RESUMO
BACKGROUND: Special AT-rich sequence-binding protein 2 is essential for the development of cerebral cortex and key molecular node for the establishment of proper neural circuitry and function. Mutations in the SATB2 gene lead to SATB2-associated syndrome, which is characterized by abnormal development of skeleton and central nervous systems. METHODS: We generated Satb2 knockout mouse model through CRISPR-Cas9 technology and performed RNA-seq and ChIP-seq of embryonic cerebral cortex. We conducted RT-qPCR, western blot, immunofluorescence staining, luciferase reporter assay and behavioral analysis for experimental verification. RESULTS: We identified 1363 downstream effector genes of Satb2 and correlation analysis of Satb2-targeted genes and neurological disease genes showed that Satb2 contribute to cognitive and mental disorders from the early developmental stage. We found that Satb2 directly regulate the expression of Ntng1, Cdh13, Kitl, genes important for axon guidance, synaptic formation, neuron migration, and Satb2 directly activates the expression of Mef2c. We also showed that Satb2 heterozygous knockout mice showed impaired spatial learning and memory. CONCLUSIONS: Taken together, our study supportsroles of Satb2 in the regulation of axonogenesis and synaptic formation at the early developmental stage and provides new insights into the complicated regulatory mechanism of Satb2 and new evidence to elucidate the pathogen of SATB2-associated syndrome. IMPACT: 1363 downstream effector genes of Satb2 were classified into 5 clusters with different temporal expression patterns. We identified Plxnd1, Ntng1, Efnb2, Ephb1, Plxna2, Epha3, Plxna4, Unc5c, and Flrt2 as axon guidance molecules to regulate axonogenesis. 168 targeted genes of Satb2 were found to regulate synaptic formation in the early development of the cerebral cortex. Transcription factor Mef2c is positively regulated by Satb2, and 28 Mef2c-targeted genes can be directly regulated by Satb2. In the Morris water maze test, Satb2+/- mice showed impaired spatial learning and memory, further strengthening that Satb2 can regulate synaptic functions.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Animais , Camundongos , Córtex Cerebral/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Receptores de Superfície Celular/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Accumulated genetic evidences indicate that the contactin associated protein-like (CNTNAP) family is implicated in autism spectrum disorders (ASD). In this study, we identified genetic mutations in the CNTNAP3 gene from Chinese Han ASD cohorts and Simons Simplex Collections. We found that CNTNAP3 interacted with synaptic adhesion proteins Neuroligin1 and Neuroligin2, as well as scaffolding proteins PSD95 and Gephyrin. Significantly, we found that CNTNAP3 played an opposite role in controlling the development of excitatory and inhibitory synapses in vitro and in vivo, in which ASD mutants exhibited loss-of-function effects. In this study, we showed that the male Cntnap3-null mice exhibited deficits in social interactionï¼ spatial learning and prominent repetitive behaviors. These evidences elucidate the pivotal role of CNTNAP3 in synapse development and social behaviors, providing mechanistic insights into ASD.
Assuntos
Transtorno do Espectro Autista/genética , Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/genética , Comportamento Social , Animais , Comportamento Animal , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , SinapsesRESUMO
Nuclear receptors (NRs) regulate gene transcription by recruiting coregulators, involved in chromatin remodeling and assembly of the basal transcription machinery. The NR-associated protein ligand-dependent corepressor (LCoR) has previously been shown to suppress hepatic lipogenesis by decreasing the binding of steroid receptor coactivators to thyroid hormone receptor. However, the role of LCoR in adipogenesis has not been established. Here, we show that LCoR expression is reduced in the early stage of adipogenesis in vitro LCoR overexpression inhibited 3T3-L1 adipocyte differentiation, whereas LCoR knockdown promoted it. Using an unbiased affinity purification approach, we identified CCAAT/enhancer-binding protein ß (C/EBPß), a key transcriptional regulator in early adipogenesis, and corepressor C-terminal binding proteins as potential components of an LCoR-containing complex in 3T3-L1 adipocytes. We found that LCoR directly interacts with C/EBPß through its C-terminal helix-turn-helix domain, required for LCoR's inhibitory effects on adipogenesis. LCoR overexpression also inhibited C/EBPß transcriptional activity, leading to inhibition of mitotic clonal expansion and transcriptional repression of C/EBPα and peroxisome proliferator-activated receptor γ2 (PPARγ2). However, LCoR overexpression did not affect the recruitment of C/EBPß to the promoters of C/EBPα and PPARγ2 in 3T3-L1 adipocytes. Of note, restoration of PPARγ2 or C/EBPα expression attenuated the inhibitory effect of LCoR on adipogenesis. Mechanistically, LCoR suppressed C/EBPß-mediated transcription by recruiting C-terminal binding proteins to the C/EBPα and PPARγ2 promoters and by modulating histone modifications. Taken together, our results indicate that LCoR negatively regulates early adipogenesis by repressing C/EBPß transcriptional activity and add LCoR to the growing list of transcriptional corepressors of adipogenesis.
Assuntos
Adipócitos/citologia , Adipogenia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Técnicas de Silenciamento de Genes , Camundongos , Mapas de Interação de Proteínas , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Mutations in the SRCAP gene are among the genetic alterations identified in autism spectrum disorders (ASD). However, the pathogenic mechanisms remain unclear. In this study, we demonstrate that Srcap+/- mice manifest deficits in social novelty response, as well as increased repetitive behaviors, anxiety, and impairments in learning and memory. Notably, a reduction in parvalbumin-positive neurons is observed in the retrosplenial cortex (RSC) and dentate gyrus (DG) of these mice. Through RNA sequencing, we identify dysregulation in 27 ASD-related genes in Srcap+/- mice. Specifically, we find that Srcap regulates expression of Satb2 via H2A.z in the promoter. Therapeutic intervention via retro-orbital injection of adeno-associated virus (AAV)-Satb2 in neonatal Srcap+/- mice leads to amelioration of the neurodevelopmental and ASD-like abnormalities. Furthermore, the expression of Satb2 only in the RSC of adolescent mice rectifies social novelty impairments. These results underscore the pivotal role of Srcap in neurodevelopment, by regulating Satb2, providing valuable insights for the pathophysiology of ASD.
Assuntos
Haploinsuficiência , Proteínas de Ligação à Região de Interação com a Matriz , Fatores de Transcrição , Animais , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Comportamento Animal , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Masculino , Comportamento Social , Camundongos Endogâmicos C57BL , Neurônios/metabolismoRESUMO
Whole-brain genome editing to correct single-base mutations and reduce or reverse behavioral changes in animal models of autism spectrum disorder (ASD) has not yet been achieved. We developed an apolipoprotein B messenger RNA-editing enzyme, catalytic polypeptide-embedded cytosine base editor (AeCBE) system for converting C·G to T·A base pairs. We demonstrate its effectiveness by targeting AeCBE to an ASD-associated mutation of the MEF2C gene (c.104T>C, p.L35P) in vivo in mice. We first constructed Mef2cL35P heterozygous mice. Male heterozygous mice exhibited hyperactivity, repetitive behavior and social abnormalities. We then programmed AeCBE to edit the mutated C·G base pairs of Mef2c in the mouse brain through the intravenous injection of blood-brain barrier-crossing adeno-associated virus. This treatment successfully restored Mef2c protein levels in several brain regions and reversed the behavioral abnormalities in Mef2c-mutant mice. Our work presents an in vivo base-editing paradigm that could potentially correct single-base genetic mutations in the brain.
Assuntos
Transtorno do Espectro Autista , Edição de Genes , Animais , Camundongos , Masculino , Transtorno do Espectro Autista/genética , Encéfalo , Mutação/genética , Fatores de Transcrição MEF2/genéticaRESUMO
Strong evidence from human genetic studies associates the thousand and one amino acid kinase 1 (TAOK1) gene with autism spectrum disorder (ASD). In this work, we discovered a de novo frameshifting mutation in TAOK1 within a Chinese ASD cohort. We found that Taok1 haploinsufficiency induces autistic-like behaviors in mice. Importantly, we observed a significant enrichment of Taok1 in the dorsal raphe nucleus (DRN). The haploinsufficiency of Taok1 considerably restrained the activation of DRN neurons during social interactions, leading to the aberrant phosphorylation of numerous proteins. Intriguingly, the genetic deletion of Taok1 in VGlut3-positive neurons of DRN resulted in mice exhibiting autistic-like behaviors. Ultimately, reintroducing wild-type Taok1, but not its kinase-dead variant, into the DRN of adult mice effectively mitigated the autistic-like behaviors associated with Taok1 haploinsufficiency. This work suggests that Taok1, through its influence in the DRN, regulates social interaction behaviors, providing critical insights into the etiology of ASD.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Humanos , Animais , Camundongos , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Núcleo Dorsal da Rafe/metabolismo , Haploinsuficiência , Comportamento Social , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND & AIMS: Transcriptional co-regulators assist nuclear receptors to control the transcription and maintain the metabolic homeostasis. Ligand-dependent corepressor (LCOR) was reported to function as a transcriptional corepressor in vitro. We found LCOR expression decreased in fatty livers of leptin-deficient (ob/ob) mice, diet-induced obese mice, as well as patients, suggesting LCOR may play a role in lipid homeostasis. We sought to investigate the physiological role of LCOR in vivo and elucidate the underlining molecular mechanisms. METHODS: The effect of LCOR on hepatic lipid accumulation and thyroid hormone receptor (TR) mediated expression of lipogenic genes was studied in vitro and in vivo. RESULTS: Ectopic expression of LCOR via intravenous infection with LCOR adenovirus decreased the hepatic triglyceride level in wild type, ob/ob, and diet-induced obese mice. Interestingly, overexpression of LCOR repressed the thyroid hormone induced expression of lipogenic genes and non-lipogenic genes, and ameliorated hepatic steatosis in obese mice, suggesting that LCOR might regulate lipogenesis as a novel TR corepressor. Furthermore, our study revealed that LCOR could interact with TRß1 in the presence of the ligand, which resulted in competitive binding and reduced recruitment of steroid receptor coactivator-1/3 (SRC-1/3) to the promoter region of TR target genes. CONCLUSIONS: Our data suggest that LCOR is likely to suppress TRß1-mediated hepatic lipogenesis by decreasing binding and recruitment of SRCs to TRß1. Our study reveals the physiological function of hepatic LCOR in lipid metabolism and the mechanism by which LCOR regulates lipogenesis. Hepatic LCOR may be a potential target for treating hepatic steatosis.
Assuntos
Proteínas Correpressoras/metabolismo , Fígado/metabolismo , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Proteínas Correpressoras/química , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Células HEK293 , Humanos , Ligantes , Lipogênese/genética , Lipogênese/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Modelos Biológicos , Domínios e Motivos de Interação entre Proteínas , Receptores alfa dos Hormônios Tireóideos/química , Receptores beta dos Hormônios Tireóideos/químicaRESUMO
Schizophrenia is a complex, severe psychiatric disorder with a high heritability that affects approximately 1% of the world's population. Numerous schizophrenia-related risk genes have been reported in large-scale studies, but the role of most genetic abnormalities in the pathogenesis of the disease is still obscure. In this study, using whole-exome sequencing, we identified a novel nonsense mutation c.1324C > T in the Interleukin 1 receptor accessory protein (IL1RAP) gene in four affected individuals with schizophrenia of a Chinese family. IL1RAP was found involved in initiating the immune responses and regulating synaptic formation. Considering that schizophrenia has been hypothesized to be neurodevelopment disorder for decades, we further explored the influence of altered expression of IL1RAP gene on neuronal growth, and assessed whether this mutation affects the function of IL1RAP protein in IL-1 signaling pathway. We used lentivirus-mediated shRNA to knockdown the IL1RAP gene expression, which suppressed the axon and dendrites growth of cultured mouse cortical neurons. These defects can be recovered by human IL1RAP wild type construct, but not the R442* mutant construct. Furthermore, this mutant even inhibited neuronal growth and IL-1ß-induced JNK phosphorylation when overexpressed in cortical neurons. Although overexpression of this mutant in HePG2 cells did not change IL1RAP protein expression, it partially prohibited the IL-1ß-induced nuclear translocation of transcript factor NF-κB, indicating that IL1RAP c.1324C > T is a loss-of-function mutation. Our findings show that IL1RAP plays an important role in early stages of neurodevelopment, and the mutation c.1324C > T may contribute to the pathogenesis of schizophrenia.
Assuntos
NF-kappa B , Esquizofrenia , Animais , Humanos , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Camundongos , Mutação , NF-kappa B/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transdução de SinaisRESUMO
Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder, causing defects of social interaction and repetitive behaviors. Here, we identify a de novo heterozygous gene-truncating mutation of the Sentrin-specific peptidase1 (SENP1) gene in people with ASD without neurodevelopmental delay. We find that Senp1+/- mice exhibit core autistic-like symptoms such as social deficits and repetitive behaviors but normal learning and memory ability. Moreover, we find that inhibitory and excitatory synaptic functions are severely affected in the retrosplenial agranular (RSA) cortex of Senp1+/- mice. Lack of Senp1 leads to increased SUMOylation and degradation of fragile X mental retardation protein (FMRP), also implicated in syndromic ASD. Importantly, re-introducing SENP1 or FMRP specifically in RSA fully rescues the defects of synaptic function and autistic-like symptoms of Senp1+/- mice. Together, these results demonstrate that disruption of the SENP1-FMRP regulatory axis in the RSA causes autistic symptoms, providing a candidate region for ASD pathophysiology.
Assuntos
Transtorno do Espectro Autista/enzimologia , Comportamento Animal , Cisteína Endopeptidases/metabolismo , Giro do Cíngulo/enzimologia , Sinapses/enzimologia , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Transtorno do Espectro Autista/psicologia , Estudos de Casos e Controles , Células Cultivadas , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores , Feminino , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Predisposição Genética para Doença , Asseio Animal , Giro do Cíngulo/fisiopatologia , Haploinsuficiência , Humanos , Potenciais Pós-Sinápticos Inibidores , Locomoção , Masculino , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Comportamento Social , SumoilaçãoRESUMO
Despite broad application of nanotechnology in neuroscience, the nanoneurotoxicity of magnetic nanoparticles in primary hippocampal neurons remains poorly characterized. In particular, understanding how magnetic nanoparticles perturb neuronal calcium homeostasis is critical when considering magnetic nanoparticles as a nonviral vector for effective gene therapy in neuronal diseases. Here, we address the pressing need to systematically investigate the neurotoxicity of magnetic nanoparticles with different surface charges in primary hippocampal neurons. We found that unlike negative and neutral nanoparticles, positively charged magnetic nanoparticles (magnetic poly(lactic-co-glycolic acid) (PLGA)-polyethylenimine (PEI) nanoparticles, MNP-PLGA-PEI NPs) rapidly elevated cytoplasmic calcium levels in primary hippocampal neurons, mainly via extracellular calcium influx regulated by voltage-gated calcium channels. We went on to show that this perturbation of intracellular calcium homeostasis elicited serious cytotoxicity in primary hippocampal neurons. However, our next experiment demonstrated that PEGylation on the surface of MNP-PLGA-PEI NPs shielded the surface charge, thereby preventing the perturbation of intracellular calcium homeostasis. That is, PEGylated MNP-PLGA-PEI NPs reduced nanoneurotoxicity. Importantly, biocompatible PEGylated MNP-PLGA-PEI NPs under an external magnetic field enhanced transfection efficiency (>7%) of plasmid DNA encoding GFP in primary hippocampal neurons compared to NPs without external magnetic field mediation. Moreover, under an external magnetic field, this system achieved gene transfection in the hippocampus of the C57 mouse. Overall, this study is the first to successfully employ biocompatible PEGylated MNP-PLGA-PEI NPs for transfection using a magnetofection strategy in primary hippocampal neurons, thereby providing a nanoplatform as a new perspective for treating neuronal diseases or modulating neuron activities.
Assuntos
Proteínas de Fluorescência Verde , Hipocampo/metabolismo , Nanopartículas/química , Neurônios/metabolismo , Plasmídeos , Transfecção , Animais , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Humanos , Camundongos , Neurônios/citologia , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoimina/química , Polietilenoimina/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease with fast progression. ALS has heavy genetic components in which a series of genetic mutations have been identified. In 2013, Mutations of the CREST gene (also known as SS18L1), which functions as a calcium-regulated transcriptional activator, were found in sporadic ALS patients. However, the pathogenic causality and mechanisms of ALS-associated mutations of CREST remain to be determined. METHODS: In this study, we constructed CREST knockout and Q394X knock-in mice with CRISPR/Cas9 system. Using biochemical and imaging tools, we illustrated core pathological phenotypes in CREST mutant mice and claimed the possible pathogenic mechanisms. Furthermore, we also observed locomotion defects in CREST mutant mice with behavioural tests. RESULTS: We demonstrate that ALS-related CREST-Q388X mutation exhibits loss-of-function effects. Importantly, the microglial activation was prevalent in CREST haploinsufficiency mice and Q394X mice mimicking the human CREST Q388X mutation. Furthermore, we showed that both CREST haploinsufficiency and Q394X mice displayed deficits in motor coordination. Finally, we identified the critical role of CREST-BRG1 complex in repressing the expression of immune-related cytokines including Ccl2 and Cxcl10 in neurons, via histone deacetylation, providing the molecular mechanisms underlying inflammatory responses within mice lack of CREST. CONCLUSION: Our findings indicate that elevated inflammatory responses in a subset of ALS may be caused by neuron-derived factors, suggesting potential therapeutic methods through inflammation pathways.
RESUMO
Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1's corepressor activity.
Assuntos
Proteínas de Homeodomínio/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Histona Desacetilases/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas Repressoras/genética , Transcrição Gênica , Proteínas Supressoras de Tumor/genéticaRESUMO
Interferon-gamma (IFN-gamma) is a major proinflammatory effector and regulatory cytokine produced by activated T cells and NK cells. IFN-gamma has been shown to play pivotal roles in fundamental immunological processes such as inflammatory reactions, cell-mediated immunity and autoimmunity. A variety of human disorders have now been linked to irregular IFN-gamma expression. In order to achieve proper IFN-gamma-mediated immunological effects, IFN-gamma expression in T cells is subject to both positive and negative regulation. In this study, we report for the first time the negative regulation of IFN-gamma expression by Prospero-related Homeobox (Prox1). In Jurkat T cells and primary human CD4+ T cells, Prox1 expression decreases quickly upon T cell activation, concurrent with a dramatic increase in IFN-gamma expression. Reporter analysis and chromatin immunoprecipitation (ChIP) revealed that Prox1 associates with and inhibits the transcription activity of IFN-,gammapromoter in activated Jurkat T cells. Co-immunoprecipitation and GST pull-down assay demonstrated a direct binding between Prox1 and the nuclear receptor peroxisome proliferator-activated receptor gamma (PPPARgamma, which is also an IFN-gamma repressor in T cells. By introducing deletions and mutations into Prox1, we show that the repression of IFN-gamma promoter by Prox1 is largely dependent upon the physical interaction between Prox1 and PPPARgamma Furthermore, PPPARgammaantagonist treatment removes Prox1 from IFN-gamma promoter and attenuates repression of IFN-gamma expression by Prox1. These findings establish Prox1 as a new negative regulator of IFN-gamma expression in T cells and will aid in the understanding of IFN-gamma transcription regulation mechanisms.
Assuntos
Proteínas de Homeodomínio/metabolismo , Interferon gama/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Anilidas/farmacologia , Animais , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Interferon gama/genética , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Camundongos , PPAR gama/antagonistas & inibidores , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Homologia de Sequência do Ácido Nucleico , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Proteínas Supressoras de Tumor/genéticaRESUMO
The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene, encoding the RNA-associated SmN protein, duplications or deletions of which are strongly associated with neurodevelopmental disabilities. SNRPN-coding protein is highly expressed in the brain. However, the role of SNRPN protein in neural development remains largely unknown. Here we showed that the expression of SNRPN increased markedly during postnatal brain development. Overexpression or knockdown of SNRPN in cortical neurons impaired neurite outgrowth, neuron migration, and the distribution of dendritic spines. We found that SNRPN regulated the expression level of Nr4a1, a critical nuclear receptor during neural development, in cultured primary cortical neurons. The abnormal spine development caused by SNRPN overexpression could be fully rescued by Nr4a1 co-expression. Importantly, we found that either knockdown of Nr4a1 or 3, 3'- Diindolylmethane (DIM), an Nr4a1 antagonist, were able to rescue the effects of SNRPN knockdown on neurite outgrowth of embryonic cortical neurons, providing the potential therapeutic methods for SNRPN deletion disorders. We thus concluded that maintaining the proper level of SNRPN is critical in cortical neurodevelopment. Finally, Nr4a1 may serve as a potential drug target for SNRPN-related neurodevelopmental disabilities, including Prader-Willi syndrome (PWS) and autism spectrum disorders (ASDs).
Assuntos
Transtorno Autístico/genética , Encéfalo/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Centrais de snRNP/genética , Transtorno Autístico/patologia , Autoantígenos/genética , Autoantígenos/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/patologia , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica/genética , Humanos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidoresRESUMO
Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Proteínas Virais/metabolismo , Animais , Chlorocebus aethiops , Dissulfetos/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Filogenia , Proteômica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Análise de Sequência de Proteína , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas ViroporinasRESUMO
The liver receptor homolog 1 (LRH-1) belongs to the Fushi tarazu factor 1 nuclear receptor subfamily, and its biological functions are just being unveiled. The molecular mechanism for the transcriptional regulation by LRH-1 is not clear yet. In this report, we use mutagenesis and reporter gene assays to carry out a detailed analysis on the hinge region and the proximal ligand binding domain (LBD) of human (h) LRH-1 that possess important regulatory functions. Our results indicate that helix 1 of the LBD is essential for the activity of hLRH-1 and that the steroid receptor coactivator (SRC)-1 interacts directly with the LBD of hLRH-1 and significantly potentiates the transcriptional activity of hLRH-1. Cotransfection assays demonstrate that overexpressed SRC-1 potentiates hLRH-1 mediated activation of the cholesterol 7-alpha-hydroxylase promoter and increases the transcription of the endogenous cholesterol 7-alpha-hydroxylase in Huh7 cells. The interaction between SRC-1 and hLRH-1 assumes a unique pattern that involves primarily a region containing the glutamine-rich domain of SRC-1, and helix 1 and activation function-2 of hLRH-1 LBD. Mutagenesis and molecular modeling studies indicate that, similar to mouse LRH-1, the coactivator-binding cleft of hLRH-1 LBD is not optimized. An interaction between helix 1 of hLRH-1 LBD and a region containing the glutamine-rich domain of SRC-1 can provide an additional stabilizing force and enhances the recruitment of SRC-1. Similar interaction is observed between hLRH-1 and SRC-2/transcriptional intermediary factor 2 or SRC-3/acetyltransferase. Moreover, transcriptional intermediary factor 2 and acetyltransferase also potentiate the transcriptional activity of hLRH-1, suggesting a functional redundancy among SRC family members. These findings collectively demonstrate an important functional role of helix 1 in cofactor recruitment and reveal a novel molecular mechanism of transcriptional regulation and cofactor recruitment mediated by hLRH-1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Colesterol 7-alfa-Hidroxilase/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Coativador 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear , Regiões Promotoras Genéticas/genética , Ligação Proteica , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Alinhamento de Sequência , Fatores de Transcrição/genéticaRESUMO
Human hepatitis B virus enhancer II B1 binding factor (hB1F also known as NR5A2, LRH-1, FTF or CPF) is an orphan nuclear receptor and belongs to the fushi tarazu factor I (FTZ-F1) subfamily. It plays important roles in the transcriptional regulation of a number of genes involved in bile acid biosynthesis pathway, hepatitis B virus (HBV) replication and liver specific regulatory network. Like other nuclear receptors, hB1F is composed of modular functional domains. We characterized a domain in its hinge region that imposes a strong repression on the transcriptional activity of hB1F, which is important for the function of hB1F on regulating the activity of HBV enhancer II/core promoter. Mutations of the core residues in this domain abrogate the repression. Bioinformatic analysis reveals that the amino acid sequence of this region is highly conserved only among members of the FTZ-F1 subfamily. The repression is observed in five cell lines tested, while the degree of the repression varies greatly, which does not parallel with the expression level of the DEAD box protein of 130 kD (DP103), a potential interacting protein of a homologous domain in the steroidogenic factor 1 (SF-1). Moreover, the repression is not affected by the silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) and steroid receptor coactivator 1 (SRC-1). Collectively, these data suggest a novel regulatory mechanism for the transcriptional activity of hB1F.