RESUMO
Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.
Assuntos
Alelos , Encefalopatias/genética , Mutação/genética , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Idade de Início , Animais , Mapeamento Encefálico , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Criança , Pré-Escolar , Neurônios Colinérgicos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Epilepsia/genética , Exoma/genética , Feminino , Fibroblastos , Genes Recessivos/genética , Humanos , Deficiência Intelectual/genética , Imageamento por Ressonância Magnética , Masculino , Microcefalia/genética , Transtornos dos Movimentos , Proteínas/genética , Transmissão Sináptica/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/deficiência , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.
Assuntos
Ciclo do Ácido Cítrico/genética , DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Proteínas de Ligação a RNA/metabolismo , Trifosfato de Adenosina/biossíntese , Sistemas CRISPR-Cas , Ciclo do Ácido Cítrico/efeitos dos fármacos , Dano ao DNA , DNA Mitocondrial/metabolismo , Etídio/toxicidade , Deleção de Genes , Células HeLa , Humanos , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Imagem Óptica , Oxirredução , Fosforilação Oxidativa/efeitos dos fármacos , Palmitoilcarnitina/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
BACKGROUND: Although estrogen receptor α (ERα) acts primarily as a transcription factor, it can also elicit membrane-initiated steroid signaling. Pharmacological tools and transgenic mouse models previously highlighted the key role of ERα membrane-initiated steroid signaling in 2 actions of estrogens in the endothelium: increase in NO production and acceleration of reendothelialization. METHODS AND RESULTS: Using mice with ERα mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ERα plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear versus membrane-initiated steroid signaling in the arterial protection conferred by ERα. ERaC451A mice were fully responsive to estrogens to prevent atheroma and angiotensin II-induced hypertension as well as to allow flow-mediated arteriolar remodeling. By contrast, ERαAF20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ERα with estetrol was able to prevent hypertension and to restore flow-mediated arteriolar remodeling. CONCLUSIONS: Altogether, these results reveal an unexpected prominent role of nuclear ERα in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ERα agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women.