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1.
Commun Biol ; 7(1): 543, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38714795

RESUMO

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.


Assuntos
Proteínas Desgrenhadas , Fatores de Troca do Nucleotídeo Guanina , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Desgrenhadas/metabolismo , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/genética , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Domínios PDZ , Motivos de Aminoácidos , Via de Sinalização Wnt , Peptídeos/metabolismo , Peptídeos/química , Sítios de Ligação , Proteínas dos Microfilamentos , Peptídeos e Proteínas de Sinalização Intracelular
2.
J Virol ; 97(10): e0124123, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37772824

RESUMO

IMPORTANCE: CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/ß-catenin pathway as an important component of viral reactivation. We further define that pUL8 and ß-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with ß-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.


Assuntos
Antígenos CD34 , Citomegalovirus , Proteínas Desgrenhadas , Células-Tronco Hematopoéticas , Proteínas Virais , Ativação Viral , beta Catenina , Humanos , Antígenos CD34/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Citomegalovirus/genética , Citomegalovirus/fisiologia , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Domínios PDZ , Proteínas Virais/química , Proteínas Virais/metabolismo , Latência Viral/genética
3.
Biochem Biophys Res Commun ; 546: 21-28, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33561744

RESUMO

SF3B1, an essential component of the U2 snRNP, is frequently mutated in cancers. Cancer-associated SF3B1 mutation causes aberrant RNA splicing, mostly at 3' splice sites (3'ss). RNA splicing of DVL2, a regulator of Notch signaling, is affected by SF3B1 mutation. Here, we report that the mutated SF3B1 use an alternative branchpoint sequence (BPS) for the aberrant splicing of DVL2, which has a higher affinity to U2 snRNA than the BPS for the canonical splicing of DVL2. Swapping the position of the alternative BPS with the position of the canonical BPS decreased the aberrant splicing of DVL2, suggesting that the mutated SF3B1 prefers to use BPS with high affinity to U2 snRNA for splicing. Additionally, swapping the positions of two BPSs associated with the canonical splicing of DVL2 demonstrated that both the affinity to the U2 snRNA and the distance to the 3'ss are important to the selection of BPS. Importantly, the aberrant splicing of DVL2 does not require the canonical 3'ss and the canonical polypyrimidine tract, which reveals a novel type of aberrant splicing induced by SF3B1 mutation. These findings provide a more comprehensive understanding of the mechanisms underlying aberrant splicing induced by SF3B1 mutation in cancer.


Assuntos
Processamento Alternativo , Proteínas Desgrenhadas/genética , Mutação , Neoplasias/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Sequência de Bases , Proteínas Desgrenhadas/química , Humanos , Fosfoproteínas/química , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA/química , RNA Nuclear Pequeno/genética
4.
Biochem Biophys Res Commun ; 532(3): 406-413, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-32888647

RESUMO

The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of ß-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1ε-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple ΔGCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1ε, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1ε trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of ß-catenin transcriptional activity. Consistent with this, wild-type Daple promoted ß-catenin transcriptional activity, following dissociation of ß-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated ß-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1ε-mediated activation of Dvl in the canonical Wnt signaling pathway.


Assuntos
Caseína Quinase 1 épsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Proteínas de Transporte/metabolismo , Proteínas Desgrenhadas/química , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células L , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Fosforilação , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
5.
Sci Rep ; 9(1): 16257, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31700102

RESUMO

Dishevelled (DVL) proteins are central mediators of the Wnt signalling pathway and are versatile regulators of several cellular processes, yet little is known about their post-translational regulation. Acetylation is a reversible post-translational modification (PTM) which regulates the function of several non-histone proteins involved in tumorigenesis. Since we previously demonstrated that lysine deacetylase, SIRT-1, regulates DVL protein levels and its function, we reasoned that DVL could potentially be a substrate for SIRT-1 mediated deacetylation. To further examine the potential role of multiple families of lysine deacetylases in the post-translational regulation of DVL, we screened for novel acetylation sites using liquid chromatography mass-spectrometry (LC-MS/MS) analysis. Herein, we report 12 DVL-1 lysine residues that show differential acetylation in response to changes in oxygen tension and deacetylase inhibition in triple-negative breast cancer (TNBC). PTMs are well documented to influence protein activity, and cellular localization. We also identify that acetylation of two key lysine residues, K69 and K285, present on the DIX and PDZ domains respectively, promote nuclear over cytoplasmic localization of DVL-1, and influences its promoter binding and regulation of genes implicated in cancer. Collectively, these findings for the first time, uncover acetylation as a novel layer of regulation of DVL-1 proteins.


Assuntos
Núcleo Celular/metabolismo , Proteínas Desgrenhadas/metabolismo , Lisina/metabolismo , Regiões Promotoras Genéticas , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Aromatase/genética , Aromatase/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos Biológicos , Oxigênio/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Neoplasias de Mama Triplo Negativas/patologia
6.
Anal Chem ; 91(21): 13501-13507, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31571476

RESUMO

The Wnt pathway is dysregulated and activated in many human malignancies. More than 90% of colon cancers have variations in the Wnt pathway. Sulindac, a drug that targets protein Dvl of the Wnt/Dvl/ß-catenin pathway, which regulates cancer gene expression, has been reported to significantly reduce the incidence and the risk of death from colorectal cancer and other types of cancer. Herein, a dual functional compound (SLN) containing Sulindac and a linked fluorophore is first reported, combining the functions of lighting up colon cancer cells as a flare and inhibiting colon tumors as a drug. SLN can not only mark the Dvl protein in the Wnt pathway to recognize tumors layer by layer but also achieve effective inhibition of colon cancer, providing a promising reagent for chemotherapy and a fluorescent indicator for surgery during the removal the colon tumors in situ.


Assuntos
Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Neoplasias/diagnóstico por imagem , Sulindaco/farmacologia , Proteínas Wnt/metabolismo , Animais , Células COS , Chlorocebus aethiops , Neoplasias do Colo , Feminino , Corantes Fluorescentes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Neoplasias/patologia , Neoplasias Experimentais , Imagem Óptica , Proteínas Wnt/química , beta Catenina/genética , beta Catenina/metabolismo
7.
Development ; 145(23)2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30389855

RESUMO

MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that regulate gene expressions by binding to the 3' untranslated region of target mRNAs thereby silencing translation. Some miRNAs are key regulators of the Wnt signaling pathways, which impact developmental processes. This study investigates miRNA regulation of different isoforms of Dishevelled (Dvl/Dsh), which encode a key component in the Wnt signaling pathway. The sea urchin Dvl mRNA isoforms have similar spatial distribution in early development, but one isoform is distinctively expressed in the larval ciliary band. We demonstrated that Dvl isoforms are directly suppressed by miRNAs. By blocking miRNA suppression of Dvl isoforms, we observed dose-dependent defects in spicule length, patterning of the primary mesenchyme cells, gut morphology, and cilia. These defects likely result from increased Dvl protein levels, leading to perturbation of Wnt-dependent signaling pathways and additional Dvl-mediated processes. We further demonstrated that overexpression of Dvl isoforms recapitulated some of the Dvl miRNATP-induced phenotypes. Overall, our results indicate that miRNA suppression of Dvl isoforms plays an important role in ensuring proper development and function of primary mesenchyme cells and cilia.


Assuntos
Proteínas Desgrenhadas/metabolismo , Embrião não Mamífero/metabolismo , MicroRNAs/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/patologia , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/genética , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , MicroRNAs/genética , Morfolinos/farmacologia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ouriços-do-Mar/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética
8.
Cell Signal ; 47: 52-64, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29559363

RESUMO

The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity [1-3]. The Dsh gene (Dsh/Dvl, in Drosophila and vertebrates respectively) gained popularity when it was discovered that it plays a key role in segment polarity during early embryonic development in Drosophila [4]. Subsequently, the vertebrate homolog of Dishevelled genes were identified in Xenopus (Xdsh), mice (Dvl1, Dvl2, Dvl3), and in humans (DVL1, DVL2, DVL3) [5-10]. Dishevelled functions as a principal component of Wnt signaling pathway and governs several cellular processes including cell proliferation, survival, migration, differentiation, polarity and stem cell renewal. This review will revisit seminal discoveries and also summarize recent advances in characterizing the role of Dishevelled in both normal and pathophysiological settings.


Assuntos
Proteínas Desgrenhadas/metabolismo , Via de Sinalização Wnt , Animais , Proteínas Desgrenhadas/química , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Domínios Proteicos , Processamento de Proteína Pós-Traducional
9.
Cell Death Differ ; 25(8): 1426-1441, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29445127

RESUMO

Identification and characterization of functional molecular targets conferring stemness properties in hepatocellular carcinoma (HCC) offers crucial insights to overcome the major hurdles of tumor recurrence, metastasis and chemoresistance in clinical management. In the current study, we investigated the significance of Cripto-1 in contributing to HCC stemness. Cripto-1 was upregulated in the sorafenib-resistant clones derived from HCC cell lines and patient-derived xenograft that we previously developed, suggesting an association between Cripto-1 and stemness. By in vitro experiments, Cripto-1 fostered cell proliferation, migration, and invasion. It also enhanced self-renewal ability and conferred chemoresistance of HCC cells. Consistently, silencing of Cripto-1 suppressed in vivo tumorigenicity on serial transplantation. On the downstream signaling mechanism, expression of major components of Wnt/ß-catenin pathway ß-catenin, AXIN2, and C-MYC, accompanied by ß-catenin activity was reduced upon Cripto-1 knockdown. The suppressive effects on stemness properties with Cripto-1 knockdown in vitro and in vivo were partially rescued by forced expression of constitutively active ß-catenin. Further elucidation revealed the binding of Cripto-1 to Frizzled-7 (FZD7), low-density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled-3 (DVL3) of the Wnt/ß-catenin pathway and stabilized DVL3 protein. Analyses with clinical samples validated Cripto-1 overexpression in HCC tissues, as well as a positive correlation between Cripto-1 and AXIN2 expressions. High Cripto-1 level in tumor was associated with poorer disease-free survival of HCC patients. Taken together, Cripto-1 binds to FZD7/LRP6 and DVL3, stabilizes DVL3 expression and activates the Wnt/ß-catenin signaling cascade to confer stemness in HCC. Our study findings substantiated the role of Cripto-1 in determining stemness phenotypes of HCC and mechanistically in modulating the Wnt/ß-catenin signaling cascade, one of the most frequently deregulated pathways in liver cancer.


Assuntos
Proteínas Desgrenhadas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Axina/genética , Proteína Axina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteínas Desgrenhadas/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sorafenibe/farmacologia
10.
Cell Signal ; 38: 85-96, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28668722

RESUMO

Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both ß-catenin-dependent and ß-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.


Assuntos
Sequência Conservada , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Embrião não Mamífero/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Polimerização , Ligação Proteica , Transdução de Sinais , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Via de Sinalização Wnt , Xenopus laevis/embriologia
11.
Biochem Biophys Res Commun ; 485(3): 584-590, 2017 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-27932247

RESUMO

Dishevelled (Dvl) plays a crucial role in Wnt signaling by interacting with membrane-bound receptors and downstream molecules through its PDZ domain. CXXC5 is one of the key molecules that interacts with Dvl and negatively regulates the Wnt/ß-catenin pathway in osteoblast differentiation. Recently, the Dvl-CXXC5 interaction has been identified as an excellent target for osteoporosis treatment. Therefore, it is desirable to have detailed structural information for the Dvl-CXXC5 interaction. Although solution structures of the Dvl1 PDZ domain have been reported, a high-resolution crystal structure would provide detailed sidechain information that is essential for drug development. Here, we determined the first crystal structure of the Dvl-1 PDZ domain at a resolution of 1.76 Å, and compared it with its previously reported solution structure. The Dvl1 PDZ domain crystal belonged to the space group H32 with unit-cell parameters a = b = 72.837, c = 120.616, α = ß = 90.00, γ = 120.00. The crystal structure of Dvl1 PDZ shared its topology with the previously reported structure determined by nuclear magnetic resonance (NMR); however, the crystal structure was quite different from the solution structure in both the secondary structural region and the ligand-binding pocket. Molecular modeling based on NMR and X-ray crystallographic data yielded detailed information about the Dvl1/CXXC5 interaction, which will be useful for designing inhibitors.


Assuntos
Proteínas Desgrenhadas/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Domínios PDZ , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Soluções , Fatores de Transcrição , Via de Sinalização Wnt
12.
J Cell Sci ; 129(20): 3892-3902, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27744318

RESUMO

Dishevelled (DVL) assembles Wnt signalosomes through dynamic head-to-tail polymerisation by means of its DIX domain. It thus transduces Wnt signals to cytoplasmic effectors including ß-catenin, to control cell fates during normal development, tissue homeostasis and also in cancer. To date, most functional studies of Dishevelled relied on its Wnt-independent signalling activity resulting from overexpression, which is sufficient to trigger polymerisation, bypassing the requirement for Wnt signals. Here, we generate a human cell line devoid of endogenous Dishevelled (DVL1- DVL3), which lacks Wnt signal transduction to ß-catenin. However, Wnt responses can be restored by DVL2 stably re-expressed at near-endogenous levels. Using this assay to test mutant DVL2, we show that its DEP domain is essential, whereas its PDZ domain is dispensable, for signalling to ß-catenin. Our results imply two mutually exclusive functions of the DEP domain in Wnt signal transduction - binding to Frizzled to recruit Dishevelled to the receptor complex, and dimerising to cross-link DIX domain polymers for signalosome assembly. Our assay avoids the caveats associated with overexpressing Dishevelled, and provides a powerful tool for rigorous functional tests of this pivotal human signalling protein.


Assuntos
Bioensaio/métodos , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Proteína Wnt3A/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Mutação/genética , Domínios PDZ , Peptídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo
13.
Mol Cell ; 64(1): 92-104, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27692984

RESUMO

Extracellular signals are often transduced by dynamic signaling complexes ("signalosomes") assembled by oligomerizing hub proteins following their recruitment to signal-activated transmembrane receptors. A paradigm is the Wnt signalosome, which is assembled by Dishevelled via reversible head-to-tail polymerization by its DIX domain. Its activity causes stabilization of ß-catenin, a Wnt effector with pivotal roles in animal development and cancer. How Wnt triggers signalosome assembly is unknown. Here, we use structural analysis, as well as biophysical and cell-based assays, to show that the DEP domain of Dishevelled undergoes a conformational switch, from monomeric to swapped dimer, to trigger DIX-dependent polymerization and signaling to ß-catenin. This occurs in two steps: binding of monomeric DEP to Frizzled followed by DEP domain swapping triggered by its high local concentration upon Wnt-induced recruitment into clathrin-coated pits. DEP domain swapping confers directional bias on signaling, and the dimerization provides cross-linking between Dishevelled polymers, illustrating a key principle underlying signalosome formation.


Assuntos
Proteínas Desgrenhadas/química , Receptores Frizzled/química , Proteínas Wnt/química , beta Catenina/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Clonagem Molecular , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
14.
Bioorg Med Chem ; 24(15): 3259-66, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27112452

RESUMO

The Dishevelled (Dvl) protein, which conveys signals from receptors to the downstream effectors, is a critical constituent of the Wnt/ß-catenin signaling pathway. Because the PDZ domain of Dvl protein functions through associations with a wide range of protein partners, Dvl protein involved in the Wnt signaling pathway has been considered to be therapeutic targets in cancers. In this study, we performed structure-based pharmacophore model of the Dvl PDZ domain to discover novel small-molecule binders and identified eight compounds with micromolar affinity. The most potent compound identified, BMD4702, efficiently bound to the Dvl PDZ domain with 11.2µM affinity and had a 0.186µM KD value according to surface plasmon resonance and fluorescence spectroscopy, respectively. Combining both structural-kinetic relationship analyses and docking studies, we fourmulated that the ligand-binding site is composed of three H-bonds and three hydrophobic features. Thus, our approach led to the identification of potent binders of the Dvl PDZ domain and the findings provide novel insights into structure-based approaches to design high-affinity binders for the Dvl PDZ domain.


Assuntos
Proteínas Desgrenhadas/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Modelos Químicos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Desgrenhadas/química , Modelos Moleculares , Terapia de Alvo Molecular/métodos , Domínios PDZ , Ligação Proteica , Espectrometria de Fluorescência/métodos , Via de Sinalização Wnt
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