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1.
Vet Microbiol ; 235: 151-163, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282373

RESUMO

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.


Assuntos
Proteínas Cdc20/metabolismo , Chaperonina com TCP-1/metabolismo , Orthoreovirus/genética , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Caspase 8/genética , Caspase 8/metabolismo , Proteínas Cdc20/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Cercopithecus aethiops , Chaperonina com TCP-1/genética , Patos/virologia , Fibroblastos/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Interferente Pequeno , Células Vero , Proteínas não Estruturais Virais/genética
2.
Eur J Med Genet ; 62(7): 103665, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31071487

RESUMO

Triple A syndrome, a multisystemic autosomal recessive disease, is characterized by the clinical triad of adrenal insufficiency, alacrima and achalasia in combination with progressive neurological impairments. The disorder is caused by homozygous or compound heterozygous mutations in the AAAS gene. Here we present the clinical and molecular data of a ten year old patient with triple A syndrome. Array CGH analysis confirmed the PCR-based assumption of a homozygous deletion of the entire AAAS gene in the patient and a heterozygous deletion in both parents. We demonstrate that the patient carries a 15 kb deletion and identified the 5' and 3' breakpoints outside the AAAS gene. This is the first report of a triple A syndrome patient with a homozygous deletion of the entire AAAS gene.


Assuntos
Insuficiência Adrenal/genética , Acalasia Esofágica/genética , Deleção de Genes , Proteínas do Tecido Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Insuficiência Adrenal/patologia , Adulto , Criança , Pré-Escolar , Acalasia Esofágica/patologia , Homozigoto , Humanos , Masculino , Linhagem
3.
Nat Commun ; 10(1): 2147, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089132

RESUMO

Cancer-relevant signalling pathways rely on bidirectional nucleocytoplasmic transport events through the nuclear pore complex (NPC). However, mechanisms by which individual NPC components (Nups) participate in the regulation of these pathways remain poorly understood. We discover by integrating large scale proteomics, polysome fractionation and a focused RNAi approach that Nup155 controls mRNA translation of p21 (CDKN1A), a key mediator of the p53 response. The underlying mechanism involves transcriptional regulation of the putative tRNA and rRNA methyltransferase FTSJ1 by Nup155. Furthermore, we observe that Nup155 and FTSJ1 are p53 repression targets and accordingly find a correlation between the p53 status, Nup155 and FTSJ1 expression in murine and human hepatocellular carcinoma. Our data suggest an unanticipated regulatory network linking translational control by and repression of a structural NPC component modulating the p53 pathway through its effectors.


Assuntos
Carcinoma Hepatocelular/patologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias Hepáticas/patologia , Metiltransferases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Metiltransferases/metabolismo , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/metabolismo
4.
BMC Cancer ; 19(1): 236, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30935371

RESUMO

BACKGROUND: Triple Negative breast cancer (TNBC) is a poor outcome subgroup of breast cancer defined based on the absence of expression of ERα and PR and HER2 amplification. These hard to treat cancers lack targeted treatment options and are therefore treated with a standard of care (SoC) generic cocktail of DNA damaging chemotherapy, with a wide range of clinical responses. While a subset of TNBC patients respond very well to this treatment, others receive no clinical benefit and die from their disease within a short time period. We currently lack biomarkers to prospectively identify patients likely to relapse and we lack alternate treatment options. METHODS: NUP98 protein expression was investigated in patient samples using two independent tissue microarrays (TMAs), as well as a normal breast TMA. Correlation with pathological response to various chemotherapy regimens was investigated. RESULTS: We have shown that high NUP98 is significantly associated with poor outcome in TNBC patient samples both by gene expression and IHC-based protein analysis. While trends linking NUP98 expression with poorer outcomes were observed in breast cancer overall (and more specifically in the LuminalB Her2- subgroup), significant correlations were observed in TNBC. This appeared to be specific to anthracycline based regimens as the association between NUP98 and response was not observed in patients treated with taxane-based chemotherapy. CONCLUSIONS: We have identified a novel biomarker, NUP98, that can predict response to anthracycline based chemotherapy in TNBC. The ability to prospectively identify patients who are less likely to respond to SoC chemotherapy is a vital step in improving the overall survival of these patients.


Assuntos
Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Adulto , Antraciclinas/farmacologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Taxoides/farmacologia , Taxoides/uso terapêutico , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
5.
Cell Mol Life Sci ; 76(17): 3407-3432, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30944974

RESUMO

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.


Assuntos
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 Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/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 , Transcriptoma
6.
Toxicol In Vitro ; 57: 226-232, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30853489

RESUMO

Alcoholic liver disease (ALD), featured by excessive hepatocyte death and inflammation, is a prevalent disease that causes heavy health burdens worldwide. Hepatocyte necroptosis is a central event that promotes inflammation in ALD. At molecular levels, inhibition of nuclear factor (erythroid - derived 2) - like 2 (NRF2) was an important trigger for cell necroptosis. The protective effects of gallic acid (GA) on liver diseases caused by multiple factors have been elucidated, however, the role of GA in ALD remained unclear. Therefore, this study was aimed to investigate the anti-ALD effects of GA and further reveal the molecular mechanisms. Results showed that GA could effectively recover cell viability and reduce the release of aspartate transaminase, alanine transaminase, and lactic dehydrogenase by ethanol-stimulated hepatocytes. More importantly, GA limited hepatocyte necroptosis under ethanol stimulation, which was characterized by reduced expression of distinct necroptotic signals receptor-interacting protein 1 (RIP1) and RIP3 and release of high mobility group box protein 1. Mechanistically, GA could induce NRF2 expression in ethanol-incubated hepatocytes, which was a molecular basis for GA to suppress ethanol-induced hepatocyte necroptosis. In conclusion, this study demonstrated that GA improved ethanol-induced hepatocyte necroptosis in vitro. Further, NRF2 activation might be requisite for GA to exert its protective effects.


Assuntos
Etanol/toxicidade , Ácido Gálico/farmacologia , Hepatócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Substâncias Protetoras/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fator 2 Relacionado a NF-E2/genética , Necrose/induzido quimicamente , Necrose/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
7.
Leukemia ; 33(8): 1868-1880, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30700838

RESUMO

T cell acute lymphoblastic leukaemia (T-ALL) cases include subfamilies that overexpress the TAL1/LMO, TLX1/3 and HOXA transcription factor oncogenes. While it has been shown that TAL1/LMO transcription factors induce self-renewal of thymocytes, whether this is true for other transcription factor oncogenes is unknown. To address this, we have studied NUP98-HOXD13-transgenic (NHD13-Tg) mice, which overexpress HOXA transcription factors throughout haematopoiesis and develop both myelodysplastic syndrome (MDS) progressing to acute myeloid leukaemia (AML) as well as T-ALL. We find that thymocytes from preleukaemic NHD13-Tg mice can serially transplant, demonstrating that they have self-renewal capacity. Transcriptome analysis shows that NHD13-Tg thymocytes exhibit a stem cell-like transcriptional programme closely resembling that induced by Lmo2, including Lmo2 itself and its critical cofactor Lyl1. To determine whether Lmo2/Lyl1 are required for NHD13-induced thymocyte self-renewal, NHD13-Tg mice were crossed with Lyl1 knockout mice. This showed that Lyl1 is essential for expression of the stem cell-like gene expression programme in thymocytes and self-renewal. Surprisingly however, NHD13 transgenic mice lacking Lyl1 showed accelerated T-ALL and absence of transformation to AML, associated with a loss of multipotent progenitors in the bone marrow. Thus multiple T cell oncogenes induce thymocyte self-renewal via Lmo2/Lyl1; however, NHD13 can also promote T-ALL via an alternative pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas de Homeodomínio/genética , Proteínas com Domínio LIM/fisiologia , Proteínas de Neoplasias/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Timócitos/fisiologia , Fatores de Transcrição/genética , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
8.
Genome Biol Evol ; 11(3): 678-687, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715330

RESUMO

The nuclear pore complex (NPC) is a large macromolecular assembly situated within the pores of the nuclear envelope. Through interactions between its subcomplexes and import proteins, the NPC mediates the transport of molecules into and out of the nucleus and facilitates dynamic chromatin regulation and gene expression. Accordingly, the NPC constitutes a highly integrated nuclear component that is ubiquitous and conserved among eukaryotes. Potential exceptions to this are nucleomorphs: Highly reduced, relict nuclei that were derived from green and red algae following their endosymbiotic integration into two lineages, the chlorarachniophytes and the cryptophyceans. A previous investigation failed to identify NPC genes in nucleomorph genomes suggesting that these genes have either been relocated to the host nucleus or lost. Here, we sought to investigate the composition of the NPC in nucleomorphs by using genomic and transcriptomic data to identify and phylogenetically classify NPC proteins in nucleomorph-containing algae. Although we found NPC proteins in all examined lineages, most of those found in chlorarachniophytes and cryptophyceans were single copy, host-related proteins that lacked signal peptides. Two exceptions were Nup98 and Rae1, which had clear nucleomorph-derived homologs. However, these proteins alone are likely insufficient to structure a canonical NPC and previous reports revealed that Nup98 and Rae1 have other nuclear functions. Ultimately, these data indicate that nucleomorphs represent eukaryotic nuclei without a canonical NPC, raising fundamental questions about their structure and function.


Assuntos
Cercozoários/genética , Criptófitas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear , Simbiose
9.
Cell Mol Life Sci ; 76(11): 2199-2216, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30762072

RESUMO

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.


Assuntos
Lamina Tipo A/genética , Lamina Tipo B/genética , Membrana Nuclear/metabolismo , Lâmina Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Lamina Tipo A/antagonistas & inibidores , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Imagem Molecular , Membrana Nuclear/ultraestrutura , Lâmina Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
11.
Genes Dev ; 33(3-4): 144-149, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30692205

RESUMO

During oncogene-induced senescence (OIS), heterochromatin is lost from the nuclear periphery and forms internal senescence-associated heterochromatin foci (SAHFs). We show that an increased nuclear pore density during OIS is responsible for SAHF formation. In particular, the nucleoporin TPR is necessary for both formation and maintenance of SAHFs. Loss of SAHFs does not affect cell cycle arrest but abrogates the senescence-associated secretory phenotype-a program of inflammatory cytokine gene activation. Our results uncover a previously unknown role of nuclear pores in heterochromatin reorganization in mammalian nuclei and demonstrate the importance of heterochromatin organization for a specific gene activation program.


Assuntos
Senescência Celular/fisiologia , Heterocromatina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Humanos , Modelos Moleculares , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/genética
12.
J Exp Clin Cancer Res ; 38(1): 33, 2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678687

RESUMO

BACKGROUND: The 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. METHODS: Western 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. RESULTS: The 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/ß-catenin signaling in cervical cancer cells. Additionally, NUSAP1-induced cervical cancer cells metastasis and the cancer stem cell phenotype were abrogated with the Wnt/ß-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. CONCLUSIONS: Our results demonstrate thatNUSAP1 upregulation contributes to metastasis of cervical cancer by promoting CSC properties and EMT via Wnt/ß-catenin signaling and XAV-939 might serve as a potential tailored therapeutic option for patients with NUSAP1-ovexpressed cervical cancer.


Assuntos
Biomarcadores Tumorais/genética , Proteínas Associadas aos Microtúbulos/genética , Prognóstico , Neoplasias do Colo do Útero/genética , Adulto , Carcinogênese/genética , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Chaperonas Moleculares/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologia , Via de Sinalização Wnt/efeitos dos fármacos
13.
Mol Cancer ; 18(1): 4, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621700

RESUMO

BACKGROUND: In recent years, circular RNAs (circRNAs), a new star of non-coding RNA, have been emerged as vital regulators and gained much attention for involvement of initiation and progression of diverse kinds of human diseases, especially cancer. However, regulatory role, clinical significance and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) still remain largely unknown. METHODS: Here, the expression profile of circRNAs in 4 pairs of TNBC tissues and adjacent non-tumor tissues was analyzed by RNA-sequencing. Quantitative real-time PCR and in situ hybridization were used to determine the level and prognostic values of circAGFG1 in two TNBC cohorts. Then, functional experiments in vitro and in vivo were performed to investigate the effects of circAGFG1 on tumor growth and metastasis in TNBC. Mechanistically, fluorescent in situ hybridization, dual luciferase reporter assay, RNA pull-down and RNA immunoprecipitation experiments were performed to confirm the interaction between circAGFG1 and miR-195-5p in TNBC. RESULTS: We found that circAGFG1 was evidently up-regulated in TNBC, and its level was correlated with clinical stage, pathological grade and poor prognosis of patients with TNBC. The results indicated that circAGFG1 could promote TNBC cell proliferation, mobility and invasion as well as tumorigenesis and metastasis in vivo. Mechanistic analysis showed that circAGFG1 may act as a ceRNA (competing endogenous RNA) of miR-195-5p to relieve the repressive effect of miR-195-5p on its target cyclin E1 (CCNE1). CONCLUSIONS: Our findings suggest that circAGFG1 promotes TNBC progression through circAGFG1/miR-195-5p/CCNE1 axis and it may serve as a new diagnostic marker or target for treatment of TNBC patients.


Assuntos
Ciclina E/genética , MicroRNAs/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Neoplasias de Mama Triplo Negativas/genética , Apoptose/genética , Carcinogênese/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Invasividade Neoplásica/genética , Prognóstico , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima/genética
15.
Hormones (Athens) ; 18(1): 109-112, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30612286

RESUMO

OBJECTIVE: Triple A syndrome is a rare autosomal recessive disorder caused by mutations in the AAAS gene on chromosome 12q13. Its main clinical features are alacrima, achalasia, and adrenal insufficiency, with most patients also having neurological symptoms and autonomic dysfunction. The neurologic manifestations are less well-understood, especially in children. Here, we examine two siblings who were found to have a novel mutation in the AAAS gene and who were found to have subtle, but important, neurologic findings. DESIGN: This is a case report of two siblings. RESULTS: We discuss two siblings exhibiting different signs of the disorder including neurologic dysfunction found at varying ages. Genetic analysis revealed that both patients have the same compound heterozygous mutations in the AAAS gene consisting of one novel mutation (c.500 C>A, A167E) and one previously described mutation (c.1331+1G> A/IVS14+1 G>A). A diagnosis of triple A syndrome was reached based on their clinical and genetic findings. CONCLUSIONS: The unique characteristic of these two cases is the novel mutation in the AAAS gene, which is likely pathogenic. In addition, they showcase the genotype-phenotype variability of the disease, as well as the importance of early identification of the neurologic abnormalities, which can result in early intervention and possibly improved outcomes.


Assuntos
Insuficiência Adrenal/genética , Acalasia Esofágica/genética , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Insuficiência Adrenal/complicações , Criança , Pré-Escolar , Acalasia Esofágica/complicações , Feminino , Humanos , Masculino , Doenças do Sistema Nervoso/etiologia , Irmãos
16.
Transplant Proc ; 50(10): 3954-3956, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30577294

RESUMO

Mutations in nucleoporin 93 (NUP93) gene have been shown recently to be one of the very rare causes of genetic steroid-resistant nephrotic syndrome (SRNS). Until now, none of the 7 published cases with NUP93-SRNS, experienced recurrence of nephrotic syndrome (NS) after transplantation. Here, we present the first case of recurrent NS in a patient with NUP93-SRNS ever reported. A 3-year-old boy with infantile SRNS was started on chronic peritoneal dialysis because of end-stage renal failure owing to biopsy-proven focal segmental glomerulosclerosis (FSGS). At the age of 6 years, the boy received a renal allograft. The posttransplant period was uncomplicated until 1.7 years after transplantation, when the patient developed nephrotic proteinuria during a respiratory tract infection. Renal graft biopsy showed subtotal fusion of podocytes, which was compatible with an early histopathologic sign of recurrence of FSGS. Immediate treatment with daily plasma exchange (PE) was started at the second day. The proteinuria disappeared completely after the second PE. However, it reappeared after stopping daily PE. It disappeared again after reintroduction of daily PE, therefore PE-dependent recurrent NS was diagnosed and treatment with rituximab was given. After the first dose, proteinuria never reappeared despite stopping PE therapy. Surprisingly, next-generation sequencing revealed compound heterozygous mutations in exons 16 and 18 of the NUP93 gene (c.1772G>T - European founder allele and 1916T>C) and his parents confirmed heterozygous asymptomatic carriers. This is the first case of recurrent NS in a patient with NUP93 gene mutations, suggesting a new pathomechanism possibly involving the nucleoporins.


Assuntos
Transplante de Rim , Síndrome Nefrótica/genética , Síndrome Nefrótica/cirurgia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Pré-Escolar , Heterozigoto , Humanos , Fatores Imunológicos/uso terapêutico , Masculino , Mutação , Síndrome Nefrótica/complicações , Plasmaferese , Recidiva , Rituximab/uso terapêutico
17.
PLoS Genet ; 14(12): e1007845, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543681

RESUMO

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.


Assuntos
Artrogripose/genética , Genes Letais , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Artrogripose/embriologia , Artrogripose/fisiopatologia , Consanguinidade , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Junção Neuromuscular/fisiopatologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Linhagem , Gravidez , Conformação Proteica , Receptores Nicotínicos/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
18.
Toxicol Lett ; 296: 39-47, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30086328

RESUMO

Dasatinib shows remarkable activity against imatinib-refractory chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL). However, severe cardiovascular toxicity limits the clinical applications of dasatinib. Since the underlying mechanism of dasatinib-induced cardiotoxicity is still elusive, we aim to clarify this. Recent studies have shown that necroptosis and apoptosis participate in multiple toxicity development. Here, we first report that dasatinib could directly induce cardiomyocytes death, as analyzed by the Sulforhodamine B (SRB) assay. This type of cardiomyocytes death was mediated by the necrosis pathway rather than apoptosis, as determined by using flow cytometry to characterize the mode of dasatinib-induced cell death. Inhibition of receptor-interacting protein kinase 1 (RIP1)activity and knockdown of receptor-interacting protein kinase 3 (RIP3)expression can block dasatinib-evoked cardiotoxicity, which further confirmed the involvement of necroptosis. We next found that the classic substrates of RIP3, mixed lineage kinase domain-like protein (MLKL) and Ca2+-calmodulin-dependent protein kinase II (CaMKII) were not involved in dasatinib-induced cardiomyocytes necroptosis. What's more, unlike the inflammation-associated necroptosis, dasatinib-triggered necroptosis was dependent on intracellular instead of secreted High-mobility group box 1 (HMGB1) protein. Collectively, our study revealed that dasatinib-induced cardiotoxicity acted via leading cardiomyocytes to HMGB1-mediated necroptosis, indicating a viable strategy for prevention of dasatinib-induced cardiotoxicity.


Assuntos
Antineoplásicos/toxicidade , Dasatinibe/toxicidade , Proteína HMGB1/metabolismo , Cardiopatias/induzido quimicamente , Necrose/induzido quimicamente , Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiotoxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Necrose/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/biossíntese , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/biossíntese , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
19.
Gastroenterology ; 155(4): 1233-1249.e22, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30009820

RESUMO

BACKGROUND & AIMS: Agents designed to block or alter cytokinesis can kill or stop proliferation of cancer cells. We aimed to identify cytokinesis-related proteins that are overexpressed in hepatocellular carcinoma (HCC) cells and might be targeted to slow liver tumor growth. METHODS: Using the Oncomine database, we compared the gene expression patterns in 16 cancer microarray datasets and assessed gene enrichment sets using gene ontology. We performed immunohistochemical analysis of an HCC tissue microarray and identified changes in protein levels that are associated with patient survival times. Candidate genes were overexpressed or knocked down with small hairpin RNAs in SMMC7721, MHCC97H, or HCCLM3 cell lines; we analyzed their proliferation, viability, and clone-formation ability and their growth as subcutaneous or orthotopic xenograft tumors in mice. We performed microarray analyses to identify alterations in signaling pathways and immunoblot and immunofluorescence assays to detect and localize proteins in tissues. Yeast 2-hybrid screens and mass spectrometry combined with co-immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co-immunoprecipitation and proximity ligation assays. Chromatin immunoprecipitation, promoter luciferase activity, and quantitative real-time polymerase chain reaction analyses were used to identify factors that regulate transcription of specific genes. RESULTS: The genes that were most frequently overexpressed in different types of cancer cells were involved in cell division processes. We identified 3 cytokinesis-regulatory proteins among the 10 genes most frequently overexpressed by all cancer cell types. Rac GTPase activating protein 1 (RACGAP1) was the cytokinesis-regulatory protein that was most highly overexpressed in multiple cancers. Increased expression of RACGAP1 in tumor tissues was associated with shorter survival times of patients with cancer. Knockdown of RACGAP1 in HCC cells induced cytokinesis failure and cell apoptosis. In microarray analyses, we found knockdown of RACGAP1 in SMMC7721 cells to reduce expression of genes regulated by yes-associated protein (YAP) and WW domain containing transcription regulator 1 (WWTR1 or TAZ). RACGAP1 reduced activation of the Hippo pathway in HCC cells by increasing activity of RhoA and polymerization of filamentous actin. Knockdown of YAP reduced phosphorylation of RACGAP1 and redistribution at the anaphase central spindle. We found transcription of the translocated promoter region, nuclear basket protein (TPR) to be regulated by YAP and coordinately expressed with RACGAP1 to promote proliferation of HCC cells. TPR redistributed upon nuclear envelope breakdown and formed complexes with RACGAP1 during mitosis. Knockdown of TPR in HCC cells reduced phosphorylation of RACGAP1 by aurora kinase B and impaired their redistribution at the central spindle during cytokinesis. STAT3 activated transcription of RACGAP in HCC cells. CONCLUSIONS: In an analysis of gene expression patterns of multiple tumor types, we found RACGAP1 to be frequently overexpressed, which is associated with shorter survival times of patients. RACGAP1 promotes proliferation of HCC cells by reducing activation of the Hippo and YAP pathways and promoting cytokinesis in coordination with TPR.


Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Citocinese , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células A549 , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Feminino , Proteínas Ativadoras de GTPase/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Carga Tumoral , Regulação para Cima , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29997211

RESUMO

Human immunodeficiency virus type 1 (HIV-1) displays the unique ability to infect nondividing cells. The capsid of HIV-1 is the viral determinant for viral nuclear import. To understand the cellular factors involved in the ability of HIV-1 to infect nondividing cells, we sought to find capsid mutations that allow the virus to infect dividing but not nondividing cells. Because the interaction of capsid with the nucleoporin protein 153 (Nup153) is important for nuclear import of HIV-1, we solved new crystal structures of hexameric HIV-1 capsid in complex with a Nup153-derived peptide containing a phenylalanine-glycine repeat (FG repeat), which we used to guide structure-based mutagenesis of the capsid-binding interface. HIV-1 viruses with mutations in these capsid residues were tested for their ability to infect dividing and nondividing cells. HIV-1 viruses with capsid N57 substitutions infected dividing but not nondividing cells. Interestingly, HIV-1 viruses with N57 mutations underwent reverse transcription but not nuclear translocation. The mutant capsids also lost the ability to interact with Nup153 and CPSF6. The use of small molecules PF74 and BI-2 prevented the interaction of FG-containing nucleoporins (Nups), such as Nup153, with the HIV-1 core. Analysis of integration sites in HIV-1 viruses with N57 mutations revealed diminished integration into transcriptionally active genes in a manner resembling that of HIV-1 in CPSF6 knockout cells or that of HIV-1-N74D. The integration pattern of the N57 mutant HIV-1 can be explained by loss of capsid interaction with CPSF6, whereas capsid interaction with Nup153 is required for HIV-1 to infect nondividing cells. Additionally, the observed viral integration profiles suggested that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.IMPORTANCE One of the key advantages that distinguish lentiviruses, such as HIV-1, from all other retroviruses is its ability to infect nondividing cells. Interaction of the HIV-1 capsid with Nup153 and CPSF6 is important for nuclear entry and integration; however, the contribution of each of these proteins to nuclear import and integration is not clear. Using genetics, we demonstrated that these proteins contribute to different processes: Nup153 is essential for the HIV-1 nuclear import in nondividing cells, and CPSF6 is important for HIV-1 integration. In addition, nuclear factors such as CPSF6 and the state of the chromatin are known to be important for integration site selection; nevertheless, the preferential determinant influencing integration site selection is not known. This work demonstrates that integration site selection is a multiparameter process that depends upon nuclear factors and the state of the cellular chromatin.


Assuntos
Capsídeo/metabolismo , Divisão Celular , HIV-1/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , HIV-1/genética , Humanos , Poro Nuclear/genética , Poro Nuclear/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
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