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BRAF(V600E) mutant colon cancers (CCs) have a characteristic gene expression signature that is also found in some tumors lacking this mutation. Collectively, they are referred to as "BRAF-like" tumors and represent some 20% of CCs. We used a shRNA-based genetic screen focused on genes upregulated in BRAF(V600E) CCs to identify vulnerabilities of this tumor subtype that might be exploited therapeutically. Here, we identify RANBP2 (also known as NUP358) as essential for survival of BRAF-like, but not for non-BRAF-like, CC cells. Suppression of RANBP2 results in mitotic defects only in BRAF-like CC cells, leading to cell death. Mechanistically, RANBP2 silencing reduces microtubule outgrowth from the kinetochores, thereby inducing spindle perturbations, providing an explanation for the observed mitotic defects. We find that BRAF-like CCs display far greater sensitivity to the microtubule poison vinorelbine both in vitro and in vivo, suggesting that vinorelbine is a potential tailored treatment for BRAF-like CCs.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Vimblastina/análogos & derivados , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Células Cultivadas , Neoplasias do Colo/classificação , Neoplasias do Colo/tratamento farmacológico , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Transplante de Neoplasias , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Proto-Oncogênicas B-raf/genética , Vimblastina/administração & dosagem , Vimblastina/farmacologia , VinorelbinaRESUMO
The neurogenetics and vision community recently mourned William L. Pak, PhD, whose pioneering work spearheaded the genetic, electrophysiological, and molecular bases of biological processes underpinning vision. This essay provides a historical background to the daunting challenges and personal experiences that carved the path to seminal findings. It also reflects on the intellectual framework, mentoring philosophy, and inspirational legacy of Bill Pak's research. An emphasis and perspectives are placed on the discoveries and implications to date of the phosphatidylinositol-specific phospholipase C (PI-PLC), NorpA, and the cyclophilin, NinaA of the fruit fly, Drosophila melanogaster, and their respective mammalian homologues, PI-PLCß4, and cyclophilin-related protein, Ran-binding protein 2 (Ranbp2) in critical biological processes and diseases of photoreceptors and other neurons.
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Ranbp2 (also known as Nup358) is a member of the nucleoporin family, which constitutes the nuclear pore complex. Ranbp2 localizes at the nuclear membrane and was recently reported at the axon initial segment (AIS). However, we show that the anti-Ranbp2 antibody used in previous studies is not specific for Ranbp2. We mapped the antibody binding site to the amino acid sequence KPLQG, which is present in both Ranbp2 and neurofascin (Nfasc), a well-known AIS protein. After silencing neurofascin expression in neurons, the AIS was not stained by the antibody. Surprisingly, an exogenously expressed N-terminal fragment of Ranbp2 localizes at the AIS. We show that this fragment interacts with stable microtubules. Finally, using CRISPR/Cas9 in primary cultured neurons, we inserted an HA-epitope tag at N-terminal, C-terminal or internal sites of the endogenously expressed Ranbp2. No matter the location of the HA-epitope, endogenous Ranbp2 was found at the nuclear membrane but not the AIS. These results show that endogenously expressed Ranbp2 is not found at AISs.This article has an associated First Person interview with the first author of the paper.
Assuntos
Segmento Inicial do Axônio , Complexo de Proteínas Formadoras de Poros Nucleares , Humanos , Neurônios , Membrana Nuclear , Poro Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
PURPOSE: Mendelian etiologies for acute encephalopathies in previously healthy children are poorly understood, with the exception of RAN binding protein 2 (RANBP2)-associated acute necrotizing encephalopathy subtype 1 (ANE1). We provide clinical, genetic, and neuroradiological evidence that biallelic variants in ribonuclease inhibitor (RNH1) confer susceptibility to a distinctive ANE subtype. METHODS: This study aimed to evaluate clinical data, neuroradiological studies, genomic sequencing, and protein immunoblotting results in 8 children from 4 families who experienced acute febrile encephalopathy. RESULTS: All 8 healthy children became acutely encephalopathic during a viral/febrile illness and received a variety of immune modulation treatments. Long-term outcomes varied from death to severe neurologic deficits to normal outcomes. The neuroradiological findings overlapped with ANE but had distinguishing features. All affected children had biallelic predicted damaging variants in RNH1: a subset that was studied had undetectable RNH1 protein. Incomplete penetrance of the RNH1 variants was evident in 1 family. CONCLUSION: Biallelic variants in RNH1 confer susceptibility to a subtype of ANE (ANE2) in previously healthy children. Intensive immunological treatments may alter outcomes. Genomic sequencing in children with unexplained acute febrile encephalopathy can detect underlying genetic etiologies, such as RNH1, and improve outcomes in the probands and at-risk siblings.
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Encefalopatia Aguda Febril , Encefalopatias , Leucoencefalite Hemorrágica Aguda , Criança , Humanos , Leucoencefalite Hemorrágica Aguda/diagnóstico , Leucoencefalite Hemorrágica Aguda/genética , Inflamassomos , Encefalopatias/genética , Fatores de Transcrição , Ribonucleases , Proteínas de TransporteRESUMO
Fetal intracranial hemorrhage (ICH) may result from a wide array of causes, either associated with maternal or fetal risk factors. In the last decade, monogenic causes of susceptibility to fetal ICH have been described, in particular in association with COL4A1 and COL4A2 genes. A peculiar form of ICH is acute necrotizing encephalitis (ANE), which is characterized by a rapid-onset severe encephalopathy following an abnormal inflammatory response to an otherwise banal infection. It usually affects healthy children and it is thought to be multifactorial, with a genetic predisposition. RANBP2 gene has been extensively associated with ANE susceptibility. We hereby present a unique case of a 42-year-old secundigravida with intrauterine fetal demise at 35 weeks of gestation. Trio-based whole-exome sequencing performed on both parents and fetal DNA showed a de novo likely pathogenic variant in the RANBP2 gene on 2q13. At the fetal autopsy, subtentorial hematoma and cerebral intraparenchymal hemorrhage were present. We speculate that this might be a new phenotypic presentation of RANBP2-associated disease. However, more similar fetal cases need to be reported in order to reinforce this hypothesis.
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Hemorragia Cerebral , Leucoencefalite Hemorrágica Aguda , Criança , Feminino , Humanos , Adulto , Leucoencefalite Hemorrágica Aguda/genética , Chaperonas Moleculares/genética , Morte FetalRESUMO
Phosphorylation is a post-translational modification that contributes to versatile protein functions in spermatogenesis, and the variations they generate usually results in abnormal spermatogenesis or sperm dysfunction. The sex-reversal phenomenon exists in Chinese tongue sole under certain conditions such that individuals with a ZW genotype can acquire a male phenotype and are thus called pseudomales. Pseudomale tongue sole can reach sexual maturity but produce only Z-type sperm, and the Z sperm carries paternal epigenetic information. Whether phosphorylation plays a role in the sperm abnormality of pseudomales is unknown. In this study, a phosphoproteomic analysis was performed to compare protein phosphorylation profiles between pseudomale and male testes. Altogether, we identified 14,253 phosphopeptides matching with 4843 proteins, with 1329 differentially phosphorylated peptides corresponding to 1045 differentially phosphorylated proteins (DPPs). Phosphorylation at 781 sites was upregulated and at 548 sites was downregulated. Four motifs were identified among differentially phosphorylated peptides, which were "SP", "SD", "RxxS", and "TP". Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the cell cycle and DNA/RNA processing were significantly enriched with the genes encoding DPPs. To analyze DPP function in depth, a protein-protein interaction network was constructed, and Ran-binding protein 2 was found to play a central role in spermatogenesis by regulating several processes such as the cell cycle, eukaryotic translation, ubiquitination, and minichromosome maintenance. In kinase-associated network analyses, two "mitogen-activated protein kinase (Mapk)-centered" clusters were identified that may account for abnormal spermatogenesis in pseudomales. One cluster was centered on Mapk6, which predominantly regulated the cell cycle by interacting with several cyclin-dependent kinases, and the other was centered on the "testis-expressed kinase 1-like (Tesk1l)/Pim1l-Mapk4l- testis-expressed 14 (Tex14)" kinase cascade, which might contribute to spermatogenesis by regulating ß-catenin. Taken together, these data suggested the new candidates involved in pseudomale sperm abnormalities and provided clues to discover the phosphorylated regulatory mechanism underlying tongue sole spermatogenesis.
Assuntos
Sêmen , Testículo , Masculino , Animais , Testículo/metabolismo , Espermatogênese/genética , Espermatozoides , PeixesRESUMO
Type I interferon (IFN-I)-induced signaling plays a critical role in host antiviral innate immune responses. Despite this, the mechanisms that regulate this signaling pathway have yet to be fully elucidated. The nucleoporin Ran Binding Protein 2 (RanBP2) (also known as Nucleoporin 358 KDa, Nup358) has been implicated in a number of cellular processes, including host innate immune signaling pathways, and is known to influence viral infection. In this study, we documented that RanBP2 mediates the sumoylation of signal transducers and activators of transcription 1 (STAT1) and inhibits IFN-α-induced signaling. Specifically, we found that RanBP2-mediated sumoylation inhibits the interaction of STAT1 and Janus kinase 1 (JAK1), as well as the phosphorylation and nuclear accumulation of STAT1 after IFN-α stimulation, thereby antagonizing the IFN-α-mediated antiviral innate immune signaling pathway and promoting viral infection. Our findings not only provide insights into a novel function of RanBP2 in antiviral innate immunity but may also contribute to the development of new antiviral therapeutic strategies.
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Interferon-alfa , Viroses , Humanos , Interferon-alfa/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares , Sumoilação , Imunidade Inata , Antivirais , Fator de Transcrição STAT1RESUMO
BACKGROUND: We report the first case of COVID-19 associated acute necrotizing encephalopathy (ANE) without pulmonary disease in a patient with an extremely high interleukin-6 (IL-6) level and Ran Binding Protein 2 (RANBP2) mutation. CASE PRESENTATION: A 29-year-old woman recently immunized with inactivated viral vaccine-BBIBP32-CorV (Sinopharm) presented with alteration of consciousness. Her body temperature was 37° Celsius, blood pressure 42/31 mmHg, heart rate 130 bpm, respiratory rate 20 per minute, and oxygen saturation 98%. Respiratory examination was unremarkable. Neurological examination revealed stupor but preserved brainstem reflexes. Non-contrast computerized tomography of the brain showed symmetrical hypodense lesions involving bilateral thalami and cerebellar hemispheres characteristic of ANE. No pulmonary infiltration was found on chest radiograph. SARS-CoV-2 was detected by PCR; whole genome sequencing later confirmed the Delta variant. RANBP2 gene analysis revealed heterozygous Thr585Met mutation. Serum IL-6 was 7390 pg/mL. Urine examination showed pyelonephritis. Her clinical course was complicated by seizure, septic shock, acute kidney injury, and acute hepatic failure. She later developed coma and passed away in 6 days. CONCLUSIONS: ANE is caused by cytokine storm leading to necrosis and hemorrhage of the brain. IL-6 was deemed as a prognostic factor and a potential treatment target of ANE in previous studies. RANBP2 missense mutation strongly predisposes this condition by affecting mitochondrial function, viral entry, cytokine signaling, immune response, and blood-brain barrier maintenance. Also, inactivated vaccine has been reported to precipitate massive production of cytokines by antibody dependent enhancement (ADE). The true incidence of COVID-19 associated ANE is not known as were the predictors of its development. We proposed these potential two factors (RANBP2 mutation and ADE) that could participate in the pathogenesis of ANE in COVID-19 apart from SARS-CoV2 infection by itself. Further study is needed to confirm this hypothesis, specifically in the post-vaccination period. Role of RANBP2 mutation and its application in COVID-19 and ANE should be further elaborated.
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Encefalopatias , COVID-19 , Leucoencefalite Hemorrágica Aguda , Adulto , Encefalopatias/complicações , Feminino , Humanos , Interleucina-6/genética , Leucoencefalite Hemorrágica Aguda/diagnóstico , Leucoencefalite Hemorrágica Aguda/genética , Chaperonas Moleculares , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares , RNA Viral , SARS-CoV-2/genética , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy occurring in otherwise healthy children after common viral infections. The condition presents as a spectrum of symptoms ranging from infections to seizures and coma, with the potential to cause long-term neurocognitive impairment or death. Familial and recurrent ANE is referred to as ANE1. A four-generation Chinese family with ANE1 was recruited for genetic analysis. A novel missense variation, c.9041A > G, p.(Glu3014Gly) in RANBP2 was identified in this family. This study is the first to identify a novel variation in RANBP2 in a Chinese family with ANE1.
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Encefalopatias , Leucoencefalite Hemorrágica Aguda , Chaperonas Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares , Encefalopatias/genética , Humanos , Leucoencefalite Hemorrágica Aguda/genética , Chaperonas Moleculares/genética , Mutação de Sentido Incorreto , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
Ran Binding Protein 2 (RanBP2 or Nucleoporin358) is one of the main components of the cytoplasmic filaments of the nuclear pore complex. Mutations in the RANBP2 gene are associated with acute necrotizing encephalopathy type 1 (ANE1), a rare condition where patients experience a sharp rise in cytokine production in response to viral infection and undergo hyperinflammation, seizures, coma, and a high rate of mortality. Despite this, it remains unclear howRanBP2 and its ANE1-associated mutations contribute to pathology. Mounting evidence has shown that RanBP2 interacts with distinct viruses to regulate viral infection. In addition, RanBP2 may regulate innate immune response pathways. This review summarizes recent advances in our understanding of how mutations in RANBP2 contribute to ANE1 and discusses how RanBP2 interacts with distinct viruses and affects viral infection. Recent findings indicate that RanBP2 might be an important therapeutic target, not only in the suppression of ANE1-driven cytokine storms, but also to combat hyperinflammation in response to viral infections.
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Encefalopatias , Leucoencefalite Hemorrágica Aguda , Viroses , Encefalopatias/genética , Humanos , Leucoencefalite Hemorrágica Aguda/tratamento farmacológico , Leucoencefalite Hemorrágica Aguda/genética , Leucoencefalite Hemorrágica Aguda/patologia , Chaperonas Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Viroses/genéticaRESUMO
Microarray-based comparative genomic hybridization (aCGH) is being increasingly applied to delineate novel genomic disorders and related syndromes in patients with developmental delay. In this study, detailed clinical and cytogenetic data of three unrelated patients with interstitial 2q12.3q13 microdeletion were described and compared with thirteen 2q12.3q13 microdeletion patients, gathered from the medical literature and public databases. 60 K aCGH analysis revealed three overlapping 2q12.3q13 microdeletions measuring 1.88 Mb in patient 1, 1.25 Mb in patient 2, and 0.41 Mb in patient 3, respectively. Confirmation and segregation studies were performed using fluorescence in situ hybridization (FISH) and quantitative real-time PCR. Variable clinical features of 2q12.3q13 microdeletion including microcephaly, prenatal growth retardation, developmental delay, short stature, behavioral problems, learning difficulties, skeletal anomalies, congenital heart defects, and features of ectodermal dysplasia were observed. The boundaries and sizes of the 2q12.3q13 deletions in the sixteen patients were different, but an overlapping region of 249 kb in 2q12.3 was defined. The SRO (smallest region of overlap) encompasses four genes, including LIMS1, RANBP2, CCDC138, and EDAR. Among these genes, RANBP2 is a strong candidate gene for neurological phenotype and genetic susceptibility to viral infections. To our knowledge, this is the first published report of 2q12.3q13 microdeletion syndrome and our observations strongly suggest that these recurrent CNVs may be a novel risk factor for developmental delay with variable expressivity and incomplete penetrance.
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Anormalidades Múltiplas/genética , Comportamento Problema , Deleção Cromossômica , Cromossomos Humanos Par 3/genética , Hibridização Genômica Comparativa/métodos , Feminino , Genômica/métodos , Genótipo , Humanos , Fenótipo , GravidezRESUMO
Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function.This article has an associated First Person interview with the first author of the paper.
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Anquirinas/metabolismo , Axônios/metabolismo , Embrião de Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Anquirinas/genética , Western Blotting , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Although GCN5L1 (also known as BLOC1S1) facilitates mitochondrial protein acetylation and controls endosomal-lysosomal trafficking, the mechanisms underpinning these disparate effects are unclear. As microtubule acetylation modulates endosome-lysosome trafficking, we reasoned that exploring the role of GCN5L1 in this biology may enhance our understanding of GCN5L1-mediated protein acetylation. We show that α-tubulin acetylation is reduced in GCN5L1-knockout hepatocytes and restored by GCN5L1 reconstitution. Furthermore, GCN5L1 binds to the α-tubulin acetyltransferase αTAT1, and GCN5L1-mediated α-tubulin acetylation is dependent on αTAT1. Given that cytosolic GCN5L1 has been identified as a component of numerous multiprotein complexes, we explored whether novel interacting partners contribute to this regulation. We identify RanBP2 as a novel interacting partner of GCN5L1 and αTAT1. Genetic silencing of RanBP2 phenocopies GCN5L1 depletion by reducing α-tubulin acetylation, and we find that RanBP2 possesses a tubulin-binding domain, which recruits GCN5L1 to α-tubulin. Finally, we find that genetic depletion of GCN5L1 promotes perinuclear lysosome accumulation and histone deacetylase inhibition partially restores lysosomal positioning. We conclude that the interactions of GCN5L1, RanBP2 and αTAT1 function in concert to control α-tubulin acetylation and may contribute towards the regulation of cellular lysosome positioning. This article has an associated First Person interview with the first author of the paper.
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Acetiltransferases/metabolismo , Fígado/metabolismo , Lisossomos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Acetilação , Animais , Células HEK293 , Células HeLa , Hepatócitos/metabolismo , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microtúbulos/metabolismo , Proteínas Mitocondriais , Cultura Primária de Células , TransfecçãoRESUMO
The nuclear pore is the gatekeeper of nucleocytoplasmic transport and signaling through which a vast flux of information is continuously exchanged between the nuclear and cytoplasmic compartments to maintain cellular homeostasis. A unifying and organizing principle has recently emerged that cements the notion that several forms of amyotrophic lateral sclerosis (ALS), and growing number of other neurodegenerative diseases, co-opt the dysregulation of nucleocytoplasmic transport and that this impairment is a pathogenic driver of neurodegeneration. The understanding of shared pathomechanisms that underpin neurodegenerative diseases with impairments in nucleocytoplasmic transport and how these interface with current concepts of nucleocytoplasmic transport is bound to illuminate this fundamental biological process in a yet more physiological context. Here, I summarize unresolved questions and evidence and extend basic and critical concepts and challenges of nucleocytoplasmic transport and its role in the pathogenesis of neurodegenerative diseases, such as ALS. These principles will help to appreciate the roles of nucleocytoplasmic transport in the pathogenesis of ALS and other neurodegenerative diseases, and generate a framework for new ideas of the susceptibility of motoneurons, and possibly other neurons, to degeneration by dysregulation of nucleocytoplasmic transport.
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Doença dos Neurônios Motores/metabolismo , Doenças Neurodegenerativas/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/metabolismo , Citoplasma/patologia , Humanos , Doença dos Neurônios Motores/patologia , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Doenças Neurodegenerativas/patologiaRESUMO
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.
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Microglia/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Células Ganglionares da Retina/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Neurônios Motores/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Nervo Óptico/anormalidades , Nervo Óptico/patologia , Comunicação Parácrina , Tamoxifeno/farmacologia , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , TranscriptomaRESUMO
Objective: To study the regulatory effects and mechanisms of deleted in lymphocytic leukemia 1 (DLEU1), microRNA-513a-5p (miR-513a-5p), and RAN binding protein 2 (RANBP2) in nephroblastoma. Methods: The GHINK-1 cells were transfected with pcDNA (pcDNA group), pcDNA-DLEU1 (pcDNA-DLEU1 group), miR-NC (miR-NC group), miR-513a-5p mimics (miR-513a-5p group), pcDNA-RANBP2 (pcDNA-RANBP2 group), pcDNA-DLEU1 and miR-NC (pcDNA-DLEU1+ miR-NC group), pcDNA-DLEU1 and miR-513a-5p mimics (pcDNA-DLEU1+ miR-513a-5p group), miR-513a-5p mimics and pcDNA (miR-513a-5p+ pcDNA group), miR-513a-5p mimics and pcDNA-RANBP2 (miR-513a-5p + pcDNA-RANBP2 group). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to detect the expressions of DLEU1, miR-513a-5p, RANBP2 in nephroblastoma tissues, normal adjacent tissues, normal kidney cell HK2, and hemangioblastoma cell GHINK-1. Western blot was used to detect the expressions of proliferating cell nuclear antigen (PCNA), B cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X (Bax). Cell counting kit 8 (CCK-8) was used to detect the cell survival rate. Flow cytometry was used to detect the apoptosis rate. Dual luciferase report test was used to detect the luciferase activity of cells. Results: The expression levels of DLEU1, miR-513a-5p and RANBP2 in adjacent tissues were 1.02±0.08, 1.01±0.06, 1.00±0.05, respectively, significantly lower than 5.16±0.24, 0.23±0.02, 1.67±0.09 in nephroblasts tumor tissues (P<0.05). Their expression levels in HK2 cells were 1.00±0.06, 1.00±0.08, 1.02±0.09, respectively, significantly lower than 3.15±0.21, 0.18±0.01, 1.54±0.10 in GHINK-1 cells (P<0.05). Overexpression of DLEU1 significantly reduced the apoptosis rate (7.35±0.41 vs 12.35±1.12, P<0.05). Overexpression of RANBP2 significantly reduced the apoptosis rate (8.89±0.48 vs 12.64±1.12, P<0.05). Compared with the miR-NC group (1.01±0.06, 0.99±0.06), the luciferase activity of DLEU1-WT (0.43±0.04) and RANBP2-WT (0.61±0.07) in miR-513a-5p group were significantly reduced (P<0.05). Compared with anti-miR-NC group (0.99±0.07, 0.98±0.05), the luciferase activity of DLEU1-WT (1.34±0.11) and RANBP2-WT (1.39 ±0.13) in anti-miR-513a-5p group was significantly increased (P<0.05). Simultaneous overexpression of pcDNA-DLEU1 and miR-513a-5p in GHINK-1 cells significantly reduced the apoptosis rate (11.34±1.03 vs 8.51±0.69, P<0.05). Simultaneous overexpression of miR-513a-5p and RANBP2 in GHINK-1 cells significantly reduced the apoptosis rate (9.96±0.72 vs 15.94±1.00, P<0.05). Conclusions: The long-chain non-coding RNA (lncRNA) DLEU1 can promote the proliferation and inhibit the apoptosis of nephroblastoma cells. The mechanism is related to the targeted regulation of miR-513a-5p and RANBP2 function, which will provide theoretical support for the nephroblastoma treatment.
Assuntos
MicroRNAs/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Supressoras de Tumor/genética , Tumor de Wilms , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA Longo não Codificante , Tumor de Wilms/genéticaRESUMO
The nuclear envelope is tethered to the cytoskeleton. The best known attachments of all elements of the cytoskeleton are via the so-called LINC complex. However, the nuclear pore complexes, which mediate the transport of soluble and membrane bound molecules, are also linked to the microtubule network, primarily via motor proteins (dynein and kinesins) which are linked, most importantly, to the cytoplasmic filament protein of the nuclear pore complex, Nup358, by the adaptor BicD2. The evidence for such linkages and possible roles in nuclear migration, cell cycle control, nuclear transport and cell architecture are discussed.
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Transporte Ativo do Núcleo Celular/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Poro Nuclear/metabolismo , HumanosRESUMO
Piwi-interacting RNAs (piRNAs) are a class of small noncoding RNAs that bind Piwi proteins to silence transposons and to regulate gene expression. In Drosophila germ cells, the Aubergine (Aub)-Argonaute 3 (Ago3)-dependent ping-pong cycle generates most germline piRNAs. Loading of antisense piRNAs amplified by this cycle enables Piwi to enter the nucleus and silence transposons. Nuclear localization is crucial for Piwi function in transposon silencing, but how this process is regulated remains unknown. It is also not known whether any of the components of the nuclear pore complex (NPC) directly function in the piRNA pathway. Here, we show that nucleoporin 358 (Nup358) and Piwi interact with each other and that a germline knockdown (GLKD) of Nup358 with short hairpin RNA prevents Piwi entry into the nucleus. The Nup358 GLKD also activated transposons, increased genomic instability, and derailed piRNA biogenesis because of a combination of decreased piRNA precursor transcription and a collapse of the ping-pong cycle. Our results point to a critical role for Nup358 in the piRNA pathway, laying the foundation for future studies to fully elucidate the mechanisms by which Nup358 contributes to piRNA biogenesis and transposon silencing.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Elementos de DNA Transponíveis , Drosophila/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Instabilidade Genômica , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Mapas de Interação de Proteínas , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2-SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-ß and CRM1 play essential roles in localising the RANBP2-SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO-conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2.
Assuntos
Carioferinas/metabolismo , Cinetocoros/metabolismo , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , beta Carioferinas/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Carioferinas/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , beta Carioferinas/genética , Proteína Exportina 1RESUMO
BACKGROUND: Cholangiocarcinoma is one of the deadly disease with poor 5-year survival and poor response to conventional therapies. Previously, we found that p27kip1 nuclear-cytoplasmic translocation confers proliferation potential to cholangiocarcinoma cell line QBC939 and this process is mediated by crm-1. However, no other post-transcriptional regulation was found in this process including sumoylation in cholangiocarcinoma. RESULTS: In this study, we explored the role of sumoylation in the nuclear-cytoplasmic translocation of p27kip1 and its involvement of QBC939 cells' proliferation. First, we identified K73 as the sumoylation site in p27kip1. By utilizing plasmid flag-p27kip1, HA-RanBP2, GST-RanBP2 and His-p27kip1 and immunoprecipitation assay, we validated that p27kip1 can serve as the sumoylation target of RanBP2 in QBC939. Furthermore, we confirmed crm-1's role in promoting nuclear-cytoplasmic translocation of p27kip1 and found that RanBP2's function relies on crm-1. However, K73R mutated p27kip1 can't be identified by crm-1 or RanBP2 in p27kip1 translocation process, suggesting sumoylation of p27kip1 via K73 site is necessary in this process by RanBP2 and crm-1. Phenotypically, the overexpression of either RanBP2 or crm-1 can partially rescue the anti-proliferative effect brought by p27kip1 overexpression in both the MTS and EdU assay. For the first time, we identified and validated the K73 sumoylation site in p27kip1, which is critical to RanBP2 and crm-1 in p27kip1 nuclear-cytoplasmic translocation process. CONCLUSION: Taken together, targeted inhibition of sumoylation of p27kip1 may serve as a potentially potent therapeutic target in the eradication of cholangiocarcinoma development and relapses.