RESUMEN
Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Disulfuros/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores Acoplados a Proteínas G/ultraestructura , Homología Estructural de Proteína , Relación Estructura-Actividad , Proteínas Wnt/química , Proteínas Wnt/aislamiento & purificación , Proteínas Wnt/ultraestructuraRESUMEN
Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post-translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured-cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC-1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild-type PANC-1 cells, but not those from Wnt5b-knockout PANC-1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b-associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco-2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco-2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b-associated exosomes from Caco-2/Wnt5b cells and inhibited Wnt5b-dependent cell proliferation. Exosomes secreted from Caco-2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner.
Asunto(s)
Movimiento Celular , Exosomas/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Comunicación Paracrina , Proteínas Wnt/metabolismo , Células A549 , Secuencia de Aminoácidos , Animales , Células CHO , Células CACO-2 , Proliferación Celular , Cricetulus , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Transducción de Señal , Proteínas Wnt/aislamiento & purificaciónRESUMEN
We describe here a technique for visualizing the lipidation status of Wnt proteins using azide-alkyne cycloaddition chemistry (click chemistry) and SDS-PAGE. This protocol incorporates in vivo labeling of a Wnt-IgG Fc fusion protein using an alkynylated palmitate probe but departs from a traditional approach by incorporating a secondary cycloaddition reaction performed on single-step purified Wnt protein immobilized on protein A resin. This approach mitigates experimental noise by decreasing the contribution of labeling from other palmitoylated proteins and by providing a robust method for normalizing labeling efficiency based on protein abundance.
Asunto(s)
Química Clic/métodos , Reacción de Cicloadición/métodos , Proteínas Wnt/aislamiento & purificación , Acilación , Alquinos/química , Catálisis , Cobre/química , Electroforesis en Gel de Poliacrilamida , Lipoilación , Proteínas Wnt/químicaRESUMEN
Wnt proteins are modified and inactivated by two extracellular enzymatic antagonists, Tiki and Notum. Tiki proteins act as membrane-tethered metalloproteases to cleave a fragment from the amino terminus of Wnt proteins. Notum is a Wnt deacylase that removes the lipid modification that is essential for Wnt activities. Here, we provide detailed procedures for preparing enzymatic active Tiki and Notum proteins and the in vitro enzymatic reactions. We also describe a metabolic labeling and click chemistry method for detection of Wnt protein acylation.
Asunto(s)
Química Clic/métodos , Biología Molecular/métodos , Proteínas Wnt/química , Acilación , Esterasas/química , Metaloproteasas/química , Procesamiento Proteico-Postraduccional/genética , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/aislamiento & purificación , Vía de Señalización WntRESUMEN
Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.
Asunto(s)
Exosomas/química , Vesículas Extracelulares/química , Biología Molecular/métodos , Proteínas Wnt/aislamiento & purificación , Animales , Retículo Endoplásmico/química , Aparato de Golgi/química , Humanos , Transducción de Señal , Proteínas Wnt/químicaRESUMEN
ß-catenin (CTNNB1) is a dual-function cell-cell adhesion/transcriptional co-activator protein and an essential transducer of canonical Wnt signals. Although a number of established techniques and reagents are available to quantify the nuclear signaling activity of ß-catenin (e.g., TCF-dependent reporter assays, nuclear accumulation of ß-catenin, and generation of N-terminally hypophosphorylated ß-catenin), there are cell-type and context-dependent limitations of these methods. Since the posttranscriptional stabilization of ß-catenin outside of the cadherin complex appears universally required for ß-catenin signaling, the following method allows for simple assessment of the cadherin-free fraction of ß-catenin in cells, using a GST-tagged form of ICAT (Inhibitor of ß-Catenin and Tcf) as an affinity matrix. This method is more sensitive and quantitative than immunofluorescence and may be useful in studies that implicate TCF-independent signaling events.
Asunto(s)
Núcleo Celular/química , Biología Molecular/métodos , Transcripción Genética , beta Catenina/aislamiento & purificación , Cadherinas/química , Cadherinas/genética , Adhesión Celular/genética , Proteínas Wnt/química , Proteínas Wnt/aislamiento & purificación , Vía de Señalización Wnt , beta Catenina/genéticaRESUMEN
In this chapter, we are presenting methods to monitor and quantify in vivo canonical Wnt signaling activities at single-cell resolution in zebrafish. Our technology is based on artificial enhancers, obtained by polymerization of TCF binding elements, cloned upstream to ubiquitous or tissue-specific promoters. The different promoter/enhancer combinations are used to drive fluorescent protein reporter constructs integrated in the zebrafish germline by microinjection of fertilized zebrafish eggs. Fish with a single integration site are selected by Mendelian analysis of fluorescent carriers, and heterozygous offspring are used to monitor and quantify canonical Wnt activities. Open source public domain software such as ImageJ/Fiji is used to calculate the integrated densities in the region of interest and compare the effect of experimental conditions on control and treated animals.
Asunto(s)
Técnicas Biosensibles/métodos , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Proteínas Wnt/genética , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Microinyecciones/métodos , Proteínas Wnt/aislamiento & purificación , Vía de Señalización Wnt/genética , Pez Cebra/genéticaRESUMEN
Functional and mechanistic studies of Wnt signaling have been severely hindered by the inaccessibility of bioactive proteins. To overcome this long-standing barrier, we engineered and characterized a panel of Chinese hamster ovary (CHO) cell lines with inducible transgenes encoding tagged and un-tagged human WNT1, WNT3A, WNT5A, WNT7A, WNT11, WNT16 or the soluble Wnt antagonist Fzd8CRD, all integrated into an identical genomic locus. Using a quantitative real-time bioluminescence assay, we show that cells expressing WNT1, 3A and 7A stimulate Wnt/beta-catenin reporter activity, while the other WNT expressing cell lines interfere with this activation. Additionally, in contrast to WNT3A, WNT1 only exhibits activity when cell-associated, and thus only signals to neighboring cells. The reporter assay also revealed a rapid decline of Wnt activity at 37°C, indicating that Wnt activity is highly labile. These engineered cell lines will reduce the cost of making and purifying Wnt proteins and serve as a continuous, reliable and regulatable source of Wnts to research laboratories around the world.
Asunto(s)
Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Células CHO , Cricetinae , Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Wnt/aislamiento & purificaciónRESUMEN
Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Matriz Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Animales , Línea Celular , Análisis por Conglomerados , Proteína 61 Rica en Cisteína/aislamiento & purificación , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteoma/aislamiento & purificación , Proteómica , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Wnt/aislamiento & purificación , Proteína Wnt-5aRESUMEN
Wnt proteins and their signaling cascades are involved in a wide variety of developmental processes, and deregulation of this pathway is frequently associated with tumorigenesis. Unlike many other growth factors, Wnts long eluded biochemical purification, in large part because of their hydrophobic nature, which is imparted by one or more lipid modifications. Here I describe a complete protocol that outlines the purification process for Wnt proteins. While this protocol has not been applied to all known Wnt proteins, it has been successfully applied to the purification of a large subset of Wnts, including the very divergent Wnt protein, Drosophila Wnt8 (Dwnt8 or WntD), indicating that this protocol is likely applicable to all Wnts.
Asunto(s)
Cromatografía/métodos , Transducción de Señal/fisiología , Proteínas Wnt/aislamiento & purificación , Animales , Línea Celular , Cromatografía/instrumentación , Ratones , Proteínas Wnt/metabolismo , Proteína Wnt3 , beta Catenina/metabolismoRESUMEN
Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.
Asunto(s)
Proteínas Wnt/biosíntesis , Proteínas Wnt/fisiología , Animales , Técnicas de Cultivo de Célula , Liposomas , Masculino , Lípidos de la Membrana/fisiología , Ratones , Ratones Endogámicos , Transducción de Señal , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/aislamiento & purificación , Proteína Wnt3 , Proteína Wnt3ARESUMEN
Plectin, a widespread intermediate filament-based cytolinker protein capable of interacting with a variety of cytoskeletal structures and plasma membrane-bound junctional complexes, serves essential functions in maintenance of cell and tissue cytoarchitecture. We have generated a mouse line bearing floxed plectin alleles and conditionally deleted plectin in stratified epithelia. This strategy enabled us to study the consequences of plectin deficiency in this particular type of tissues in the context of the whole organism without plectin loss affecting other tissues. Conditional knockout mice died early after birth, showing signs of starvation and growth retardation. Blistering was observed on their extremities and on the oral epithelium after initial nursing, impairing food uptake. Knockout epidermis was very fragile and showed focal epidermal barrier defects caused by the presence of small skin lesions. Stratification, proliferation and differentiation of knockout skin seemed unaffected by epidermis-restricted plectin deficiency. In an additionally generated mouse model, tamoxifen-induced Cre-ER(T)-mediated recombination led to mice with a mosaic plectin deletion pattern in adult epidermis, combined with microblister formation and epidermal barrier defects. Our study explains the early lethality of plectin-deficient mice and provides a model to ablate plectin in adult animals which could be used for developing gene or pharmacological therapies.
Asunto(s)
Epidermis/metabolismo , Queratina-5/metabolismo , Queratinocitos/patología , Plectina/genética , Plectina/metabolismo , Enfermedades de la Piel/patología , Animales , Vesícula/patología , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Distonina , Epidermis/patología , Marcación de Gen , Integrasas/genética , Integrasas/metabolismo , Queratina-15 , Queratina-5/genética , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Plectina/aislamiento & purificación , Enfermedades de la Piel/genética , Enfermedades de la Piel/metabolismo , Proteínas Wnt/aislamiento & purificación , Proteínas Wnt/metabolismo , Proteína Wnt3RESUMEN
The Wnt signal acts by binding to Frizzled receptors, with the subsequent activation of two different signal transduction cascades, the canonical and the non-canonical Wnt pathways, involved in cell growth, differentiation, migration and fate. The canonical pathway functions through the translocation of beta-catenin to the nucleus and the activation of TCF/LEF transcription factors; it plays an important role in developmental patterning and cell fate decisions during embryogenesis. The non-canonical Wnt pathway is responsible for the planar cell polarity process in invertebrates, and for the convergent-extension movements during vertebrate gastrulation. The final effect of the non-canonical Wnt pathway is the rearrangement of the cell cytoskeleton, through the activation of the subfamily of Ras-like small GTPases. In a recent report we described for the first time the isolation of a Wnt-related gene, Sd-Frizzled, from the most basal animal phylum, the Porifera. In the present study we report the isolation and phylogenetic characterization of several Wnt pathway-related genes from the sponge Suberites domuncula: Sd-TCF/LEF, Sd-GSK3, a recently discovered molecule with a putative function as a Wnt regulator (Sd-LZIC), the small Rho GTPases Sd-RhoA, Sd-Cdc42, and their effector Sd-mrlc. Also the isolation of a secreted frizzled related protein sFRP from another sponge species (Lubomirskia baicalensis) is reported.
Asunto(s)
Poríferos/genética , Proteínas Wnt/genética , Proteínas Wnt/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Células Cultivadas , Receptores Frizzled/química , Receptores Frizzled/genética , Receptores Frizzled/aislamiento & purificación , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Datos de Secuencia Molecular , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Filogenia , Poríferos/citología , Factores de Transcripción TCF/química , Factores de Transcripción TCF/genética , Proteínas Wnt/química , Proteína de Unión al GTP cdc42/química , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rhoA/química , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Wnt-5a is a representative ligand that activates a beta-catenin-independent pathway in Wnt signalling. In the present paper, the roles of the post-translational modifications in the actions of Wnt-5a were investigated. We found that Wnt-5a is modified with palmitate at Cys104 and glycans at Asn114, Asn120, Asn311 and Asn325. The palmitoylation was not essential for the secretion of Wnt-5a, but was necessary for its ability to suppress Wnt-3a-dependent T-cell factor transcriptional activity and to stimulate cell migration. Wnt-5a activated focal adhesion kinase and this activation also required palmitoylation. Wild-type Wnt-5a induced the internalization of Fz (Frizzled) 5, but a Wnt-5a mutant that lacks the palmitoylation site did not. Furthermore, the binding of Wnt-5a to the extracellular domain of Fz5 required palmitoylation of Wnt-5a. These results indicate that palmitoylation of Wnt-5a is important for the triggering of signalling at the cell surface level and, therefore, that the lipid-unmodified form of Wnt-5a cannot activate intracellular signal cascades. In contrast, glycosylation was necessary for the secretion of Wnt-5a, but not essential for the actions of Wnt-5a. Thus the post-translational palmitoylation and glycosylation of Wnt-5a are important for the actions and secretion of Wnt-5a.
Asunto(s)
Ácidos Grasos Monoinsaturados/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Movimiento Celular , Glicosilación , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/aislamiento & purificación , Alineación de Secuencia , Factores de Transcripción TCF/metabolismo , Proteína 2 Similar al Factor de Transcripción 7 , Proteínas Wnt/química , Proteínas Wnt/genética , Proteínas Wnt/aislamiento & purificación , Proteína Wnt-5aRESUMEN
The Wnts comprise a large class of secreted proteins that control essential developmental processes such as embryonic patterning, cell growth, migration, and differentiation. In the most well-understood "canonical" Wnt signaling pathway, Wnt binding to Frizzled receptors induces beta-catenin protein stabilization and entry into the nucleus, where it complexes with T-cell factor/lymphoid enhancer factor transcription factors to affect the transcription of target genes. In addition to the canonical pathway, evidence for several other Wnt signaling pathways has accumulated, in particular for Wnt5a, which has therefore been classified as a noncanonical Wnt family member. To study the alternative mechanisms by which Wnt proteins signal, we purified the Wnt5a protein to homogeneity. We find that purified Wnt5a inhibits Wnt3a protein-induced canonical Wnt signaling in a dose-dependent manner, not by influencing beta-catenin levels but by downregulating beta-catenin-induced reporter gene expression. The Wnt5a signal is mediated by the orphan tyrosine kinase Ror2, is pertussis toxin insensitive, and does not influence cellular calcium levels. We show that in addition to its inhibitory function, Wnt5a can also activate beta-catenin signaling in the presence of the appropriate Frizzled receptor, Frizzled 4. Thus, this study shows for the first time that a single Wnt ligand can initiate discrete signaling pathways through the activation of two distinct receptors. Based on these and additional observations, we propose a model wherein receptor context dictates Wnt signaling output. In this model, signaling by different Wnt family members is not intrinsically regulated by the Wnt proteins themselves but by receptor availability.