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1.
Mol Cell ; 77(5): 1032-1043.e4, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31924447

RESUMO

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.


Assuntos
Cromatina/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Ribonuclease H/metabolismo , Terminação da Transcrição Genética , Animais , Cromatina/genética , Éxons , Exorribonucleases/genética , Exorribonucleases/metabolismo , Feminino , Células HCT116 , Humanos , Íntrons , Camundongos Endogâmicos C57BL , Modelos Genéticos , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Oligonucleotídeos Antissenso/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Ribonuclease H/genética , Fatores de Tempo
2.
Nat Commun ; 11(1): 433, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974380

RESUMO

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Erastin, the ferroptosis activator, binds to voltage-dependent anion channels VDAC2 and VDCA3, but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels. Depletion of Nedd4 limits the protein degradation of VDAC2/3, which increases the sensitivity of cancer cells to erastin. By understanding the molecular mechanism of erastin-induced cellular resistance, we can discover how cells adapt to new molecules to maintain homeostasis. Furthermore, erastin-induced resistance mediated by FOXM1-Nedd4-VDAC2/3 negative feedback loop provides an initial framework for creating avenues to overcome the drug resistance of ferroptosis activators.


Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Piperazinas/farmacologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Proteína Forkhead Box M1/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Camundongos Nus , Proteínas de Transporte da Membrana Mitocondrial/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitinação/efeitos dos fármacos , Canal de Ânion 2 Dependente de Voltagem/genética , Canais de Ânion Dependentes de Voltagem/genética , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Cell Physiol Biochem ; 54(1): 1-14, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31916733

RESUMO

BACKGROUND/AIMS: Deubiquitinating enzymes (DUBs) are crucially involved in controlling signal transductions, and reverse ubiquitination by removing the ubiquitin from protein substrates. The Hippo signaling has an important role in tissue growth, cell proliferation, differentiation, and apoptosis. Since disruption of the Hippo signaling is associated with a number of diseases, it is imperative to investigate the molecular mechanism of the Hippo signaling. METHODS: DUB screening was performed using the kidney of the mouse unilateral ureteric obstruction (UUO) model to identify the cellular mechanism of the DUB-regulated Hippo signaling. In addition, kidney cells were used to confirm cell proliferation and protein levels in the Hippo signaling pathway. Densitometric analysis was conducted to compare the expression level of proteins using Image J. RESULTS: We found that YOD1, also known as OTU1, is downregulated in the mouse UUO model. We also demonstrated that YOD1 binds to and deubiquitinates neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4). Furthermore, we observed that YOD1 suppresses NEDD4-induced cell proliferation. CONCLUSION: YOD1 regulates the Hippo signaling pathway through NEDD4, and the K63-linked polyubiquitin chain of NEDD4 plays an important role. Also, our results indicate that YOD1 plays an important role in kidney diseases.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Camundongos , Mutagênese , Ubiquitina-Proteína Ligases Nedd4/química , Ubiquitina-Proteína Ligases Nedd4/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Tioléster Hidrolases/química , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
4.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 41-43, 2020 Jan 10.
Artigo em Chinês | MEDLINE | ID: mdl-31922594

RESUMO

OBJECTIVE: To explore the genetic basis of a patient featuring global developmental delay, intellectual disability, cleft palate, seizures and hypotonia. METHODS: Clinical examination and laboratory tests were carried out. Peripheral blood samples were obtained from the patient and his parents. Whole genomic DNA was extracted and subjected to next generation sequencing. Candidate variation was analyzed by using bioinformatic software and validated by Sanger sequencing. RESULTS: The proband was found to carry a heterozygous c.2117T>C (p.Leu706Pro) variant of the NEDD4L gene, which was a de novo variant validated by Sanger sequencing and predicted to be likely pathogenic according to the American College of Medical Genetics Guidelines. CONCLUSION: The heterozygous variant of c.2117T>C (p.Leu706Pro) of the NEDD4L gene probably underlies the disorders in the patient.


Assuntos
Ubiquitina-Proteína Ligases Nedd4 , Heterotopia Nodular Periventricular , Testes Genéticos , Heterozigoto , Humanos , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Masculino , Mutação , Ubiquitina-Proteína Ligases Nedd4/genética , Heterotopia Nodular Periventricular/complicações , Heterotopia Nodular Periventricular/genética
5.
J Biochem Mol Toxicol ; 34(2): e22423, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31729781

RESUMO

MicroRNAs are endogenous small noncoding RNAs that posttranscriptionally regulate the expression of target genes and play crucial roles in diverse physiopathologic processes. In the current study, we examined the microRNA (miRNA) expression profile of high-glucose-treated neonatal rat cardiomyocytes and the potential mechanisms. Differentially expressed miRNAs were analyzed by a miRNA microarray and validated by a quantitative real-time polymerase chain reaction in high-glucose-treated rat cardiomyocytes. Based on the results of our previous study and the bioinformatics prediction, we identified miR-195-5p/SGK1/Nedd4-2/hERG as the top-ranked signal pathway in diabetes cell model in vitro. In summary, our present study provides novel insights into the regulatory mechanism of miR-195-5p/SGK1/Nedd4-2/hERG in rat cardiomyocytes under high-glucose stress, which may provide a novel idea for the development of diagnostic and therapeutic strategies for diabetic cardiomyopathy in the future.


Assuntos
Cardiomiopatias Diabéticas/metabolismo , Glucose/farmacologia , MicroRNAs/genética , Miócitos Cardíacos/efeitos dos fármacos , Transcriptoma , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção
6.
Int J Cancer ; 146(7): 1963-1978, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390487

RESUMO

E3 ubiquitin ligases primarily determine the substrate specificity of the ubiquitin-proteasome system and play an essential role in the resistance to bortezomib in multiple myeloma (MM). Neural precursor cell-expressed developmentally downregulated gene 4-1 (NEDD4-1, also known as NEDD4) is a founding member of the NEDD4 family of E3 ligases and is involved in the proliferation, migration, invasion and drug sensitivity of cancer cells. In the present study, we investigated the role of NEDD4-1 in MM cells and explored its underlying mechanism. Clinically, low NEDD4-1 expression has been linked to poor prognosis in patients with MM. Functionally, NEDD4-1 knockdown (KD) resulted in bortezomib resistance in MM cells in vitro and in vivo. The overexpression (OE) of NEDD4-1, but not an enzyme-dead NEDD4-1-C867S mutant, had the opposite effect. Furthermore, the overexpression of NEDD4-1 in NEDD4-1 KD cells resensitized the cells to bortezomib in an add-back rescue experiment. Mechanistically, pAkt-Ser473 levels and Akt signaling were elevated and decreased by NEDD4-1 KD and OE, respectively. NEDD4-1 ubiquitinated Akt and targeted pAkt-Ser473 for proteasomal degradation. More importantly, the NEDD4-1 KD-induced upregulation of Akt expression sensitized MM cells to growth inhibition after treatment with an Akt inhibitor. Collectively, our results suggest that high NEDD4-1 levels may be a potential new therapeutic target in MM.


Assuntos
Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/patologia , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Animais , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Ubiquitina-Proteína Ligases Nedd4/genética , Cultura Primária de Células , Prognóstico , Proteólise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
7.
PLoS One ; 14(12): e0225913, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31805126

RESUMO

Temozolomide (TMZ) is a first-line alkylating agent for glioblastoma multiforme (GBM). Clarifying the mechanisms inducing TMZ insensitivity may be helpful in improving its therapeutic effectiveness against GBM. Insulin-like growth factor (IGF)-1 signaling and micro (mi)RNAs are relevant in mediating GBM progression. However, their roles in desensitizing GBM cells to TMZ are still unclear. We aimed to identify IGF-1-mediated miRNA regulatory networks that elicit TMZ insensitivity for GBM. IGF-1 treatment attenuated TMZ cytotoxicity via WNT/ß-catenin signaling, but did not influence glioma cell growth. By miRNA array analyses, 93 upregulated and 148 downregulated miRNAs were identified in IGF-1-treated glioma cells. miR-513a-5p from the miR-513a-2 gene locus was upregulated by IGF-1-mediated phosphoinositide 3-kinase (PI3K) signaling. Its elevated levels were also observed in gliomas versus normal cells, in array data of The Cancer Genome Atlas (TCGA), and the GSE61710, GSE37366, and GSE41032 datasets. In addition, lower levels of neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin protein ligase that inhibits WNT signaling, were found in gliomas by analyzing cells, arrays, and RNA sequencing data of TCGA glioma patients. Furthermore, a negative correlation was identified between miR-513a-5p and NEDD4L in glioma. NEDD4L was also validated as a direct target gene of miR-513a-5p, and it was reduced by IGF-1 treatment. Overexpression of NEDD4L inhibited glioma cell viability and reversed IGF-1-repressed TMZ cytotoxicity. In contrast, miR-513a-5p significantly affected NEDD4L-inhibited WNT signaling and reduced TMZ cytotoxicity. These findings demonstrate a distinct role of IGF-1 signaling through miR-513a-5p-inhibited NEDD4L networks in influencing GBM's drug sensitivity to TMZ.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Glioma/genética , Glioma/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , MicroRNAs/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Temozolomida/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Interferência de RNA
8.
J Exp Clin Cancer Res ; 38(1): 490, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831018

RESUMO

BACKGROUND: N-myc downstream-regulated gene 1 (NDRG1) has been shown to play a key role in tumor metastasis. Recent studies demonstrate that NDRG1 can suppress tumor growth and is related to tumor proliferation; however, the mechanisms underlying these effects remain obscure. METHODS: Immunohistochemistry (IHC) was used to detect NDRG1 and p21 protein expression in colorectal cancer tissue, and clinical significance of NDRG1 was also analyzed. CCK-8 assay, colony formation assay, flow cytometry, and xenograft model were used to assess the effect of NDRG1 on tumor proliferation in vivo and in vitro. The mechanisms underlying the effect of NDRG1 were investigated using western blotting, immunofluorescence, immunoprecipitation, and ubiquitylation assay. RESULTS: NDRG1 was down-regulated in CRC tissues and correlated with tumor size and patient survival. NDRG1 inhibited tumor proliferation through increasing p21 expression via suppressing p21 ubiquitylation. NDRG1 and p21 had a positive correlation both in vivo and in vitro. Mechanistically, E3 ligase NEDD4 could directly interact with and target p21 for degradation. Moreover, NDRG1 could emulatively antagonize NEDD4-mediated ubiquitylation of p21, increasing p21 expression and inhibit tumor proliferation. CONCLUSION: Our study could fulfill potential mechanisms of the NDRG1 during tumorigenesis and metastasis, which may serve as a tumor suppressor and potential target for new therapies in human colorectal cancer.


Assuntos
Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Prognóstico , Proteólise , Análise de Sobrevida , Carga Tumoral , Ubiquitinação
9.
BMC Psychiatry ; 19(1): 405, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31849325

RESUMO

BACKGROUND: Schizophrenia is a complex psychiatric disorder with unknown etiology. A number of recent studies have shown that the polymorphism of the neural precursor cell expressed developmentally down-regulated 4 (NEDD4) gene is associated with a variety of neuropsychiatric disorders, such as schizophrenia, and may also be associated with cognitive dysfunction in these diseases. METHODS: A case-control study was carried out, the alleles and genotypes distributions of five loci (rs3088077, rs2303579, rs7162435, rs11550869, rs62043855) of the NEDD4 gene from 296 schizophrenia patients and 320 healthy controls were detected by using Taqman single-nucleotide polymorphism (SNP) genotyping technology. The clinical data of case and control group members were collected by self-made questionnaire and the psychotic symptoms of case group members were assessed by the Positive and Negative Syndrome Scale (PANSS). The Matrics Consensus Cognitive Battery (MCCB) was used to test the cognitive function of case group members. RESULTS: The alleles and genotypes frequency of two loci (rs3088077, rs2303579) between case and control group showed significant differences (P <  0.05). There was no significant difference in MCCB scores of patients with different genotypes at rs3088077, rs11550869 and rs7162435 loci in case group. The study of rs2303579 locus showed that, patients' scores with CT genotype were significantly lower than those with CC and TT genotypes (P <  0.05) in the test of Wechsler Memory Scale-Third Edition (WMS-III): Spatial Span, the scores of patients with TT genotype were significantly higher than those with CT genotype (P < 0.05) in the test of Hopkins Verbal Learning Test-Revised (HVLT-R). The study of rs62043855 locus showed that patients with TG genotype had significantly lower scores than those with GG genotype (P < 0.05) in the test of Neuropsychological Assessment Battery (NAB): Mazes. CONCLUSIONS: Our study showed that in schizophrenia patients of Chinese Han population, the polymorphisms of rs3088077 and rs2303579 loci were related to the pathogenesis of schizophrenia, while the polymorphisms of rs2303579 and rs62043855 loci were associated with cognitive dysfunction.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Disfunção Cognitiva/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Polimorfismo de Nucleotídeo Único/genética , Esquizofrenia/genética , Adulto , Estudos de Casos e Controles , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Esquizofrenia/diagnóstico , Esquizofrenia/epidemiologia
10.
Breast Cancer Res ; 21(1): 148, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856858

RESUMO

BACKGROUND: A role for neural precursor cell-expressed developmentally downregulated gene 4 (NEDD4) in tumorigenesis has been suggested. However, information is lacking on its role in breast tumor biology. The purpose of this study was to determine the role of NEDD4 in the promotion of the growth and progression of breast cancer (BC) and to evaluate the clinicopathologic and prognostic significance of NEDD4. METHODS: The impact of NEDD4 expression in BC cell growth was determined by Cell Counting Kit-8 and colony formation assays. Formalin-fixed paraffin-embedded specimens were collected from 133 adjacent normal tissues (ANTs), 445 BC cases composed of pre-invasive ductal carcinoma in situ (DCIS, n = 37), invasive ductal carcinomas (IDC, n = 408, 226 without and 182 with lymph node metastasis), and 116 invaded lymph nodes. The expression of NEDD4 was analyzed by immunohistochemistry. The association between NEDD4 expression and clinicopathological characteristics was analyzed by chi-square test. Survival was evaluated using the Kaplan-Meier method, and curves were compared using a log-rank test. Univariate and multivariate analyses were performed using the Cox regression method. RESULTS: NEDD4 promoted BC growth in vitro. In clinical retrospective studies, 16.5% of ANTs (22/133) demonstrated positive NEDD4 staining. Strikingly, the proportion of cases showing NEDD4-positive staining increased to 51.4% (19/37) in DCIS, 58.4% (132/226) in IDC without lymph node metastasis, and 73.1% (133/182) in BC with lymph node metastasis (BCLNM). In addition, NEDD4-positive staining was associated with clinical parameters, including tumor size (P = 0.030), nodal status (P = 0.001), estrogen receptor status (P = 0.035), and progesterone receptor status (P = 0.023). Moreover, subset analysis in BCLNM revealed that high NEDD4 expression correlated with an elevated risk of relapse (P = 0.0276). Further, NEDD4 expression was an independent prognostic predictor. Lastly, the rates for 10-year overall survival and disease-free survival were significantly lower in patients with positive NEDD4 staining than those in BC patients with negative NEDD4 staining BC (P = 0.0024 and P = 0.0011, respectively). CONCLUSIONS: NEDD4 expression is elevated in BC and is associated with BC growth. NEDD4 correlated with clinicopathological parameters and predicts a poor prognosis. Thus, NEDD4 is a potential biomarker of poor prognosis and a potential therapeutic target for BC treatment.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Expressão Gênica , Ubiquitina-Proteína Ligases Nedd4/genética , Adulto , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor IGF Tipo 1/metabolismo , Adulto Jovem
11.
Epigenetics Chromatin ; 12(1): 78, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856907

RESUMO

BACKGROUND: While the role of Polycomb group protein-mediated "cell memory" is well established in developmental contexts, little is known about their role in adult tissues and in particular in post-mitotic cells. Emerging evidence assigns a pivotal role in cell plasticity and adaptation. PRC2-Ezh1α/ß signaling pathway from cytoplasm to chromatin protects skeletal muscle cells from oxidative stress. However, detailed mechanisms controlling degradation of cytoplasmic Ezh1ß and assembly of canonical PRC2-Ezh1α repressive complex remain to be clarified. RESULTS: Here, we report NEDD4 ubiquitin E3 ligase, as key regulator of Ezh1ß. In addition, we report that ubiquitination and degradation of Ezh1ß is controlled by another layer of regulation, that is, one specific phosphorylation of serine 560 located at Ezh1ß-specific C terminal. Finally, we demonstrate that also Ezh1α needs to be stabilized under stress condition and this stabilization process requires decreased association pattern between another E3 ubiquitin ligase HUWE1. CONCLUSIONS: Together, these results shed light on key components that regulate PRC2-Ezh1α/ß pathway to direct modulation of epigenome plasticity and transcriptional output in skeletal muscle cells.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Estresse Oxidativo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular , Histonas/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Ubiquitinação
12.
PLoS Pathog ; 15(11): e1008100, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710650

RESUMO

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitinação , Montagem de Vírus , Replicação Viral , Humanos , Coriomeningite Linfocítica/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas
13.
Int J Med Sci ; 16(11): 1517-1524, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31673244

RESUMO

NEDD4L (neural precursor cell expressed developmentally down-regulated 4-like) protein is a member of ubiquitin ligases Nedd4 family. Although studies have shown that Nedd4L may act as a tumor suppressor in various cancers, including gastric cancer (GC), its clinical significance and the diagnostic value in GC is not well defined. HIF-1α (hypoxia inducible factor family of transcription factors) is actively involved in the metabolism of many tumors, although the relationship between its expression levels and clinical significance in GC still need to be established. In this study, the level of HIF-1α and NEDD4L mRNA and protein in 25 freshly frozen GC- and matched normal-tissues were determined by western blot and quantitative PCR (qPCR). Additionally, immunohistochemistry assay was performed to measure the protein level of NEDD4L and HIF-1α in 124 GC and 25 normal control tissues. We observed that the NEDD4L mRNA and protein levels decreased significantly (P < 0.001) in GC tissues, while that of HIF-1α increased (P < 0.001), and they both were associated with a poor prognosis, as was the case in patients with lower NEDD4L and higher HIF-1α expression (P < 0.001). On correlation analysis, a significantly negative relationship (r = 0.288, P < 0.01) was revealed between NEDD4L and HIF-1α expressions. Multivariate analysis revealed that co-expression of NEDD4L (P < 0.05) and HIF-1α (P < 0.001) were independent predictors of GC prognosis. Thus, the correlation of NEDD4L and HIF-1α levels may act as a prognostic marker of GC.


Assuntos
Biomarcadores Tumorais/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Neoplasias Gástricas/genética , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia
14.
Bull Exp Biol Med ; 168(2): 219-223, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31776946

RESUMO

We analyzed the expression of molecular targets of natriuretic action of prolactin in different layers of the kidney in the rat model of cholestasis of pregnancy. Sodium bicarbonate cotransporter NBCe1 was most sensitive to the conditions of cholestasis and cholestasis of pregnancy: the expression NBCe1 mRNA and protein in the renal outer medulla decreased in comparison with the normal. All forms of cholestasis affected the mRNA expression of sodium-potassium chloride co-transporter NCC, α-subunit of the ENaCα epithelial sodium channel, and Nedd4-2 ubiquitin ligase in different layers of the kidney. The obtained data suggest that prolactin provides fine tuning of various sodium transporters in different parts of the nephron under pathological conditions.


Assuntos
Colestase/patologia , Transporte de Íons/fisiologia , Medula Renal/metabolismo , Prolactina/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Modelos Animais de Doenças , Canais Epiteliais de Sódio/biossíntese , Canais Epiteliais de Sódio/genética , Feminino , Ubiquitina-Proteína Ligases Nedd4/biossíntese , Ubiquitina-Proteína Ligases Nedd4/genética , Gravidez , RNA Mensageiro/biossíntese , Ratos , Simportadores de Sódio-Bicarbonato/biossíntese , Simportadores de Sódio-Bicarbonato/genética , Membro 3 da Família 12 de Carreador de Soluto/biossíntese , Membro 3 da Família 12 de Carreador de Soluto/genética
15.
Am J Physiol Renal Physiol ; 317(6): F1513-F1525, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566435

RESUMO

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-ß1 ß1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional ß1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates ß1Pix in vitro. Among several AMPK phosphorylation sites on ß1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type ß1Pix, overexpression of a phosphorylation-deficient ß1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both ß1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a ß1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to ß1Pix is critical for the formation of a ß1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both ß1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of ß1Pix at Ser71, which promotes 14-3-3 interactions with ß1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Regulação Enzimológica da Expressão Gênica/genética , Células HEK293 , Humanos , Túbulos Renais Coletores/metabolismo , Camundongos , Mutação/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosforilação , Fatores de Troca de Nucleotídeo Guanina Rho/genética
16.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614475

RESUMO

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the "ubiquitylation reserve" of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.


Assuntos
Substituição de Aminoácidos , Síndrome do QT Longo/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Motivos de Aminoácidos , Animais , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ligação Proteica , Ubiquitinação , Adulto Jovem
17.
Elife ; 82019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31373553

RESUMO

The metabotropic glutamate receptor 7 (mGlu7) is a class C G protein-coupled receptor that modulates excitatory neurotransmitter release at the presynaptic active zone. Although post-translational modification of cellular proteins with ubiquitin is a key molecular mechanism governing protein degradation and function, mGlu7 ubiquitination and its functional consequences have not been elucidated yet. Here, we report that Nedd4 ubiquitin E3 ligase and ß-arrestins regulate ubiquitination of mGlu7 in heterologous cells and rat neurons. Upon agonist stimulation, ß-arrestins recruit Nedd4 to mGlu7 and facilitate Nedd4-mediated ubiquitination of mGlu7. Nedd4 and ß-arrestins regulate constitutive and agonist-induced endocytosis of mGlu7 and are required for mGlu7-dependent MAPK signaling in neurons. In addition, Nedd4-mediated ubiquitination results in the degradation of mGlu7 by both the ubiquitin-proteasome system and the lysosomal degradation pathway. These findings provide a model in which Nedd4 and ß-arrestin act together as a complex to regulate mGlu7 surface expression and function at presynaptic terminals.


Assuntos
Ubiquitina-Proteína Ligases Nedd4/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Ubiquitinação , beta-Arrestinas/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Transporte Proteico , Ratos
18.
Arch Biochem Biophys ; 672: 108065, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31394088

RESUMO

Recently, we reported that treatment with the mouse agonist of the constitutive androstane receptor (CAR), 1,4-bis benzene[2-(3,5-dichloropyridyloxy)] (TCPOBOP; a well-known hepatomitogen), reduced PTEN protein levels, leading to Akt activation. Hence, the present study was performed to demonstrate the role of CAR in PTEN regulation and liver growth. Liver hyperplasia caused by CAR activation was confirmed to be mediated by a decrease in PTEN protein level and the activation of the Akt signalling pathway in the liver of mice. Treatment with the CAR agonist decreased the PTEN levels and increased Foxm1 levels, which correlate with the elevated expression of the FoxM1 target gene, Nedd4-1, an E3 ligase involved in PTEN ubiquitination, and the promotion of degradation. The increase in Nedd4-1 levels was accompanied by an increase in CAR-mediated accumulation of Foxm1 on the Nedd4-1 gene promoter. Therefore, these results provide evidence that a notable function of CAR is its liver growth promotion effect, which is accompanied by FoxM1-Nedd4-mediated repression of PTEN and Akt pathway activation.


Assuntos
Proteína Forkhead Box M1/metabolismo , Fígado/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Família 2 do Citocromo P450/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Oximas/farmacologia , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Transdução de Sinais/fisiologia , Esteroide Hidroxilases/metabolismo , Tiazóis/farmacologia
19.
Adv Exp Med Biol ; 1152: 365-375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456194

RESUMO

Massively parallel sequencing, genomic and proteomic technologies have provided near complete resolution of signaling landscape of breast cancer (BCa). NEDD4 family of E3-ubiquitin ligases comprises a large family of proteins particularly, SMURFs (SMURF1, SMURF2), WWPs and NEDD4 which are ideal candidates for targeted therapy. However, it is becoming progressively more understandable that SMURFs and NEDD4 have "split-personalities". These molecules behave dualistically in breast cancer and future studies must converge on detailed identification of context specific role of these proteins in BCa. Finally, we provide scattered clues of regulation of SMURF2 by oncogenic miRNAs, specifically considering longstanding questions related to regulation of SMURF1 and WWPs by miRNAs in BCa. SMURFS, WWPs and NEDD4 are versatile regulators and represent a fast-growing field in cancer research and better understanding of the underlying mechanisms will be helpful in transition of our knowledge from a segmented view to a more conceptual continuum.


Assuntos
Neoplasias da Mama/enzimologia , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases/genética , Neoplasias da Mama/genética , Feminino , Humanos , MicroRNAs/genética , Oncogenes , Proteômica , Transdução de Sinais , Ubiquitinação
20.
Am J Physiol Renal Physiol ; 317(4): F825-F838, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31364380

RESUMO

Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg2+ excretion and, via activity of the Na+-Cl- cotransporter (NCC), also plays a key role in K+ homeostasis by metering Na+ delivery to distal segments. Little is known about the mechanisms by which plasma Mg2+ concentration regulates NCC activity and how low-plasma Mg2+ concentration and K+ concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg2+ restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg2+ restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K+ restriction. Mg2+ restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg2+ or combined Mg2+-K+ restriction but were dramatically lower compared with a low-K+ diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na+ delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg2+-K+ restriction, but, surprisingly, NCC was not activated after long-term K+ restriction despite lower plasma K+ concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K+ restriction.


Assuntos
Hipopotassemia/metabolismo , Deficiência de Magnésio/metabolismo , Ubiquitina-Proteína Ligases Nedd4/biossíntese , Animais , Dieta , Regulação para Baixo , Túbulos Renais Distais/metabolismo , Magnésio/sangue , Deficiência de Magnésio/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4/genética , Fosforilação , Potássio/sangue , Deficiência de Potássio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/biossíntese , Membro 3 da Família 12 de Carreador de Soluto/genética
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