RESUMO
Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.
Assuntos
Íntrons , Mutação , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Alelos , Processamento Alternativo , Substituição de Aminoácidos , Pré-Escolar , Ativação Enzimática , Éxons , Fibroblastos , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Imagem Molecular , Síndrome Oculocerebrorrenal/diagnóstico , FenótipoRESUMO
A number of solute carrier (SLC) proteins are subject to changes in expression and activity during carcinogenesis. Whether these changes play a role in carcinogenesis is unclear, except for some nutrients and ion carriers whose deregulation ensures the necessary reprogramming of energy metabolism in cancer cells. In this study, we investigated the functional role in tumor progression of the sodium/iodide symporter (NIS; aka SLC5A5), which is upregulated and mislocalized in many human carcinomas. Notably, we found that NIS enhanced cell migration and invasion without ion transport being involved. These functions were mediated by NIS binding to leukemia-associated RhoA guanine exchange factor, a Rho guanine exchange factor that activates the small GTPase RhoA. Sequestering NIS in intracellular organelles or impairing its targeting to the cell surface (as observed in many cancers) led to a further increase in cell motility and invasiveness. In sum, our results established NIS as a carrier protein that interacts with a major cell signaling hub to facilitate tumor cell locomotion and invasion.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Invasividade Neoplásica/patologia , Transdução de Sinais/fisiologia , Simportadores/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução Genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.
Assuntos
Relógios Circadianos/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Hipocampo/citologia , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Análise de Variância , Animais , Bicuculina/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Chlorocebus aethiops , Relógios Circadianos/genética , Inibidores de Cisteína Proteinase/farmacologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Dendritos/metabolismo , Interações Medicamentosas , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Mutação/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Quinoxalinas/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Transfecção/métodos , Técnicas do Sistema de Duplo-Híbrido , Valina/análogos & derivados , Valina/farmacologiaRESUMO
MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56epsilon, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.