RESUMO
The Hedgehog (Hh) pathway depends on primary cilia in vertebrates, but the signaling machinery within cilia remains incompletely defined. We report the identification of a complex between two ciliary proteins, EFCAB7 and IQCE, which positively regulates the Hh pathway. The EFCAB7-IQCE module anchors the EVC-EVC2 complex in a signaling microdomain at the base of cilia. EVC and EVC2 genes are mutated in Ellis van Creveld and Weyers syndromes, characterized by impaired Hh signaling in skeletal, cardiac, and orofacial tissues. EFCAB7 binds to a C-terminal disordered region in EVC2 that is deleted in Weyers patients. EFCAB7 depletion mimics the Weyers cellular phenotype-the mislocalization of EVC-EVC2 within cilia and impaired activation of the transcription factor GLI2. Evolutionary analysis suggests that emergence of these complexes might have been important for adaptation of an ancient organelle, the cilium, for an animal-specific signaling network.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cílios/fisiologia , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Cromatografia de Afinidade , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Células NIH 3T3 , Fenótipo , Filogenia , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor Smoothened , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Gli2 com Dedos de ZincoRESUMO
Anemia of inflammation (AI) is common in patients with infection, autoimmune diseases, cancer, and chronic kidney disease. Unless the underlying condition can be reversed, treatment options are limited to erythropoiesis-stimulating agents with or without intravenous iron therapy, modalities that are not always effective and can cause serious adverse effects. Hepcidin, the iron regulatory hormone, has been identified as a pathogenic factor in the development of AI. To explore new therapeutic options for AI and other iron-related disorders caused by hepcidin excess, we developed a cell-based screen to identify hepcidin antagonists. Of the 70,000 small molecules in the library, we identified 14 compounds that antagonized the hepcidin effect on ferroportin. One of these was fursultiamine, a Food and Drug Administration (FDA)-approved thiamine derivative. Fursultiamine directly interfered with hepcidin binding to its receptor, ferroportin, by blocking ferroportin C326 thiol residue essential for hepcidin binding. Consequently, fursultiamine prevented hepcidin-induced ferroportin ubiquitination, endocytosis, and degradation in vitro and allowed continuous cellular iron export despite the presence of hepcidin, with IC(50) in the submicromolar range. Thiamine, the fursultiamine metabolite, and benfotiamine, another thiamine derivative, did not interfere with the effect of hepcidin on ferroportin. Other FDA-approved thiol-reactive compounds were at least 1000-fold less potent than fursultiamine in antagonizing hepcidin. In vivo, fursultiamine did not reproducibly antagonize the effect of hepcidin on serum iron, likely because of its rapid conversion to inactive metabolites. Fursultiamine is a unique antagonist of hepcidin in vitro that could serve as a template for the development of drug candidates that inhibit the hepcidin-ferroportin interaction.
Assuntos
Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Endocitose/efeitos dos fármacos , Endocitose/genética , Fursultiamina/farmacologia , Células HEK293 , Hepcidinas , Humanos , Ferro/metabolismo , Distúrbios do Metabolismo do Ferro/tratamento farmacológico , Distúrbios do Metabolismo do Ferro/genética , Distúrbios do Metabolismo do Ferro/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética , Proteólise/efeitos dos fármacos , Receptores de LDL/genética , Receptores de LDL/metabolismo , Tiamina/genética , Tiamina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genéticaRESUMO
Ferroportin exports iron into plasma from absorptive enterocytes, erythrophagocytosing macrophages, and hepatic stores. The hormone hepcidin controls cellular iron export and plasma iron concentrations by binding to ferroportin and causing its internalization and degradation. We explored the mechanism of hepcidin-induced endocytosis of ferroportin, the key molecular event in systemic iron homeostasis. Hepcidin binding caused rapid ubiquitination of ferroportin in cell lines overexpressing ferroportin and in murine bone marrow-derived macrophages. No hepcidin-dependent ubiquitination was observed in C326S ferroportin mutant which does not bind hepcidin. Substitutions of lysines between residues 229 and 269 in the third cytoplasmic loop of ferroportin prevented hepcidin-dependent ubiquitination and endocytosis of ferroportin, and promoted cellular iron export even in the presence of hepcidin. The human ferroportin mutation K240E, previously associated with clinical iron overload, caused hepcidin resistance in vitro by interfering with ferroportin ubiquitination. Our study demonstrates that ubiquitination is the functionally relevant signal for hepcidin-induced ferroportin endocytosis.