune-therapies, DNA-damaging agents, such as platinum-based drugs, still represent the first-line of treatment. However, side effects and drug resistance limit their efficacy and use. We recently demonstrated that RAN Binding Protein 9 (RANBP9) plays a critical role in the DNA-damage response (DDR) of lung cancer cells. However, how it affects the mechanisms and outcome of the DDR is not known. In this regard, p53 activity is crucial for cell cycle arrest or programmed cell death. In particular, acetylation of p53 at lysine 120 by the Tat-Interacting Protein of 60 KDa (Tip60) is dispensable for p53-mediated growth arrest but is essential for p21 induction and p53-dependent apoptosis. It has been reported that RANBP9 and Tip60 co-localize. Here, we investigate whether RANBP9 affects Tip60-dependent activation of p53 and its relevance in deciding cell faith in response to DNA damage. For our study we used H1299, A549 and HeLa cells. By CRISPR/Cas9, we generated RANBP9 wild type (WT) and knockout (KO) A549 clones. We also established KO clones stably re-expressing RANBP9-FLAG. Cell response to DNA damage was assessed upon treatment with Ionizing Radiation (IR) and/or Cisplatin (CDDP). Interaction between RANBP9 with Tip60 was assessed by immunoprecipitation of transfected HeLa cell lysates. We show that RANBP9 and Tip60 co-immunoprecipitate either by pulling-down RANBP9 or Tip60. Using different deletion mutants of Tip60, we found that a region encompassing the Zinc Finger and part of the Histone Acetyl-Transferase domain is required for the interaction with RANBP9. Further, the enzymatic-dead Tip60 retained its ability to interact with RANBP9. Using p53 null H1299 cells, we established that this interaction is independent of p53. However, RANBP9 depletion affects both total and activated p53 (p-p53s15) levels after DNA damage. Re-expressing RANBP9 in stable KO clones rescued p53 levels. Upon CDDP treatment, p53 protein levels were higher in the clones stably re-expressing RANBP9 compared to the KO ones. Upon IR or CDDP treatment, p21 protein levels were higher in the presence of RANBP9. Finally, we found that Tip60 levels were higher in CDDP-treated and untreated WT cells comparing to the KO ones. Our results indicate that RANBP9 and Tip60 constitutively interact. The decrease of p53 in RANBP9 absence suggests that RANBP9 might favor p53 acetylation by Tip60 upon DNA damage. Further investigations will elucidate how RANBP9 affects the levels and the functions of Tip60. Also, we will measure the levels of p53-AcK120 both in the WT and KO clones. If p53-AcK120 is impaired when RANBP9 is absent, RANBP9 KO cells might undergo cell death instead of cell cycle arrest. Altogether, our results shed light on a potential RANBP9-Tip60-p53 axis at play during the DDR. Citation Format: Shimaa Soliman, Arturo Orlacchio, Anna Tessari, Marina Capece, Rosa Visone, Carlo Croce, Dario Palmieri, Vincenzo Coppola. RANBP9 presence affects levels of Tip60 and activated p53 in lung cancer cells in response to DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3514.

Comparative Analysis of Soluble Proteins in Four Medicinal Aloe Species by Two-Dimensional Electrophoresis and MALDI-TOF-MS.

Background: Aloe barbadensis Miller 1768, A. vera L. var. chinensis (Haw.) Berger 1908, A. ferox Miller 1768, and A. arborescens Miller 1768 are the most widely cultivated species of Aloe and are used in Asia along with 400 other Aloe species worldwide because of their potent and potential bioactivity. Objective: The objective was to analyze and compare the soluble proteins of four commonly used medicinal Aloe species. Methods: Aloe protein samples were obtained by TCA/acetone-saturated phenol-methanol/ammonium acetate combined extraction (phenol extraction), and then were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Finally, the differentially expressed proteins of four Aloe species were identified by matrix-assisted laser desorption ionization-time-of-flight-MS analysis. Results: The phenol extraction method was the most suitable method for the protein extraction of Aloe. Fifty differentially expressed proteins in four Aloe species were successfully identified and divided into eight functional categories. Furthermore, Malate dehydrogenase and ran-binding protein in A. barbadensis, cytoskeletal-related protein tubulin in A. vera var. chinensis and auxin-induced protein PCNT-115 in A. arborescens are closely related to their morphological characteristics. Conclusions: There are differences in the soluble proteins of the four Aloe species. Those proteins, related to the difference of their morphology of Aloe, might be used to identify different species. Highlights: Fifty differentially expressed proteins in four medicinal Aloe species were identified, and these proteins were classified into eight categories according to their biological functions. Four special proteins closely related to the morphological characteristics of Aloe were found and might be used to identify these four Aloe species.

Microglial activation in an amyotrophic lateral sclerosis-like model caused by Ranbp2 loss and nucleocytoplasmic transport impairment in retinal ganglion neurons.

Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ran-binding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1+-motoneurons develop cardinal ALS-like motor traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This will help to understand the role of nucleocytoplasmic transport in the differential vulnerability of neuronal cell types to ALS and to uncover non-motor endophenotypes with pathognomonic signs of ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1+-RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified CD11b+ and CD45+-microglia, increase of F4\80+-microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80+-microglia intermingled between RGNs of naive mice. Further, there was the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Hence, Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neuronal-type selective substrates. Immune-modulators underpinning RGN-to-microglial signaling are regulated by Ranbp2, and this neuronal-glial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of motoneuron diseases, such as ALS.

RANBP9 suppresses tumor proliferation in colorectal cancer.

RAN binding protein 9 (RANBP9) is widely expressed in mammalian tissues, including osteosarcoma, lung, gastric and breast cancer tissues. However, currently, not much is known about the role of RANBP9 in colorectal cancer (CRC). In the present study, RANBP9 expression in CRC tissues and cell lines was measured by immunohistochemistry and western blotting, respectively. Subsequently, RANBP9-short hairpin RNA (shRNA) and RANBP9 plasmids were constructed and transfected into HCT116 and HT29 cells. The effects of RANBP9 knockdown were assessed by Cell Counting kit-8 and colony formation assays, and its effects on tumorigenicity in a nude mouse animal model were investigated. The effect of RANBP9-shRNA on cell cycle progression was analyzed by flow cytometry, while cell cycle-associated protein expression levels were examined by western blotting. Compared with in paired normal mucosa, RANBP9 was overexpressed in CRC tissues. Inhibition of RANBP9 in HCT116 and HT29 cells significantly promoted cell growth, colony formation and S phase transition, and increased tumorigenesis in vivo. Accordingly, RANBP9 overexpression inhibited cell growth and colony formation. Knockdown of RANBP9 was associated with upregulated cyclin A2 in the two cell lines. In conclusion, RANBP9 served an inhibitory role in CRC in vitro and in vivo. Therefore, RANBP9 may be considered a potential target for treatment of CRC.

Regulation of c-Raf Stability through the CTLH Complex.

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.

Nucleolar and spindle associated protein 1 promotes metastasis of cervical carcinoma cells by activating Wnt/\u03b2-catenin signaling

BackgroundThe primary obstacle to treat cervical cancer is its high prevalence of metastasis, which severely affects patients’ quality of life and survival time. Nucleolar and spindle associated protein 1 (NUSAP1) has been implicated in the development, progression, and metastasis in several types of cancer. However, its oncogenic role in cervical cancer remains unclear.MethodsWestern blot assay and immunohistochemistry were used to determine the expression of NUSAP1 in 21 clinical fresh Cervical cancer tissues and 233 clinicopathologically characterized cervical cancer specimens. The biological roles of NUSAP1 in the metastasis of cervical cancer were investigated both in vitro by EMT, Side population analysis and Transwell assays and so on, and in vivo using a mouse 4w model of hematogenous metastasis and lymph node metastasis. Bioinformatics analysis, luciferase reporter analysis, immunoprecipitation and immunoblotting of nuclear and cytoplasmic cellular fractions were applied to discern and examine the relationshipbetween NUSAP1 and its potential targets.ResultsThe results demonstrated that NUSAP1 was upregulated in cervical cancer cells and tissues, correlated positively with metastasis and poor clinical outcome of patients. High expression of NUSAP1 promoted metastasis by enhancing cancer stem cell (CSC) traits and epithelial-mesenchyme transition (EMT) progression, while silencing of NUSAP1 reduced CSC traits and EMT progression. Mechanistically, upregulation of NUSAP1 induced SUMOylation of TCF4 via interacting with SUMO E3 ligase Ran-binding protein 2 (RanBP2) and hyperactivated Wnt/β-catenin signaling in cervical cancer cells. Additionally, NUSAP1-induced cervical cancer cells metastasis and the cancer stem cell phenotype were abrogated with the Wnt/β-catenin signaling inhibitor XAV-939 treatment. Importantly, co-therapy of conventional treatment and XAV-939 will provide a novel and effective treatment for NUSAP1-ovexpressed cervical cancer patients.ConclusionsOur results demonstrate thatNUSAP1 upregulation contributes to metastasis of cervical cancer by promoting CSC properties and EMT via Wnt/β-catenin signaling and XAV-939 might serve as a potential tailored therapeutic option for patients with NUSAP1-ovexpressed cervical cancer.

Child with Susceptibility to Infection-Induced Acute Encephalopathy 3

AbstractA 10-month-old Bahraini boy born full-term to first-degree cousins, initially had normal developmental milestones, but presented with recurrent encephalopathy and seizures associated with upper respiratory tract infection. With each attack, the patient developmentally regressed further. Brain magnetic resonance imaging showed multiple old and new infarcts. Cerebrospinal fluid biochemistry, microbiology, and amino acids were unremarkable. Tandems mass spectrometry of urine organic acids was unremarkable as well. Electroencephalogram showed asymmetry with cortical irritability. Whole exome sequencing revealed a homozygous mutation of RAN-binding protein 2 gene, suggesting a diagnosis of susceptibility to infection-induced acute encephalopathy 3, which is an autosomal dominant condition. This is the first case to be reported in Bahrain.

Mechanistic insights into the interaction of the MOG1 protein with the cardiac sodium channel Nav1.5 clarify the molecular basis of Brugada syndrome

Nav1.5 is the α-subunit of the cardiac sodium channel complex. Abnormal expression of Nav1.5 on the cell surface because of mutations that disrupt Nav1.5 trafficking causes Brugada syndrome (BrS), sick sinus syndrome (SSS), cardiac conduction disease, dilated cardiomyopathy, and sudden infant death syndrome. We and others previously reported that Ran-binding protein MOG1 (MOG1), a small protein that interacts with Nav1.5, promotes Nav1.5 intracellular trafficking to plasma membranes and that a substitution in MOG1, E83D, causes BrS. However, the molecular basis for the MOG1/Nav1.5 interaction and how the E83D substitution causes BrS remains unknown. Here, we assessed the effects of defined MOG1 deletions and alanine-scanning substitutions on MOG1's interaction with Nav1.5. Large deletion analysis mapped the MOG1 domain required for the interaction with Nav1.5 to the region spanning amino acids 146-174, and a refined deletion analysis further narrowed this domain to amino acids 146-155. Site-directed mutagenesis further revealed that Asp-148, Arg-150, and Ser-151 cluster in a peptide loop essential for binding to Nav1.5. GST pulldown and electrophysiological analyses disclosed that the substitutions E83D, D148Q, R150Q, and S151Q disrupt MOG1's interaction with Nav1.5 and significantly reduce its trafficking to the cell surface. Examination of MOG1's 3D structure revealed that Glu-83 and the loop containing Asp-148, Arg-150, and Ser-151 are spatially proximal, suggesting that these residues form a critical binding site for Nav1.5. In conclusion, our findings identify the structural elements in MOG1 that are crucial for its interaction with Nav1.5 and improve our understanding of how the E83D substitution causes BrS.

A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export.

Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis.

RanBP1 Couples Nuclear Export and Golgi Regulation through LKB1 to Promote Cortical Neuron Polarity

SummaryNeuronal polarity in the developing cortex begins during the early stages of neural progenitor migration toward the cortical plate and culminates with the specification of the axon and dendrites. Here, we demonstrate that the Ran-dependent nucleocytoplasmic transport machinery is essential for the establishment of cortical neuron polarity. We found that Ran-binding protein 1 (RanBP1) regulates axon specification and dendritic arborization in cultured neurons in vitro and radial neural migration in vivo. During axonogenesis, RanBP1 regulates the cytoplasmic levels of the polarity protein LKB1/Par4, and this is dependent on the nuclear export machinery. Our results show that downstream of RanBP1, LKB1 function is mediated by the STK25-GM130 pathway, which promotes axonogenesis through Golgi regulation. Our results indicate that the nucleocytoplasmic transport machinery is a main regulator of neuron polarity, including radial migration, and that the regulated export of LKB1 through RanBP1 is a limiting step of axonogenesis.

RAN/RANBP2 polymorphisms and neuroblastoma risk in Chinese children: a three-center case-control study.

The genetic etiology of sporadic neuroblastoma remains largely obscure. RAN and RANBP2 genes encode Ras-related nuclear protein and Ran-binding protein 2, respectively. These two proteins form Ran-RanBP2 complex that regulate various cellular activities including nuclear transport. Aberrant functions of the two proteins are implicated in carcinogenesis. Given the unknown role of RAN/RANBP2 single nucleotide polymorphisms (SNPs) in neuroblastoma risk, we performed a multi-center case-control study in Chinese children to assess the association of the RAN/RANBP2 SNPs with neuroblastoma risk. We analyzed three potentially functional SNPs in RAN gene (rs56109543 C>T, rs7132224 A>G, rs14035 C>T) and one in RANBP2 (rs2462788 C>T) in 429 cases and 884 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to access the association between these four polymorphisms and neuroblastoma risk. No single variant was found to statistically significantly associate with neuroblastoma risk. However, individuals with 3 protective genotypes were less likely to develop neuroblastoma, in comparison to non-carriers (adjusted OR=0.33; 95% CI=0.12-0.96; P=0.042), as well as those with 0-2 protective genotypes (adjusted OR=0.33; 95% CI=0.11-0.94; P=0.038). Stratified analysis revealed no significant association for any of the four polymorphisms. Further studies are warranted to validate the weak impact of RAN/RANBP2 SNPs on neuroblastoma risk.

Soluble syntaxin 3 functions as a transcriptional regulator

Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that Stx3 (syntaxin 3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform that we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RanBP5 (RAN-binding protein 5), targets to the nucleus, and interacts physically and functionally with several transcription factors, including ETV4 (ETS variant 4) and ATF2 (activating transcription factor 2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified, suggesting that nuclear signaling is a conserved, novel function common among these membrane-trafficking proteins.

A comprehensive analysis of the predicted targets of miR-642b-3p associated with the long non-coding RNA HOXA11-AS in NSCLC cells.

Long non-coding RNA HOXA11 antisense RNA (HOXA11-AS) has been previously reported to be involved in the tumorigenesis and progression of ovarian cancer and glioma. However, the function of HOXA11-AS in lung cancer remains unclear. Following the knockdown of HOXA11-AS in A549 cells, a microarray analysis was performed in order to detect the differences in microRNA (miRNA/miR) profiles. Subsequently, miR-642b-3p was selected for further analysis. Four miRNA target prediction algorithms were used to identify potential target genes of miR-642b-3p. Bioinformatics analyses, including Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes, protein-protein interactions (PPIs) and network analysis, were performed to investigate the potential functions, pathways and networks of the target genes. Furthermore, the differential expression of miR-642b-3p and its target genes between normal lung and non-small cell lung cancer (NSCLC) tissues was verified using The Cancer Genome Atlas (TCGA) database. Six target genes [zinc finger protein 350, heterogeneous nuclear ribonucleoprotein U, high mobility group box 1, phosphodiesterase 4D (PDE4D), synaptotagmin binding cytoplasmic RNA interacting protein and basic helix-loop-helix family member B9] of miR-642b-3p were predicted using all 4 algorithms. It was revealed that miR-642b-3p was overexpressed in adenocarcinoma and squamous cell carcinoma tissues compared with non-cancerous lung tissues based on the TCGA database. From the 6 target genes, PDE4D was downregulated in lung adenocarcinoma and squamous cell carcinoma tissues, and a weak negative correlation between HOXA11-AS and PDE4D was identified. The area under the curve of PDE4D was 0.905 [95% confidence interval (CI), 0.879–0.931] for patients with lung adenocarcinoma and 0.665 (95% CI, 0.606–0.725) for patients with squamous cell carcinoma. Additionally, GO analysis of the target genes revealed that miR-642b-3p was specifically involved in complex cellular pathways. The target gene RAN binding protein 2 possessed the highest degree of interactions in the PPI network (degree=40). It was hypothesized that HOXA11-AS may have a function in NSCLC by regulating the expression of miR-642b-3p and PDE4D, which laid the foundation for the further elucidation of the potential molecular mechanisms of NSCLC.

EGFR feedback-inhibition by Ran-binding protein 6 is disrupted in cancer

Transport of macromolecules through the nuclear pore by importins and exportins plays a critical role in the spatial regulation of protein activity. How cancer cells co-opt this process to promote tumorigenesis remains unclear. The epidermal growth factor receptor (EGFR) plays a critical role in normal development and in human cancer. Here we describe a mechanism of EGFR regulation through the importin β family member RAN-binding protein 6 (RanBP6), a protein of hitherto unknown functions. We show that RanBP6 silencing impairs nuclear translocation of signal transducer and activator of transcription 3 (STAT3), reduces STAT3 binding to the EGFR promoter, results in transcriptional derepression of EGFR, and increased EGFR pathway output. Focal deletions of the RanBP6 locus on chromosome 9p were found in a subset of glioblastoma (GBM) and silencing of RanBP6 promoted glioma growth in vivo. Our results provide an example of EGFR deregulation in cancer through silencing of components of the nuclear import pathway.