RESUMO
The Wnt receptor Frizzled3 (FZD3) is important for brain axonal development and cancer progression. We report structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nb). The crystal structure of Nb8 in complex with the FZD3 cysteine-rich domain (CRD) reveals that the nanobody binds at the base of the lipid-binding groove and can compete with Wnt5a. Nb8 fused with the Dickkopf-1 C-terminal domain behaves as a FZD3-specific Wnt surrogate, activating ß-catenin signalling. The cryo-EM structure of FZD3 in complex with Nb9 reveals partially resolved density for the CRD, which exhibits positional flexibility, and a transmembrane conformation that resembles active GPCRs. Nb9 binds to the cytoplasmic region of FZD3 at the putative Dishevelled (DVL) or G protein-binding site, competes with DVL binding, and inhibits GαS coupling. In combination, our FZD3 structures with nanobody modulators map extracellular and intracellular interaction surfaces of functional, and potentially therapeutic, relevance.
Assuntos
Receptores Frizzled , Anticorpos de Domínio Único , Receptores Frizzled/metabolismo , Receptores Frizzled/química , Humanos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Ligação Proteica , Cristalografia por Raios X , Células HEK293 , Sítios de Ligação , Microscopia Crioeletrônica , Animais , Modelos Moleculares , Domínios Proteicos , Proteínas Desgrenhadas/metabolismo , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/genética , Via de Sinalização Wnt , beta Catenina/metabolismo , beta Catenina/químicaRESUMO
The Wnt signaling pathway is a huge network governing development and homeostasis, dysregulation of which is associated with a myriad of human diseases. The Frizzled receptor (FZD) family comprises receptors for Wnt ligands, which indispensably mediate Wnt signaling jointly with a variety of co-receptors. Studies of FZDs have revealed that 10 FZD subtypes play diverse roles in physiological processes. At the same time, dysregulation of FZDs is also responsible for various diseases, in particular human cancers. Enormous attention has been paid to the molecular understanding and targeted therapy of FZDs in the past decade. In this review, we summarize the latest research on FZD structure, function, regulation and targeted therapy, providing a basis for guiding future research in this field.
Assuntos
Receptores Frizzled , Neoplasias , Via de Sinalização Wnt , Receptores Frizzled/metabolismo , Receptores Frizzled/química , Humanos , Animais , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologiaRESUMO
WNT morphogens trigger signaling pathways fundamental for embryogenesis, regeneration, and cancer. WNTs are modified with palmitoleate, which is critical for binding Frizzled (FZD) receptors and activating signaling. However, it is unknown how WNTs are released and spread from cells, given their strong lipid-dependent membrane attachment. We demonstrate that secreted FZD-related proteins and WNT inhibitory factor 1 are WNT carriers, potently releasing lipidated WNTs and forming active soluble complexes. WNT release occurs by direct handoff from the membrane protein WNTLESS to the carriers. In turn, carriers donate WNTs to glypicans and FZDs involved in WNT reception and to the NOTUM hydrolase, which antagonizes WNTs by lipid moiety removal. WNT transfer from carriers to FZDs is greatly facilitated by glypicans that serve as essential co-receptors in Wnt signaling. Thus, an extracellular network of carriers dynamically controls secretion, posttranslational regulation, and delivery of WNT morphogens, with important practical implications for regenerative medicine.
Assuntos
Glipicanas , Proteínas Wnt , Proteínas Wnt/metabolismo , Glipicanas/metabolismo , Via de Sinalização Wnt , Desenvolvimento Embrionário , Lipídeos , Receptores Frizzled/química , Receptores Frizzled/metabolismoRESUMO
The lack of incorporating epitope information into the selection process makes the conventional antibody screening method less effective in identifying antibodies with desired functions. Here, we developed an epitope-directed antibody selection method by designing a directed library favoring the target epitope and a precise "counter" antigen for clearing irrelevant binders in the library. With this method, we successfully isolated an antibody, pF7_A5, that targets the less conserved region on the FZD2/7 CRD as designed. Guided by the structure of pF7_A5-FZD2CRD, a further round of evolution was conducted together with the "counter" antigen selection strategy, and ultimately, an FZD2-specific antibody and an FZD7-preferred antibody were obtained. Because of targeting the predefined functional site, all these antibodies exhibited the expected modulatory activity on the Wnt pathway. Together, the method developed here will be useful in antibody drug discovery, and the identified FZD antibodies will have clinical potential in FZD-related cancer therapy.
Assuntos
Anticorpos Monoclonais , Evolução Molecular Direcionada , Mapeamento de Epitopos , Epitopos , Receptores Frizzled , Via de Sinalização Wnt , Descoberta de Drogas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Receptores Frizzled/química , Receptores Frizzled/genética , Receptores Frizzled/imunologia , Via de Sinalização Wnt/imunologia , Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos/métodos , Humanos , Conformação Proteica , Evolução Molecular Direcionada/métodosRESUMO
Expression of Frizzled 9 (FZD9) is critical to the activity of the lung cancer chemoprevention agent and prostacyclin analogue, iloprost. FZD9 is required in lung epithelial cells for iloprost to activate peroxisome proliferator activated receptor gamma (PPARG) and related anti-tumor signaling. We aimed to investigate which miRNA regulate FZD9 in the context of cigarette smoke exposure and iloprost treatment. We found that miR-520a-5p binds the FZD9 3'UTR in lung cell lines and alters activity and expression of FZD9 downstream targets. Cigarette smoke condensate (CSC) increases expression of miR-520a-5p, while iloprost decreases expression. Cancer promoting effects of a miR-520a-5p mimic were rescued with iloprost treatment, and effects of cigarette smoke were partially rescued with a miR-520a-5p inhibitor. Here we confirm miR-520a-5p as a regulator of FZD9 activity and a mediator of CSC and iloprost effects in the lung. Targeting miR-520a-5p could be an approach to restoring FZD9 expression and improving response to iloprost lung cancer chemoprevention.
Assuntos
Fumar Cigarros/efeitos adversos , Receptores Frizzled/genética , Iloprosta/farmacologia , Neoplasias Pulmonares/genética , MicroRNAs/genética , Linhagem Celular Tumoral , Quimioprevenção , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevenção & controle , MicroRNAs/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
cWnt-signalling plays a crucial role in stem cell maintenance and tissue homeostasis. Secreted frizzled-related proteins(SFRP), Wnt inhibitors consist of the N-terminal cysteine rich domain(CRD) and the C-terminal netrin(NTR) domain. SFRP1 binds to the Wnt ligands and frizzled receptors(FZ) either through its SFRP1CRD or through its SFRP1Netrin domains; however, very little is known on these binding affinities. Here, we attempted to understand the interactions and binding affinities of SFRP1-Wnt5B, SFRP1-FZ(2, 3 & 7) and Wnt5B-FZ(2, 3 & 7) that are mainly expressed in murine hair follicle stem cells. SFRP1CRD, SFRP1Netrin, Wnt5B and FZ(2, 3 & 7) structures were built using homology modelling, followed by their molecular dynamics simulations. SFRP1CRD showed lower fluctuation when in complex with FZ2, FZ3 and FZ7 and Wnt5B as compared to SFRP1Netrin using RMSF and RMSD. However, free energy showed SFRP1Netrin was energetically more stable than SFRP1CRD. SFRP1Netrin formed more number of interactions with FZ as compared to SFRP1CRD. Importantly, SFRP1Netrin favoured binding to the FZ receptors(FZ3 > FZ7 > FZ2) as compared to Wnt5B ligand. Conversely, the SFRP1CRD showed more affinity towards the Wnt5B ligand as compared to FZ receptors. Wnt5B showed the best binding affinity with FZ3 followed by SFRP1CRD and SFRP1Netrin. Therefore, SFRP1Netrin can bind to the FZ3 with higher binding affinity and may inhibit non-canonical Wnt-signalling pathway. Our study provides the comprehensive information on the binding affinities among the Wnt5B, SFRP1CRD/Netrin and FZ(2, 3 & 7). Thus, this information might also help in designing novel strategies to inhibit aberrant Wnt-signalling.Communicated by Ramaswamy H. Sarma.
Assuntos
Receptores Frizzled , Proteínas Wnt , Animais , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Ligantes , Proteínas de Membrana , Camundongos , Netrinas , Transdução de Sinais , Proteínas Wnt/química , Proteínas Wnt/metabolismoRESUMO
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.
Assuntos
Receptores Frizzled/química , Receptores Frizzled/metabolismo , Receptor Smoothened/química , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Compostos de Boro/química , Microscopia Crioeletrônica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores Frizzled/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Fosfoproteínas/metabolismo , Conformação Proteica , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/metabolismoRESUMO
WNT-Frizzled (FZD) signaling plays a critical role in embryonic development, stem cell regulation and tissue homeostasis. FZDs are linked to severe human pathology and are seen as a promising target for therapy. Despite intense efforts, no small molecule drugs with distinct efficacy have emerged. Here, we identify the Smoothened agonist SAG1.3 as a partial agonist of FZD6 with limited subtype selectivity. Employing extensive in silico analysis, resonance energy transfer- and luciferase-based assays we describe the mode of action of SAG1.3. We define the ability of SAG1.3 to bind to FZD6 and to induce conformational changes in the receptor, recruitment and activation of G proteins and dynamics in FZD-Dishevelled interaction. Our results provide the proof-of-principle that FZDs are targetable by small molecules acting on their seven transmembrane spanning core. Thus, we provide a starting point for a structure-guided and mechanism-based drug discovery process to exploit the potential of FZDs as therapeutic targets.
Assuntos
Proteínas Desgrenhadas/metabolismo , Descoberta de Drogas/métodos , Receptores Frizzled/agonistas , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Piridinas/química , Tiofenos/química , Via de Sinalização Wnt/efeitos dos fármacos , Membrana Celular/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular/métodos , Morfolinas/farmacologia , Estudo de Prova de Conceito , Purinas/farmacologia , Piridinas/farmacologia , Receptor Smoothened/agonistas , Relação Estrutura-Atividade , Tiofenos/farmacologiaRESUMO
Triple-negative breast cancer is the leading worldwide cause of cancer-related deaths in women. The prospection and development of new substances with antitumoral potential is of great importance for the treatment of this disease. The objective of this work was to identify a commercial drug or ligand that could potentially bind to the FZD7 transmembrane protein and inactivate the Wnt signaling pathway in triple-negative breast cancer cells. We aimed at computationally modeling the FZD7, Wnt3, and Wnt3a proteins, at making them available in protein model databases, and at conducting docking analysis to assess the binding free energy between FZD7 and the selected ligands. The Wnt3 and Wnt3a proteins were modeled by homology modeling, and the FZD7 protein was modeled by homology modeling and ab initio modeling. The ligands were selected based on their similarity to the palmitoleic acid and were gathered from the ZINC database. A total of 30 commercially available ligands were found in the ZINC database. The docking results show that the ligands zinc08221009, zinc13546050, zinc05260769, zinc04529321, and zinc05972969 are good candidates for novel drug development. The created models and conducted analysis by this work will most certainly help in future research on the Wnt signaling pathway and its components.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Receptores Frizzled/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Simulação por Computador , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Receptores Frizzled/antagonistas & inibidores , Receptores Frizzled/química , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Proteína Wnt3/química , Proteína Wnt3/metabolismoRESUMO
Several proteins other than the frizzled receptors (Fzd) and the secreted Frizzled-related proteins (sFRP) contain Fzd-type cysteine-rich domains (CRD). We have termed these domains "putative Fzd-type CRDs", as the relevance of Wnt signalling in the majority of these is unknown; the RORs, an exception to this, are well known for mediating non-canonical Wnt signalling. In this study, we have predicted the likely binding affinity of all Wnts for all putative Fzd-type CRDs. We applied both our previously determined WntâFzd CRD binding affinity prediction model, as well as a newly devised model wherein the lipid term was forced to contribute favourably to the predicted binding energy. The results obtained from our new model indicate that certain putative Fzd CRDs are much more likely to bind Wnts, in some cases exhibiting selectivity for specific Wnts. The results of this study inform the investigation of Wnt signalling modulation beyond Fzds and sFRPs.
Assuntos
Cisteína , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Wnt/metabolismo , Cisteína/química , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Transdução de SinaisRESUMO
Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/química , Células Epiteliais/efeitos dos fármacos , Receptores Frizzled/antagonistas & inibidores , Peptídeos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Células Epiteliais/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/crescimento & desenvolvimento , Receptores Frizzled/metabolismo , Proteínas Ligadas por GPI/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Receptores Frizzled/química , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Células HEK293 , Humanos , Ligantes , Luciferases/metabolismo , Camundongos , Mutação/genética , Neovascularização Fisiológica , Domínios Proteicos , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/químicaRESUMO
Wnts are lipid-modified proteins that regulate stem cell signaling via Frizzled receptors on the cell surface. Determination of binding interactions between lipid-modified Wnt proteins and their Frizzled receptors has been challenging due to the lack of availability of facile detection methods and technical hurdles associated with generating the relevant reagents. Here we report an enzyme-linked immunosorbent assay to measure the binding of a biotinylated form of lipid-modified Wnt3a to the extracellular cysteine-rich domain of Frizzled receptor. The method described herein is robust and rapid, uses minimum volumes of reagents, and can be conducted in a high-throughput format. Because of these attributes, the method could find utility in drug discovery applications such as characterizing the effect of pharmacological inhibitors on Wnt signaling without the need for sophisticated biophysical instrumentation.
Assuntos
Receptores Frizzled , Via de Sinalização Wnt , Proteína Wnt3A , Animais , Ensaio de Imunoadsorção Enzimática , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Ligação Proteica , Proteína Wnt3A/química , Proteína Wnt3A/metabolismoRESUMO
To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.
Assuntos
Receptores Frizzled/antagonistas & inibidores , Receptores Frizzled/metabolismo , Simulação de Acoplamento Molecular , Animais , Anquirinas/química , Anquirinas/metabolismo , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Duodeno/citologia , Duodeno/metabolismo , Receptores Frizzled/química , Humanos , Camundongos Endogâmicos C57BL , Ligação Proteica , Conformação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Recently, molecular dynamics simulations, from all atom and coarse grained to hybrid methods bridging the two scales, have provided exciting functional insights into class F (Frizzled and Taste2) GPCRs (about 40 members in humans). Findings include: (i) The activation of one member of the Frizzled receptors (FZD4) involves a bending of transmembrane helix TM7 far larger than that in class A GPCRs. (ii) The affinity of an anticancer drug targeting another member (Smoothened receptor) decreases in a specific drug-resistant variant, because the mutation ultimately disrupts the binding cavity and affects TM6. (iii) A novel two-state recognition mechanism explains the very large agonist diversity for at least one member of the Taste2 GPCRs, hTAS2R46.
Assuntos
Receptores Frizzled/química , Receptores Acoplados a Proteínas G/química , Sítios de Ligação , Humanos , Ligantes , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
BACKGROUND: Frizzled family members belong to G-protein coupled receptors and encode proteins accountable for cell signal transduction, cell proliferation and cell death. Members of Frizzled receptor family are considered to have critical roles in causing various forms of cancer, cardiac hypertrophy, familial exudative vitreoretinopathy (FEVR) and schizophrenia. RESULTS: This study investigates the evolutionary and structural aspects of Frizzled receptors, with particular focus on FEVR associated FZD4 gene. The phylogenetic tree topology suggests the diversification of Frizzled receptors at the root of metazoans history. Moreover, comparative structural data reveals that FEVR associated missense mutations in FZD4 effect the common protein region (amino acids 495-537) through a well-known phenomenon called epistasis. This critical protein region is present at the carboxyl-terminal domain and encompasses the K-T/S-XXX-W, a PDZ binding motif and S/T-X-V PDZ recognition motif. CONCLUSION: Taken together these results demonstrate that during the course of evolution, FZD4 has acquired new functions or epistasis via complex patter of gene duplications, sequence divergence and conformational remodeling. In particular, amino acids 495-537 at the C-terminus region of FZD4 protein might be crucial in its normal function and/or pathophysiology. This critical region of FZD4 protein may offer opportunities for the development of novel therapeutics approaches for human retinal vascular disease.
Assuntos
Evolução Molecular , Oftalmopatias Hereditárias/genética , Receptores Frizzled/química , Receptores Frizzled/genética , Doenças Retinianas/genética , Vitreorretinopatias Exsudativas Familiares , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto/genética , Filogenia , Domínios ProteicosRESUMO
Colorectal cancer (CRC) is a common malignancy and the leading cause of cancer death worldwide. According to previous studies, vanillin possesses pharmacological and anticancer activities. In this work, we have modified the structure of vanillin to obtain a vanillin derivative called 4-(1H-imidazo [4,5-f][1,10]-phenanthrolin-2-yl)-2-methoxyphenol (IPM711), which has improved anticancer activity. The present study is intended to explore the anti-colorectal cancer activity of IPM711 in HT29 and HCT116 cells. The results of this study suggest that IPM711 can inhibit the growth, invasion and migration of HT29 and HCT116 cells. Western blot and molecular docking showed that IPM711 could bind to a Wnt/ß-catenin signaling receptor to inhibit cell growth, invasion and migration in HT29 cells. Based on these results, IPM711 is a promising anticancer drug candidate for human colorectal cancer therapy.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Benzaldeídos/química , Benzaldeídos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Antineoplásicos/metabolismo , Benzaldeídos/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HCT116 , Células HT29 , Humanos , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
BACKGROUND: Autosomal recessive nail dysplasia is characterized by thick and hard nails with a very slow growth on the hands and feet. Mutations in FZD6 gene were found to be associated with autosomal recessive nail dysplasia in 2011. Presently, only seven mutations have been reported in FZD6 gene; five mutations are clustered in the C-terminus, one is at the seventh transmembrane domain, and another is at the very beginning of third extracellular loop. METHODS: Whole exome sequencing (WES) was applied to the index case, her one affected sister and her healthy consanguineous parents. The mutation was verified via Sanger sequencing. Molecular dynamics simulations of the predicted structures of native and mutant proteins were compared to gain insight into the pathogenicity mechanism of the mutation. RESULTS: Here, we report a homozygous 8 bp deletion mutation, p.Gly559Aspfs*16; c.1676_1683delGAACCAGC, in FZD6 gene which causes a frameshift and creates a premature stop codon at position 16 of the new reading frame. Our molecular dynamics calculations predict that the pathogenicity of this frameshift mutation may be caused by the change in entropy of the protein with negative manner, disturbing the C-terminal domain structure, and hence interaction partners of FZD6. CONCLUSION: We identified a homozygous deletion mutation in FZD6 in a consanguineous Turkish family with nail dysplasia. We also provide a molecular mechanism about the effects of the deletion on the protein structure and its possible motions. This study provides a pathogenicity mechanism for this mutation in nail dysplasia for the first time.
Assuntos
Receptores Frizzled/genética , Predisposição Genética para Doença , Mutação , Unhas Malformadas/genética , Adulto , Sequência de Aminoácidos , Códon sem Sentido , Consanguinidade , Feminino , Mutação da Fase de Leitura , Receptores Frizzled/química , Estudos de Associação Genética , Homozigoto , Humanos , Modelos Moleculares , Linhagem , Conformação Proteica , Análise de Sequência , Deleção de Sequência , TurquiaRESUMO
Wnt signaling regulates physiological processes ranging from cell differentiation to bone formation. Dysregulation of Wnt signaling is linked to several human ailments, including colorectal, pancreatic, and breast cancers. As such, modulation of this pathway has been an attractive strategy for therapeutic development of anticancer agents. Since the discovery of Wnt proteins more than 35 years ago, research efforts continue to focus on understanding the biochemistry of their molecular interactions and their biological functions. Wnt is a secreted glycoprotein covalently modified with a cis-unsaturated fatty acyl group at a conserved serine residue, and this modification is required for Wnt secretion and activity. To initiate signaling, Wnt proteins bind to cell-surface Frizzled (FZD) receptors, but the molecular basis for recognition of Wnt's fatty acyl moiety by the extracellular cysteine-rich domain of FZD has become clear only very recently. Here, we review the most recent developments in the field, focusing on structural and biochemical studies of the FZD receptor family and highlighting new insights into their molecular arrangement and mode of regulation by cis-unsaturated fatty acids. Additionally, we examine how other lipid-binding proteins recognize fatty acyl chains on Wnt proteins in the regulation of Wnt secretion and activities. Altogether, this perspective expands our understanding of fatty acid-protein interactions in the FZD system and provides a basis for guiding future research in the field.
Assuntos
Ácidos Graxos/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Família Multigênica , Animais , Sítios de Ligação , Cristalografia por Raios X , Ácidos Graxos/química , Receptores Frizzled/genética , Humanos , Modelos Moleculares , Transdução de SinaisRESUMO
Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the ß-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.