RESUMEN
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.
Asunto(s)
Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Conducta Social , Animales , Conducta Animal , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , SinapsisRESUMEN
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.
Asunto(s)
Trastorno del Espectro Autista , Edición Génica , Animales , Ratones , Masculino , Trastorno del Espectro Autista/genética , Encéfalo , Mutación/genética , Factores de Transcripción MEF2/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Línea Celular , Histona Desacetilasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Proteínas Represoras/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
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.
Asunto(s)
Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Virales/metabolismo , Animales , Chlorocebus aethiops , Disulfuros/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Filogenia , Proteómica , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Análisis de Secuencia de Proteína , Glicoproteína de la Espiga del Coronavirus , Células Vero , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas ViroporinasRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histona Acetiltransferasas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Coactivador 1 de Receptor Nuclear , Coactivador 2 del Receptor Nuclear , Regiones Promotoras Genéticas/genética , Unión Proteica , Conformación Proteica , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/genética , Alineación de Secuencia , Factores de Transcripción/genéticaRESUMEN
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.