RESUMEN
Wnts comprise a family of lipid-modified, secreted signaling proteins that control embryogenesis, as well as tissue homeostasis in adults. Post-translational attachment of palmitoleate (C16:1) to a conserved Ser in Wnt proteins is catalyzed by Porcupine (Porcn), a member of the membrane bound O-acyltransferase (MBOAT) family, and is required for Wnt secretion and signaling. Moreover, genetic alterations in the PORCN gene lead to focal dermal hypoplasia, an X-linked developmental disorder. Despite its physiological importance, the biochemical mechanism governing Wnt acylation by Porcn is poorly understood. Here, we use a cell-based fatty acylation assay that is a direct readout of Porcn acyltransferase activity to perform structure-function analysis of highly conserved residues in Porcn and Wnt3a. In total, 16-point mutations in Porcn and 13 mutations in Wnt3a were generated and analyzed. We identified key residues within Porcn required for enzymatic activity, stability, and Wnt3a binding and mapped these active site residues to predicted transmembrane domain 9. Analysis of focal dermal hypoplasia-associated mutations in Porcn revealed that loss of enzymatic activity arises from altered stability. A consensus sequence within Wnt3a was identified (CXCHGXSXXCXXKXC) that contains residues that mediate Porcn binding, fatty acid transfer, and Wnt signaling. We also showed that Ser or Thr, but not Cys, can serve as a fatty acylation site in Wnt, establishing Porcn as an O-acyltransferase. This analysis sheds light into the mechanism by which Porcn transfers fatty acids to Wnt proteins and provides insight into the mechanisms of fatty acid transfer by MBOAT family members.
Asunto(s)
Aciltransferasas/metabolismo , Dominio Catalítico , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Proteína Wnt3A/metabolismo , Acilación , Aciltransferasas/química , Aciltransferasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Estabilidad de Enzimas , Ácidos Grasos Monoinsaturados/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Proteína Wnt3A/química , Proteína Wnt3A/genéticaRESUMEN
Inhibition of Sonic hedgehog (Shh) signaling is of great clinical interest. Here we exploit Hedgehog acyltransferase (Hhat)-mediated Shh palmitoylation, a modification critical for Shh signaling, as a new target for Shh pathway inhibition. A target-oriented high-throughput screen was used to identify small-molecule inhibitors of Hhat. In cells, these Hhat inhibitors specifically block Shh palmitoylation and inhibit autocrine and paracrine Shh signaling.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Inhibidores Enzimáticos/química , Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Lipoilación , Luciferasas , Ratones , Puercoespines , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , TransfecciónRESUMEN
Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
Asunto(s)
Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Transducina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Sistema Libre de Células , Humanos , Ratones , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transducina/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMEN
Wnt proteins contain palmitoleic acid, an unusual lipid modification. Production of an active Wnt signal requires the acyltransferase Porcupine and depends on the attachment of palmitoleic acid to Wnt. The source of this monounsaturated fatty acid has not been identified, and it is not known how Porcupine recognizes its substrate and whether desaturation occurs before or after fatty acid transfer to Wnt. Here, we show that stearoyl desaturase (SCD) generates a monounsaturated fatty acid substrate that is then transferred by Porcupine to Wnt. Treatment of cells with SCD inhibitors blocked incorporation of palmitate analogs into Wnt3a and Wnt5a and reduced Wnt secretion as well as autocrine and paracrine Wnt signaling. The SCD inhibitor effects were rescued by exogenous addition of monounsaturated fatty acids. We propose that SCD is a key molecular player responsible for Wnt biogenesis and processing and that SCD inhibition provides an alternative mechanism for blocking Wnt pathway activation.