RESUMEN
Sonic hedgehog (Shh) signaling is a key component of embryonic development and is a driving force in several cancers. Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase family of enzymes, catalyzes the attachment of palmitate to the N-terminal cysteine of Shh, a posttranslation modification critical for Shh signaling. The activity of Hhat has been assayed in cells and in vitro, and cryo-EM structures of Hhat have been reported, yet several unanswered questions remain regarding the enzyme's reaction mechanism, substrate specificity, and the impact of the latter on Shh signaling. Here, we present an in vitro acylation assay with purified Hhat that directly monitors attachment of a fluorescently tagged fatty acyl chain to Shh. Our kinetic analyses revealed that the reaction catalyzed by Hhat proceeds through a random sequential mechanism. We also determined that Hhat can utilize multiple fatty acyl-CoA substrates for fatty acid transfer to Shh, with comparable affinities and turnover rates for myristoyl-CoA, palmitoyl-CoA, palmitoleoyl-CoA, and oleoyl-CoA. Furthermore, we investigated the functional consequence of differential fatty acylation of Shh in a luciferase-based Shh reporter system. We found that the potency of the signaling response in cells was higher for Shh acylated with saturated fatty acids compared to monounsaturated fatty acids. These findings demonstrate that Hhat can attach fatty acids other than palmitate to Shh and suggest that heterogeneous fatty acylation has the potential to impact Shh signaling in the developing embryo and/or cancer cells.
Asunto(s)
Proteínas Hedgehog , Lipoilación , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Lipoilación/fisiología , Aciltransferasas/metabolismo , Palmitatos , Coenzima ARESUMEN
Wnt proteins are a family of secreted signaling proteins that play key roles in regulating cell proliferation in both embryonic and adult tissues. Production of active Wnt depends on attachment of palmitoleate, a monounsaturated fatty acid, to a conserved serine by the acyltransferase Porcupine (PORCN). Studies of PORCN activity relied on cell-based fatty acylation and signaling assays as no direct enzyme assay had yet been developed. Here, we present the first in vitro assay that accurately recapitulates PORCN-mediated fatty acylation of a Wnt substrate. The critical feature is the use of a double disulfide-bonded Wnt peptide that mimics the two-dimensional structure surrounding the Wnt acylation site. PORCN-mediated Wnt acylation was abolished when the Wnt peptide was treated with DTT, and did not occur with a linear (non-disulfide-bonded) peptide, or when the double disulfide-bonded Wnt peptide contained Ala substituted for the Ser acylation site. We exploited this in vitro Wnt acylation assay to provide direct evidence that the small molecule LGK974, which is in clinical trials for managing Wnt-driven tumors, is a bona fide PORCN inhibitor whose IC50 for inhibition of Wnt fatty acylation in vitro closely matches that for inhibition of Wnt signaling. Side-by-side comparison of PORCN and Hedgehog acyltransferase (HHAT), two enzymes that attach 16-carbon fatty acids to secreted proteins, revealed that neither enzyme will accept the other's fatty acyl-CoA or peptide substrates. These findings illustrate the unique enzyme-substrate selectivity exhibited by members of the membrane-bound O-acyl transferase family.
Asunto(s)
Aciltransferasas/metabolismo , Hipoplasia Dérmica Focal/genética , Proteínas de la Membrana/metabolismo , Mutación Puntual , Procesamiento Proteico-Postraduccional , Proteína Wnt3A/metabolismo , Acilación/efectos de los fármacos , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/química , Aciltransferasas/genética , Sustitución de Aminoácidos , Animales , Cistina/química , Cistina/metabolismo , Inhibidores Enzimáticos/farmacología , Hipoplasia Dérmica Focal/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt3A/químicaRESUMEN
Post-translational modification of proteins by attachment of palmitate serves as a mechanism to regulate protein localization and function in both normal and malignant cells. Given the essential role that palmitoylation plays in cancer cell signaling, approaches that target palmitoylated proteins and palmitoyl acyltransferases (PATs) have the potential for therapeutic intervention in cancer. Highlighted here are recent advances in understanding the importance of protein palmitoylation in tumorigenic pathways. A new study has uncovered palmitoylation sites within the epidermal growth factor receptor that regulate receptor trafficking, signaling and sensitivity to tyrosine kinase inhibitors. Global data analysis from nearly 150 cancer studies reveals genomic alterations in several PATs that may account for their ability to function as tumor suppressors or oncogenes. Selective inhibitors have recently been developed that target hedgehog acyltransferase (Hhat) and Porcupine (Porcn), the acyltransferases that modify hedgehog and Wnt proteins, respectively. These inhibitors, coupled with targeted knockdown of Hhat and Porcn, reveal the essential functions of fatty acylation of secreted morphogens in a wide variety of human tumors.
Asunto(s)
Aciltransferasas/metabolismo , Mutación , Neoplasias/genética , Palmitatos/metabolismo , Aciltransferasas/genética , Animales , Receptores ErbB/química , Predisposición Genética a la Enfermedad , Proteínas Hedgehog/metabolismo , Humanos , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/metabolismoRESUMEN
The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.
Asunto(s)
Aciltransferasas/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Proteínas Hedgehog/metabolismo , Lipoilación/genética , Mutación Missense , Transducción de Señal/genética , Aciltransferasas/química , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Homocigoto , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Testículo/embriologíaRESUMEN
Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Hedgehog/metabolismo , Palmitatos/metabolismo , Animales , Células COS , Catálisis , Chlorocebus aethiops , Biología Computacional , Retículo Endoplásmico/metabolismo , Epítopos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lipoilación , Microscopía Fluorescente , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
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
BACKGROUND: Hedgehog acyltransferase (Hhat) catalyzes the transfer of the fatty acid palmitate onto Sonic Hedgehog (Shh), a modification that is essential for Shh signaling activity. The Shh signaling pathway has been implicated in the progression of breast cancer. METHODS: To determine the functional significance of Hhat expression in breast cancer, we used a panel of breast cancer cell lines that included estrogen receptor (ER) positive, HER2 amplified, triple negative, and tamoxifen resistant cells. We monitored both anchorage dependent and independent proliferation of these cells following depletion of Hhat with lentiviral shRNA and inhibition of Hhat activity with RU-SKI 43, a small molecule inhibitor of Hhat. RESULTS: Depletion of Hhat decreased anchorage-dependent and anchorage-independent proliferation of ER positive, but not triple negative, breast cancer cells. Treatment with RU-SKI 43 also reduced ER positive cell proliferation, whereas a structurally related, inactive compound had no effect. Overexpression of Hhat in ER positive cells not only rescued the growth defect in the presence of RU-SKI 43 but also resulted in increased cell proliferation in the absence of drug. Furthermore, depletion or inhibition of Hhat reduced proliferation of HER2 amplified as well as tamoxifen resistant cells. Inhibition of Smoothened had no effect on proliferation, indicating that canonical Shh signaling was not operative. Moreover, Hhat regulated the proliferation of both Shh responsive and non-responsive ER positive cells, suggesting a Shh independent function for Hhat. CONCLUSIONS: These data suggest that Hhat plays a critical role in ER positive, HER2 amplified, and hormone resistant breast cancer proliferation and highlights the potential promise of Hhat inhibitors for therapeutic benefit in breast cancer.
Asunto(s)
Aciltransferasas/metabolismo , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/enzimología , Receptor alfa de Estrógeno/metabolismo , Receptor ErbB-2/genética , Tamoxifeno/farmacología , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/genética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Amplificación de Genes , Proteínas Hedgehog/metabolismo , Humanos , Lapatinib , Transporte de Proteínas , Quinazolinas/farmacología , Transducción de SeñalRESUMEN
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
Sonic Hedgehog (Shh) is a secreted morphogen that regulates embryonic development. After removal of the signal peptide, Shh is processed to the mature, active form through autocleavage and a series of lipid modifications, including the attachment of palmitate. Covalent attachment of palmitate to the N-terminal cysteine of Shh is catalyzed by Hedgehog acyltransferase (Hhat) and is critical for proper signaling. The sequences within Shh that are responsible for palmitoylation by Hhat are not known. Here we show that the first six amino acids of mature Shh (CGPGRG) are sufficient for Hhat-mediated palmitoylation. Alanine scanning mutagenesis was used to determine the role of each amino acid and the positional sequence requirement in a cell-based Shh palmitoylation assay. Mutation of residues in the GPGR sequence to Ala had no effect on palmitoylation, provided that a positively charged residue was present within the first seven residues. The N-terminal position exhibited a strong but not exclusive requirement for Cys. Constructs with an N-terminal Ala were not palmitoylated. However, an N-terminal Ser served as a substrate for Hhat, but not the Drosophila melanogaster ortholog Rasp, highlighting a critical difference between the mammalian and fly enzymes. These findings define residues and regions within Shh that are necessary for its recognition as a substrate for Hhat-mediated palmitoylation. Finally, we report the results of a bioinformatics screen to identify other potential Hhat substrates encoded in the human genome.
Asunto(s)
Aciltransferasas/metabolismo , Aminoácidos/metabolismo , Proteínas de Drosophila/química , Proteínas Hedgehog/química , Lipoilación , Secuencia de Aminoácidos , Animales , Biología Computacional , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Señales de Clasificación de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Several assays have been developed to monitor the in vitro catalytic activity of Hedgehog acyltransferase (Hhat), an enzyme critical to the Hedgehog signaling pathway in cells. However, the majority of these previously reported assays involve radioactive fatty acyl donor substrates, multiple steps to achieve product readout, or specialized equipment. To increase safety, efficiency, and convenience, we developed a direct, fluorescent in vitro assay to monitor Hhat activity. Our assay utilizes purified Hhat, a fluorescently labeled fatty acyl-CoA donor substrate, and a Sonic hedgehog (Shh) peptide recipient substrate sufficient for fatty acylation. The protocol is a straightforward process that yields direct readout of fatty acylated Shh peptide via fluorescence detection of the transferred fatty acyl group. This protocol was validated in: J Biol Chem (2022), DOI: 10.1016/j.jbc.2022.102422 Graphical abstract Graphical abstract adapted from Schonbrun and Resh (2022).
RESUMEN
Lipid modifications such as palmitoylation or myristoylation target intracellular proteins to cell membranes. Secreted ligands of the Hedgehog and Wnt families are also palmitoylated; this modification, which requires the related transmembrane acyltransferases Rasp and Porcupine, can enhance their secretion, transport, or activity. We show here that rasp is also essential for the developmental functions of Spitz, a ligand for the Drosophila epidermal growth factor receptor (EGFR). In cultured cells, Rasp promotes palmitate addition to the N-terminal cysteine residue of Spitz, and this cysteine is required for Spitz activity in vivo. Palmitoylation reduces Spitz secretion and enhances its plasma membrane association, but does not alter its ability to activate the EGFR in vitro. In vivo, overexpressed unpalmitoylated Spitz has an increased range of action but reduced activity. These data suggest a role for palmitoylation in restricting Spitz diffusion, allowing its local concentration to reach the threshold required for biological function.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Drosophila/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas de la Membrana/metabolismo , Aciltransferasas/genética , Animales , Secuencia de Bases , Transporte Biológico Activo , Línea Celular , Membrana Celular/metabolismo , Cisteína/química , ADN/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Femenino , Genes de Insecto , Técnicas In Vitro , Ligandos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Ácido Palmítico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by approximately 98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2(-/-) mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.
Asunto(s)
Anexina A2/fisiología , VIH-1/fisiología , VIH-1/patogenicidad , Animales , Anexina A2/antagonistas & inhibidores , Anexina A2/deficiencia , Anexina A2/genética , Secuencia de Bases , Células COS , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/fisiología , Humanos , Células Jurkat , Macrófagos/fisiología , Macrófagos/virología , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Proteínas S100/fisiología , Virión/fisiología , Virulencia/fisiología , Ensamble de Virus/fisiología , Liberación del Virus/fisiología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Fatty acylation is a widespread form of protein modification that occurs on specific intracellular and secreted proteins. Beyond increasing hydrophobicity and the affinity of the modified protein for lipid bilayers, covalent attachment of a fatty acid exerts effects on protein localization, inter- and intramolecular interactions and signal transduction. As such, research into protein fatty acylation has been embraced by an extensive community of biologists. This special issue highlights advances at the forefront of the field, by focusing on two families of enzymes that catalyse post-translational protein fatty acylation, zDHHC palmitoyl acyltransferases and membrane-bound O-acyl transferases, and signalling pathways regulated by their fatty acylated protein substrates. The collected contributions catalogue the tremendous progress that has been made in enzyme and substrate identification. In addition, articles in this special issue provide insights into the pivotal functions of fatty acylated proteins in immune cell, insulin and EGF receptor-mediated signalling pathways. As selective inhibitors of protein fatty acyltransferases are generated, the future holds great promise for therapeutic targeting of fatty acyltransferases that play key roles in human disease.
Asunto(s)
Ácidos Grasos/química , Proteínas de la Membrana/química , Procesamiento Proteico-Postraduccional , Acilación , Animales , Humanos , Transducción de Señal , Especificidad por SustratoRESUMEN
Hedgehog acyltransferase (Hhat), a member of the membrane-bound O-acyltransferase (MBOAT) family, catalyses the covalent attachment of palmitate to the N-terminus of Hedgehog proteins. Palmitoylation is a post-translational modification essential for Hedgehog signalling. This review explores the mechanisms involved in Hhat acyltransferase enzymatic activity, similarities and differences between Hhat and other MBOAT enzymes, and the role of palmitoylation in Hedgehog signalling. In vitro and cell-based assays for Hhat activity have been developed, and residues within Hhat and Hedgehog essential for palmitoylation have been identified. In cells, Hhat promotes the transfer of palmitoyl-CoA from the cytoplasmic to the luminal side of the endoplasmic reticulum membrane, where Shh palmitoylation occurs. Palmitoylation is required for efficient delivery of secreted Hedgehog to its receptor Patched1, as well as for the deactivation of Patched1, which initiates the downstream Hedgehog signalling pathway. While Hhat loss is lethal during embryogenesis, mutations in Hhat have been linked to disease states or abnormalities in mice and humans. In adults, aberrant re-expression of Hedgehog ligands promotes tumorigenesis in an Hhat-dependent manner in a variety of different cancers, including pancreatic, breast and lung. Targeting hedgehog palmitoylation by inhibition of Hhat is thus a promising, potential intervention in human disease.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Hedgehog/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Humanos , Lipoilación , Neoplasias/metabolismo , Transducción de SeñalRESUMEN
Retroviral Gag proteins are synthesized as soluble, myristoylated precursors that traffic to the plasma membrane and promote viral particle production. The intracellular transport of human immunodeficiency virus type 1 (HIV-1) Gag to the plasma membrane remains poorly understood, and cellular motor proteins responsible for Gag movement are not known. Here we show that disrupting the function of KIF4, a kinesin family member, slowed temporal progression of Gag through its trafficking intermediates and inhibited virus-like particle production. Knockdown of KIF4 also led to increased Gag degradation, resulting in reduced intracellular Gag protein levels; this phenotype was rescued by reintroduction of KIF4. When KIF4 function was blocked, Gag transiently accumulated in discrete, perinuclear, nonendocytic clusters that colocalized with endogenous KIF4, with Ubc9, an E2 SUMO-1 conjugating enzyme, and with SUMO. These studies identify a novel transit station through which Gag traffics en route to particle assembly and highlight the importance of KIF4 in regulating HIV-1 Gag trafficking and stability.
Asunto(s)
VIH-1/metabolismo , Cinesinas/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citoplasma/metabolismo , VIH-1/genética , Humanos , Cinesinas/genética , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.
Asunto(s)
Compartimento Celular/fisiología , Integrina alfa6beta4/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Microdominios de Membrana/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Ácido Palmítico/metabolismo , Ratas , Transducción de Señal/fisiología , Familia-src Quinasas/metabolismoRESUMEN
c-Src is a non-receptor tyrosine kinase that associates with both the plasma membrane and endosomal compartments. In many human cancers, especially breast cancer, c-Src and the EGF receptor (EGFR) are overexpressed. Dual overexpression of c-Src and EGFR correlates with a Src-dependent increase in activation of EGFR, and synergism between these two tyrosine kinases increases the mitogenic activity of EGFR. Despite extensive studies of the functional interaction between c-Src and EGFR, little is known about the interactions in the trafficking pathways for the two proteins and how that influences signaling. Given the synergism between c-Src and EGFR, and the finding that EGFR is internalized and can signal from endosomes, we hypothesized that c-Src and EGFR traffic together through the endocytic pathway. Here we use a regulatable c-SrcGFP fusion protein that is a bona fide marker for c-Src to show that c-Src undergoes constitutive macropinocytosis from the plasma membrane into endocytic compartments. The movement of c-Src was dependent on its tyrosine kinase activity. Stimulation of cells with EGF revealed that c-Src traffics into the cell with activated EGFR and that c-Src expression and kinase activity prolongs EGFR activation. Surprisingly, even in the absence of EGF addition, c-Src expression induced activation of EGFR and of EGFR-mediated downstream signaling targets ERK and Shc. These data suggest that the synergy between c-Src and EGFR also occurs as these two kinases traffic together, and that their co-localization promotes EGFR-mediated signaling.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Células COS , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Pollos , Chlorocebus aethiops , Endosomas/efectos de los fármacos , Endosomas/enzimología , Activación Enzimática/efectos de los fármacos , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/enzimología , Ligandos , Ratones , Células 3T3 NIH , Pinocitosis/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers. It is not known how palmitoyl-CoA crosses the ER membrane to access the active site of Hhat. Here, we use fluorescent and radiolabeled palmitoyl-CoA probes to demonstrate that Hhat promotes the uptake of palmitoyl-CoA across the ER membrane in microsomes and semi-intact cells. Reconstitution of purified Hhat into liposomes provided further evidence that palmitoyl-CoA uptake activity is an intrinsic property of Hhat. Palmitoyl-CoA uptake was regulated by and could be uncoupled from Hhat enzymatic activity, implying that Hhat serves a dual function as a palmitoyl acyltransferase and a conduit to supply palmitoyl-CoA to the luminal side of the ER.
Asunto(s)
Aciltransferasas/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Hedgehog/metabolismo , Microsomas/metabolismo , Palmitoil Coenzima A/metabolismo , Procesamiento Proteico-Postraduccional , Aciltransferasas/genética , Animales , Transporte Biológico , Células COS , Línea Celular , Chlorocebus aethiops , Retículo Endoplásmico/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Liposomas/metabolismo , Liposomas/ultraestructura , Lipoilación , Ratones , Microsomas/ultraestructura , Transducción de Señal , Coloración y Etiquetado/métodosRESUMEN
Hedgehog and Wnt proteins are modified by covalent attachment of the fatty acids palmitate and palmitoleate, respectively. These lipid modifications are essential for Hedgehog and Wnt protein signaling activities and are catalyzed by related, but distinct fatty acyltransferases: Hedgehog acyltransferase (Hedgehog) and Porcupine (Wnt). In this chapter, we provide detailed methods to directly monitor Hedgehog and Wnt protein fatty acylation in vitro. Palmitoylation of Sonic hedgehog (Shh), a representative Hedgehog family member, is assayed using purified Hedgehog acyltransferase (Hhat) or Hhat-enriched membranes, a recombinant 19 kDa Shh protein or C-terminally biotinylated Shh 10-mer peptide, and 125I-iodopalmitoyl CoA as the donor fatty acyl CoA substrate. The radiolabeled reaction products are quantified by SDS-PAGE and phosphorimaging or by γ-counting. To assay Wnt acylation, the reaction consists of a biotinylated, double disulfide-bonded Wnt peptide containing the sequence surrounding the Wnt3a acylation site, [125I] iodo-cis-9-pentadecenoyl CoA, and Porcupine-enriched membranes. Radiolabeled, biotinylated Wnt3a peptide is captured on streptavidin coated beads and the reaction product is quantified by γ-counting.
Asunto(s)
Aciltransferasas/química , Proteínas Hedgehog/química , Proteínas de la Membrana/química , Palmitoil Coenzima A/química , Procesamiento Proteico-Postraduccional , Proteínas Wnt/química , Acilación , Aciltransferasas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Radioisótopos de Yodo/química , Proteínas de la Membrana/metabolismo , Membranas Artificiales , Palmitoil Coenzima A/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.