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BACKGROUND: Satellite cells are tissue-specific stem cells primarily responsible for the regenerative capacity of skeletal muscle. Satellite cell function and maintenance are regulated by extrinsic and intrinsic mechanisms, including the ubiquitin-proteasome system, which is key for maintaining protein homeostasis. In this context, it has been shown that ubiquitin-ligase NEDD4-1 targets the transcription factor PAX7 for proteasome-dependent degradation, promoting muscle differentiation in vitro. Nonetheless, whether NEDD4-1 is required for satellite cell function in regenerating muscle remains to be determined. RESULTS: Using conditional gene ablation, we show that NEDD4-1 loss, specifically in the satellite cell population, impairs muscle regeneration resulting in a significant reduction of whole-muscle size. At the cellular level, NEDD4-1-null muscle progenitors exhibit a significant decrease in the ability to proliferate and differentiate, contributing to the formation of myofibers with reduced diameter. CONCLUSIONS: These results indicate that NEDD4-1 expression is critical for proper muscle regeneration in vivo and suggest that it may control satellite cell function at multiple levels.
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Músculo Esquelético/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Células Madre , Ubiquitinas/metabolismo , Diferenciación Celular , Desarrollo de Músculos/fisiología , Proliferación Celular/fisiología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/metabolismoRESUMEN
Epilepsy is a disorder of the brain charac-terized by abnormal neuron excitability.However,the underlying molecular mechanism of neuron excitability modulation remains elusive.With the help of bioinformatic methods,we have identified receptor-type tyrosine-pro-tein phosphatase-like N(PTPRN)as a critical gene dur-ing epileptogenesis.PTPRN recruits NEDD4L ubiquitin E3 ligase to NaV1.2 sodium channels,facilitating NEDD4L-mediated ubiquitination and endocytosis.Knockout of PTPRN endows hippocampal granule cells with augmented depolarization currents and higher intrinsic excitability,which is reflected by increased seizure susceptibility of transgenic mice.On the contrary,reduced neuron excit-ability and decreased seizure susceptibility are observed after PTPRN overexpression.Meanwhile,we find that a 133 aa fragment recaptures modulation effect of PTPRN full-length,and this fragment shows therapeutic potential towards epilepsy caused by NaV1.2 gain of function vari-ants.In brief,our results demonstrate PTPRN playsa criti-calroleinregulatingneuronexcitability,providing a poten-tial therapeutic approach for epilepsy.
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Stroke is the second leading cause of death worldwide,and oxidative stress plays a crucial role.Celastrol exhibits strong antioxidant properties in several diseases;however,whether it can affect oxidation in cerebral ischemic-reperfusion injury(CIRI)remains unclear.This study aimed to determine whether celastrol could reduce oxidative damage during CIRI and to elucidate the underlying mechanisms.Here,we found that celastrol attenuated oxidative injury in CIRI by upregulating nuclear factor E2-related factor 2(Nrf2).Using alkynyl-tagged celastrol and liquid chromatography-tandem mass spectrometry,we showed that celastrol directly bound to neuronally expressed developmentally downregulated 4(Nedd4)and then released Nrf2 from Nedd4 in astrocytes.Nedd4 promoted the degradation of Nrf2 through K48-linked ubiquitination and thus contributed to astrocytic reactive oxygen species production in CIRI,which was significantly blocked by celastrol.Furthermore,by inhibiting oxidative stress and astrocyte activation,celastrol effectively rescued neurons from axon damage and apoptosis.Our study uncovered Nedd4 as a direct target of celastrol,and that celastrol exerts an antioxidative effect on as-trocytes by inhibiting the interaction between Nedd4 and Nrf2 and reducing Nrf2 degradation in CIRI.
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Acetaminophen (APAP) overdose is a major cause of liver injury. Neural precursor cell expressed developmentally downregulated 4-1 (NEDD4-1) is an E3 ubiquitin ligase that has been implicated in the pathogenesis of numerous liver diseases; however, its role in APAP-induced liver injury (AILI) is unclear. Thus, this study aimed to investigate the role of NEDD4-1 in the pathogenesis of AILI. We found that NEDD4-1 was dramatically downregulated in response to APAP treatment in mouse livers and isolated mouse hepatocytes. Hepatocyte-specific NEDD4-1 knockout exacerbated APAP-induced mitochondrial damage and the resultant hepatocyte necrosis and liver injury, while hepatocyte-specific NEDD4-1 overexpression mitigated these pathological events both in vivo and in vitro. Additionally, hepatocyte NEDD4-1 deficiency led to marked accumulation of voltage-dependent anion channel 1 (VDAC1) and increased VDAC1 oligomerization. Furthermore, VDAC1 knockdown alleviated AILI and weakened the exacerbation of AILI caused by hepatocyte NEDD4-1 deficiency. Mechanistically, NEDD4-1 was found to interact with the PPTY motif of VDAC1 through its WW domain and regulate K48-linked ubiquitination and degradation of VDAC1. Our present study indicates that NEDD4-1 is a suppressor of AILI and functions by regulating the degradation of VDAC1.
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【Objective】 4-like protein with down-regulated expression and development in neural precursor cells (NEDD4L) plays an important role in blood pressure (BP) regulation and sodium homeostasis by regulating epithelial sodium channel protein. In this study, we aimed to explore the relationship of NEDD4L gene polymorphisms with BP responses to sodium and potassium intake. 【Methods】 In 2004, 514 subjects from 124 families in Meixian County, Shaanxi Province, were recruited to establish a salt-sensitive hypertension study cohort. All the subjects received a 3-day baseline survey, a 7-day low-salt diet, a 7-day high-salt diet, and finally a 7-day high-salt and potassium supplementation. Their BP was measured and peripheral blood samples were collected at different intervention periods. The 14 gene polymorphisms of NEDD4L gene were genotyped and analyzed by MassARRAY platform. 【Results】 BP decreased on a low-salt diet, and significantly increased on a high-salt diet, and decreased again after potassium supplementation. NEDD4L SNPs rs74408486 were significantly associated with systolic BP, diastolic BP and mean arterial pressure responses to the low-salt diet. SNPs rs292449 and rs2288775 were significantly associated with pulse pressure response to the high-salt diet. In addition, SNPs rs563283 and rs292449 were significantly associated with diastolic BP, mean arterial pressure, and pulse pressure responses to high-salt and potassium supplementation diet. 【Conclusion】 NEDD4L gene polymorphisms were significantly associated with BP responses to sodium and potassium intake, suggesting that NEDD4L gene may be involved in the development of salt sensitivity and potassium sensitivity.
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Neural precursor cell Expressed‚Developmentally Down-regulated protein 4 (NEDD4-1‚ also known as NEDD4 in some papers) is a tumor-related protein that has attracted much attention in recent years. It belongs to the E3 HECT (homologous to E6 associated protein C terminus) ubiquitin ligase‚ which could ubiquitinate various proteins that are subsequently degraded in lysosomes or proteasomes‚ or mediate their nuclear-cytoplasmic translocation‚ or indirectly affect various signaling pathways of different malignant tumors. With the deepening of a large number of tumor-related experiments‚ it has been found that NEDD4-1 can affect the biological behavior of tumors by regulating cell cycle‚ invasion and metastasis of cancer cells‚ antagonize drug resistance and many other pathways. In digestive system tumors‚ NEDD4-1 mainly promotes the proliferation‚ invasion and migration of hepatocellular carcinoma through multiple pathways such as PTEN/ PI3K/ Akt‚ TGF-β‚ Hippo and LDLRAD4. In pancreatic cancer‚ NEDD4-1 acted as an oncogene in the PI3K/ Akt signaling pathway‚ but acted as a tumor suppressor gene in the Myc-Sirt2 signaling circuit. In gastric and colorectal cancer‚ the NEDD4-1-related signaling pathways are different from other digestive system tumors. NEDD4-1 promotes gastric cancer progression and metastasis (via the EGFR signaling pathway) and inhibits colorectal cancer tumor growth (via the Wnt signaling pathway) independently of the PTEN/ PI3K/ Akt pathway. NEDD4-1 has become a hot research direction for therapeutic purposes. In this paper‚ we summarize the functions‚ signaling pathways and potential inhibitors of NEDD4-1 in different digestive system tumors‚ and discuss the relationship between NEDD4-1 and different signaling pathways‚ aiming to provide important reference data for cancer therapy.
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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.
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Objective::To observe the expression of divalent metal transporter 1 (DMT1) in neurons under the condition of Ndfip1 overexpression, and to explore the regulation effect of Ndfip1 on DMT1. Methods:The Ndfip1 eukaryotic expression plasmid was constructed, and the human neuroblastoma SH-SY5Y cells were transfected with Ndfip1 plasmid; the transfection efficiency was detected. The SH-SY5Y cells transfected with Ndfip1 plasmid were used as experimental group,and the SH-SY5Y cells transfected with empty plasmid were used as control group. The expression intensities of DMT1 protein in the SH-SY5Y cells in two groups were observed by immunofluorescence method,and the expression levels of DMT1 protein in the SH-SY5Y cells in two groups were detected by Western blotting method. Results:The plasmid was amplified, electrophoretically separated and sequenced, and the results proved that the plasmid was successfully constructed. After transfection of SH-SY5Y cells with Ndfip1 plasmid for 48 h, the expression level of Ndfip1 was significantly increased compared with before transfection (P<0.01). The immunofluorescence results showed that the fluorescence intensity of DMT1 in the cells in experimental group was significantly decreased compared with control group. The Western blotting results showed that the expression level of DMT1 in the cells in experimental group was decreased (P<0.05) compared with control group. Conclusion:The overexpression of Ndfip1 can down-regulate the expression of DMT1 protein in the nerve cells, and Ndfip1 nerve has a negatively regulatory effect.
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Objective To construct Nedd4 knockout bone marrow-derived macrophages(BMDM) cell line by CRISPR/Cas9 technology and to provide an effective tool for studying the function and mechanism of Nedd4 in macrophage.Methods First,three high-grade sgRNAs targeting Nedd4 gene exons were screened using the online tool before synthesized sgRNAs were inserted into the PX330 plasmid respectively.Secondly,the recombinant plasmids were transferred into BMDM cells and monoclonal cells were obtained by limiting dilution method.The protein levels of NEDD4 in monoclonal cells were detected by Western blotting.Finally,the DNA sequence of the monoclonal cells was confirmed by sequence analysis.Results One Nedd4 knockout BMDM cell line was obtained.The sequencing result showed that the Nedd4 gene had 16bp deletion mutation in this cell line.Conclusion The Nedd4 knockout BMDM macrophage cell line constructed by CRISPR/Cas9 technology will be a useful tool for studying the function and mechanism of Nedd4 in BMDM cells.
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Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (72PPLP75) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (72PPLP75 was changed to 72APLA75) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.
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Humanos , Proteínas Adaptadoras Transductoras de Señales/química , Línea Celular , Regulación hacia Abajo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación del Desarrollo de la Expresión Génica , Inmunoprecipitación , Microscopía Confocal , Mutación , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/fisiología , Transfección , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
OBJECTIVES: Alteration of hippocampus was demonstrated in the maternal social separation(MSS) pups, separated from dams on postnatal day(pnd) 14 and placed alone. Therefore, to understand the molecular events involved in the MSS, we have initiated a search for gene profiles that are up or down-regulated in the hippocampus of MSS pups. METHODS: Analysis of cDNA microarray was performed by using total RNA extracted from the hippocampus of control and MSS pups on pnd 17. Also, passive-avoidance test was demonstrated on pnd 35. RESULTS: Up-regulation of Nedd4a was observed in the hippocampus of MSS pups. Also, MSS rats showed less elongation of latency in passive avoidance test. CONCLUSION: We suggest that environmental effects of MSS may be altered the neural and/or glial differentiation and synapse formation-related genes which may lead cognitive alterations in MSS rats.
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Animales , Ratas , Expresión Génica , Hipocampo , Memoria , Análisis por Micromatrices , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN , Sinapsis , Regulación hacia ArribaRESUMEN
No abstract available.