RESUMO
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.
Assuntos
Aciltransferases/metabolismo , Domínio Catalítico , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Wnt3A/metabolismo , Acilação , Aciltransferases/química , Aciltransferases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Estabilidade Enzimática , Ácidos Graxos Monoinsaturados/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Proteína Wnt3A/química , Proteína Wnt3A/genéticaRESUMO
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.
Assuntos
Aciltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/química , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Lipoilação , Luciferases , Camundongos , Porcos-Espinhos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , TransfecçãoRESUMO
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.
Assuntos
Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Transducina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Sistema Livre de Células , Humanos , Camundongos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Transducina/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
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.