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1.
Biochemistry (Mosc) ; 84(4): 358-369, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228927

RESUMO

Cytoplasmic actin structures are essential components of the eukaryotic cytoskeleton. According to the classic concepts, actin structures perform contractile and motor functions, ensuring the possibility of cell shape changes during cell spreading, polarization, and movement both in vitro and in vivo, from the early embryogenesis stages and throughout the life of a multicellular organism. Intracellular organization of actin structures, their biochemical composition, and dynamic properties play a key role in the realization of specific cellular and tissue functions and vary in different cell types. This paper is a review of recent studies on the organization and properties of actin structures in endotheliocytes, interaction of these structures with other cytoskeletal components and elements involved in cell adhesion, as well as their role in the functional activity of endothelial cells.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/genética , Caderinas/química , Caderinas/metabolismo , Citosol/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo
2.
Dev Cell ; 48(6): 780-792.e4, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30853441

RESUMO

FAT4 mutations lead to several human diseases that disrupt the normal development of the kidney. However, the underlying mechanism remains elusive. In studying the duplex kidney phenotypes observed upon deletion of Fat4 in mice, we have uncovered an interaction between the atypical cadherin FAT4 and RET, a tyrosine kinase receptor essential for kidney development. Analysis of kidney development in Fat4-/- kidneys revealed abnormal ureteric budding and excessive RET signaling. Removal of one copy of the RET ligand Gdnf rescues Fat4-/- kidney development, supporting the proposal that loss of Fat4 hyperactivates RET signaling. Conditional knockout analyses revealed a non-autonomous role for Fat4 in regulating RET signaling. Mechanistically, we found that FAT4 interacts with RET through extracellular cadherin repeats. Importantly, expression of FAT4 perturbs the assembly of the RET-GFRA1-GDNF complex, reducing RET signaling. Thus, FAT4 interacts with RET to fine-tune RET signaling, establishing a juxtacrine mechanism controlling kidney development.


Assuntos
Caderinas/metabolismo , Rim/embriologia , Rim/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais , Animais , Caderinas/química , Caderinas/deficiência , Deleção de Genes , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Rim/anormalidades , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Regulação para Cima
4.
Biophys J ; 115(12): 2368-2385, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30527337

RESUMO

The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) "repeats" that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of nonclassical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has 11 EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size-exclusion chromatography coupled to multiangle laser light scattering and small-angle x-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the "giant" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.


Assuntos
Caderinas/química , Espaço Extracelular/metabolismo , Fenômenos Mecânicos , Multimerização Proteica , Precursores de Proteínas/química , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Domínios Proteicos , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Suínos
5.
PLoS Pathog ; 14(12): e1007477, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30532249

RESUMO

Viruses in the rhinovirus C species (RV-C) are more likely to cause severe wheezing illnesses and asthma exacerbations in children than related isolates of the RV-A or RV-B. The RV-C capsid is structurally distinct from other rhinoviruses and does not bind ICAM-1 or LDL receptors. The RV-C receptor is instead, human cadherin-related family member 3 (CDHR3), a protein unique to the airway epithelium. A single nucleotide polymorphism (rs6967330, encoding C529Y) in CDHR3 regulates the display density of CDHR3 on cell surfaces and is among the strongest known genetic correlates for childhood virus-induced asthma susceptibility. CDHR3 immunoprecipitations from transfected or transduced cell lysates were used to characterize the RV-C interaction requirements. The C529 and Y529 variations in extracellular repeat domain 5 (EC5), bound equivalently to virus. Glycosylase treatment followed by mass spectrometry mapped 3 extracellular N-linked modification sites, and further detected surface-dependent, α2-6 sialyation unique to the Y529 format. None of these modifications were required for RV-C recognition, but removal or even dilution of structurally stabilizing calcium ions from the EC junctions irreversibly abrogated virus binding. CDHR3 deletions expressed in HeLa cells or as bacterial recombinant proteins, mapped the amino-terminal EC1 unit as the required virus contact. Derivatives containing the EC1 domain, could not only recapitulate virus:receptor interactions in vitro, but also directly inhibit RV-C infection of susceptible cells for several virus genotypes (C02, C15, C41, and C45). We propose that all RV-C use the same EC1 landing pad, interacting with putative EC3-mediated multimerization formats of CDHR3.


Assuntos
Caderinas/química , Infecções por Enterovirus , Enterovirus/patogenicidade , Proteínas de Membrana/química , Ligação Viral , Linhagem Celular , Humanos , Imunoprecipitação/métodos , Conformação Proteica
6.
Elife ; 72018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30024850

RESUMO

In vivo, the primary molecular mechanotransductive events mechanically initiating cell differentiation remain unknown. Here we find the molecular stretching of the highly conserved Y654-ß-catenin-D665-E-cadherin binding site as mechanically induced by tissue strain. It triggers the increase of accessibility of the Y654 site, target of the Src42A kinase phosphorylation leading to irreversible unbinding. Molecular dynamics simulations of the ß-catenin/E-cadherin complex under a force mimicking a 6 pN physiological mechanical strain predict a local 45% stretching between the two α-helices linked by the site and a 15% increase in accessibility of the phosphorylation site. Both are quantitatively observed using FRET lifetime imaging and non-phospho Y654 specific antibody labelling, in response to the mechanical strains developed by endogenous and magnetically mimicked early mesoderm invagination of gastrulating Drosophila embryos. This is followed by the predicted release of 16% of ß-catenin from junctions, observed in FRAP, which initiates the mechanical activation of the ß-catenin pathway process.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo/química , Sítios de Ligação , Caderinas/química , Proteínas de Drosophila/química , Transferência Ressonante de Energia de Fluorescência , Mecanotransdução Celular , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Homologia de Sequência , Fatores de Transcrição/química
7.
J Pept Sci ; 24(6): e3083, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29737576

RESUMO

Cell-penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure-activity relationship and sequence alignment studies have suggested that the hydrophobic N-terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N-terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Peptídeos/farmacologia , Antibacterianos/química , Anti-Infecciosos/química , Caderinas/química , Caderinas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Simulação de Dinâmica Molecular , Peptídeos/química , Relação Estrutura-Atividade
8.
Oncol Rep ; 39(6): 2837-2844, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29693128

RESUMO

Vascular endothelial cadherin (VE­cadherin) was first found in vascular endothelial cells to maintain normal vascular structures and regulate endothelial cell permeability by homology adhesion. New evidence indicates that certain invasive tumor cells also express VE­cadherin, which is involved in vasculogenic mimicry to provide a blood supply required for tumor growth and metastasis. EC1 and EC3 domains of VE­cadherin were reported to be important for intercellular homology adhesion. In the present study, a monoclonal antibody specific to the outer-membrane immunoglobulin-like domains of VE­cadherin was generated and the binding epitope was identified as peptide LDREVVPWYNLTVEA in the EC4 domain. This antibody inhibited proliferation and capillary-like structure formation of lung cancer Glc­82 cells in 3D Matrigel culture in vitro. This effect was mediated by the inhibition of AKT phosphorylation. Our results suggested that the EC4 domain participates in VE­cadherin clustering and the antibody targeting the EC4 domain of VE­cadherin may be a promising anti­vasculogenic mimicry agent for cancer treatment.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD/química , Caderinas/química , Epitopos/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Chem Commun (Camb) ; 54(31): 3851-3854, 2018 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-29594291

RESUMO

An efficient surface modification for upconversion nanoparticles (UCNPs) is reported via supramolecular host-guest self-assembly. Cucurbit[7]uril (CB) can provide a hydrophilic surface and cavities for most biomolecules. High biological efficiency, activity and versatility of the approach enable UCNPs to be significantly applied in bio-imaging, early disease detection, and bio-sensing.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Imidazóis/química , Nanopartículas Metálicas/química , Adamantano/análogos & derivados , Adamantano/química , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Caderinas/química , Európio/química , Fluoretos/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/toxicidade , Imunoglobulina G/química , Ligantes , Nanopartículas Metálicas/toxicidade , Tamanho da Partícula , Itérbio/química , Ítrio/química
10.
EBioMedicine ; 28: 168-179, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29409727

RESUMO

Dyslexia is a heritable neurodevelopmental disorder characterized by difficulties in reading and writing. In this study, we describe the identification of a set of 17 polymorphisms located across 1.9Mb region on chromosome 5q31.3, encompassing genes of the PCDHG cluster, TAF7, PCDH1 and ARHGAP26, dominantly inherited with dyslexia in a multi-incident family. Strikingly, the non-risk form of seven variations of the PCDHG cluster, are preponderant in the human lineage, while risk alleles are ancestral and conserved across Neanderthals to non-human primates. Four of these seven ancestral variations (c.460A>C [p.Ile154Leu], c.541G>A [p.Ala181Thr], c.2036G>C [p.Arg679Pro] and c.2059A>G [p.Lys687Glu]) result in amino acid alterations. p.Ile154Leu and p.Ala181Thr are present at EC2: EC3 interacting interface of γA3-PCDH and γA4-PCDH respectively might affect trans-homophilic interaction and hence neuronal connectivity. p.Arg679Pro and p.Lys687Glu are present within the linker region connecting trans-membrane to extracellular domain. Sequence analysis indicated the importance of p.Ile154, p.Arg679 and p.Lys687 in maintaining class specificity. Thus the observed association of PCDHG genes encoding neural adhesion proteins reinforces the hypothesis of aberrant neuronal connectivity in the pathophysiology of dyslexia. Additionally, the striking conservation of the identified variants indicates a role of PCDHG in the evolution of highly specialized cognitive skills critical to reading.


Assuntos
Caderinas/genética , Dislexia/genética , Predisposição Genética para Doença , Variação Genética , Família Multigênica , Alelos , Sequência de Aminoácidos , Gânglios da Base/metabolismo , Sequência de Bases , Caderinas/química , Segregação de Cromossomos/genética , Família , Genes Dominantes , Humanos , Padrões de Herança/genética , Modelos Moleculares , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Especificidade da Espécie , Homologia Estrutural de Proteína
11.
Biochemistry ; 57(11): 1702-1710, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29443515

RESUMO

Human hearing relies upon the tip-to-tip interaction of two nonclassical cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23). Together, these proteins form a filament called the tip link that connects neighboring stereocilia of mechanosensitive hair cells. As sound waves enter the cochlea, the stereocilia deflect and tension is applied to the tip link, opening nearby transduction channels. Disruption of the tip link by loud sound or calcium chelators eliminates transduction currents and illustrates that tip-link integrity is critical for mechanosensing. Tip-link remodeling after disruption is a dynamic process, which can lead to the formation of atypical complexes that incorporate alternatively spliced variants of PCDH15. These variants are categorized into six groups (N1-N6) based upon differences in the first two extracellular cadherin (EC) repeats. Here, we characterized the two N-terminal EC repeats of all PCDH15 variants (pcdh15(N1) to pcdh15(N6)) and combined these variants to test complex formation. We solved the crystal structure of a new complex composed of CDH23 EC1-2 (cdh23) and pcdh15(N2) at 2.3 Šresolution and compared it to the canonical cdh23-pcdh15(N1) complex. While there were subtle structural differences, the binding affinity between cdh23 and pcdh15(N2) is ∼6 times weaker than cdh23 and pcdh15(N1) as determined by surface plasmon resonance analysis. Steered molecular dynamics simulations predict that the unbinding force of the cdh23-pcdh15(N2) complex can be lower than the canonical tip link. Our results demonstrate that alternative heterophilic tip-link structures form stable protein-protein interactions in vitro and suggest that homophilic PCDH15-PCDH15 tip links form through the interaction of additional EC repeats.


Assuntos
Processamento Alternativo , Caderinas/genética , Orelha Interna/metabolismo , Precursores de Proteínas/genética , Animais , Caderinas/química , Caderinas/metabolismo , Cristalografia por Raios X , Orelha Interna/fisiologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/fisiologia , Humanos , Mecanotransdução Celular , Camundongos , Ligação Proteica , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Ressonância de Plasmônio de Superfície
12.
Biomaterials ; 154: 134-146, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29128842

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is a worldwide leading malignancy with poor prognoses. Aggressive HNSCC is manifested by forming tumor satellites in the invasive front, which is closely associated with epithelial-mesenchymal transition, local invasion, and metastasis. Limited by the pathological analyses of static cancer specimens conducted in most previous investigations, the dynamic processes and the decisive factors of tumor satellite formation in HNSCC cannot be monitored and studied. The establishment of a system to recapitulate the phenomenon in vitro may be instrumental to explicitly address the question. In this study, we explored the feasibility of establishing an in vitro system to induce tumor satellite formation of different HNSCC cells by applying a system composed of three-dimensional collagen scaffolds. The real-time dynamic process of tumor satellite formation could be monitored in detail, and the pivotal factors accounting for HNSCC tumor satellite formation were evaluated. E-cadherin remodeling, vimentin aggregation, invadopodia formation, and extracellular matrix degradation occurred in the HNSCC cells when they were cultured in an environment created with a low calcium concentration and steric collagen scaffolds, which altogether contributed to tumor satellite formation and spreading. The phenomena of HNSCC cell transition observed in the current system were confirmed in vivo with compatible findings in surgical specimens. Through the use of this system, we illustrated a novel method to study tumor satellite formation in HNSCC. It may serve as a platform for further investigation of underlying mechanisms for tumor satellites of cancer.


Assuntos
Colágeno/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/patologia , Tecidos Suporte/química , Caderinas/química , Caderinas/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Humanos , Domínios Proteicos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo
13.
Microbiol Immunol ; 62(2): 80-89, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29266585

RESUMO

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Células Epiteliais/metabolismo , Hemaglutininas/metabolismo , Mucosa Intestinal/metabolismo , Antígenos CD , Sítios de Ligação , Toxinas Botulínicas/química , Toxinas Botulínicas Tipo A/química , Células CACO-2 , Caderinas/química , Caderinas/metabolismo , Carboidratos , Clostridium botulinum tipo B/genética , DNA Bacteriano/genética , Hemaglutininas/química , Hemaglutininas/genética , Humanos , Absorção Intestinal , Mutagênese Sítio-Dirigida , Plasmídeos , Ligação Proteica , Proteínas Recombinantes , Deleção de Sequência
14.
RNA Biol ; 15(4-5): 649-658, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28910573

RESUMO

Tryptophanyl-tRNA synthetase (TrpRS) in vertebrates contains a N-terminal extension in front of the catalytic core. Proteolytic removal of the N-terminal 93 amino acids gives rise to T2-TrpRS, which has potent anti-angiogenic activity mediated through its extracellular interaction with VE-cadherin. Zinc has been shown to have anti-angiogenic effects and can bind to human TrpRS. However, the connection between zinc and the anti-angiogenic function of TrpRS has not been explored. Here we report that zinc binding can induce structural relaxation in human TrpRS to facilitate the proteolytic generation of a T2-TrpRS-like fragment. The zinc-binding site is likely to be contained within T2-TrpRS, and the zinc-bound conformation of T2-TrpRS is mimicked by mutation H130R. We determined the crystal structure of H130R T2-TrpRS at 2.8 Å resolution, which reveals drastically different conformation from that of wild-type (WT) T2-TrpRS. The conformational change creates larger binding surfaces for VE-cadherin as suggested by molecular dynamic simulations. Surface plasmon resonance analysis indicates more than 50-fold increase in binding affinity of H130R T2-TrpRS for VE-cadherin, compared to WT T2-TrpRS. The enhanced interaction is also confirmed by a cell-based binding analysis. These results suggest that zinc plays an important role in activating TrpRS for angiogenesis regulation.


Assuntos
Inibidores da Angiogênese/química , Antígenos CD/química , Caderinas/química , Triptofano-tRNA Ligase/química , Zinco/química , Inibidores da Angiogênese/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Caderinas/genética , Caderinas/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Triptofano-tRNA Ligase/genética , Triptofano-tRNA Ligase/metabolismo , Zinco/metabolismo
15.
Eur Biophys J ; 47(1): 59-67, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28620741

RESUMO

E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution.


Assuntos
Caderinas/química , Multimerização Proteica , Simulação de Dinâmica Molecular , Estabilidade Proteica , Estrutura Quaternária de Proteína , Soluções
16.
PLoS One ; 12(11): e0188963, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190819

RESUMO

During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In Xenopus laevis, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in X.laevis CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation in vivo by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration in vivo. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors.


Assuntos
Encéfalo/citologia , Caderinas/metabolismo , Movimento Celular , Crista Neural/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Animais , Encéfalo/metabolismo , Caderinas/química , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Xenopus laevis/embriologia
17.
PLoS One ; 12(12): e0189546, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29261728

RESUMO

Protein-protein interactions play a crucial role in biological processes such as cell-cell adhesion, immune system-pathogen interactions, and sensory perception. Understanding the structural determinants of protein-protein complex formation and obtaining quantitative estimates of their dissociation constant (KD) are essential for the study of these interactions and for the discovery of new therapeutics. At the same time, it is equally important to characterize protein-protein interactions in a high-throughput fashion. Here, we use a modified thermal scanning assay to test interactions of wild type (WT) and mutant variants of N-terminal fragments (EC1+2) of cadherin-23 and protocadherin-15, two proteins essential for inner-ear mechanotransduction. An environmentally sensitive fluorescent dye (SYPRO orange) is used to monitor melting temperature (Tm) shifts of protocadherin-15 EC1+2 (pcdh15) in the presence of increasing concentrations of cadherin-23 EC1+2 (cdh23). These Tm shifts are absent when we use proteins containing deafness-related missense mutations known to disrupt cdh23 binding to pcdh15, and are increased for some rationally designed mutants expected to enhance binding. In addition, surface plasmon resonance binding experiments were used to test if the Tm shifts correlated with changes in binding affinity. We used this approach to find a double mutation (cdh23(T15E)- pcdh15(G16D)) that enhances binding affinity of the cadherin complex by 1.98 kJ/mol, roughly two-fold that of the WT complex. We suggest that the thermal scanning methodology can be used in high-throughput format to quickly compare binding affinities (KD from nM up to 100 µM) for some heterodimeric protein complexes and to screen small molecule libraries to find protein-protein interaction inhibitors and enhancers.


Assuntos
Caderinas/metabolismo , Mapeamento de Interação de Proteínas/métodos , Precursores de Proteínas/metabolismo , Animais , Caderinas/química , Orelha Interna/metabolismo , Cinética , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Desnaturação Proteica , Precursores de Proteínas/química , Soluções , Ressonância de Plasmônio de Superfície , Temperatura Ambiente
18.
Proc Natl Acad Sci U S A ; 114(46): E9829-E9837, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087338

RESUMO

Clustered protocadherins (Pcdhs) mediate numerous neural patterning functions, including neuronal self-recognition and non-self-discrimination to direct self-avoidance among vertebrate neurons. Individual neurons stochastically express a subset of Pcdh isoforms, which assemble to form a stochastic repertoire of cis-dimers. We describe the structure of a PcdhγB7 cis-homodimer, which includes the membrane-proximal extracellular cadherin domains EC5 and EC6. The structure is asymmetric with one molecule contributing interface surface from both EC5 and EC6, and the other only from EC6. Structural and sequence analyses suggest that all Pcdh isoforms will dimerize through this interface. Site-directed mutants at this interface interfere with both Pcdh cis-dimerization and cell surface transport. The structure explains the known restrictions of cis-interactions of some Pcdh isoforms, including α-Pcdhs, which cannot form homodimers. The asymmetry of the interface approximately doubles the size of the recognition repertoire, and restrictions on cis-interactions among Pcdh isoforms define the limits of the Pcdh recognition unit repertoire.


Assuntos
Caderinas/química , Caderinas/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neurônios/metabolismo , Isoformas de Proteínas/genética , Multimerização Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína
19.
PLoS One ; 12(9): e0184574, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28926625

RESUMO

Receptor-type protein tyrosine phosphatases (RPTPs) of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, dimerization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC) in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascular integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with controls. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a membrane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane domains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that removal of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and transmembrane domain of R3-PTPs in interaction with VE-cadherin.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Antígenos CD/química , Antígenos CD/genética , Caderinas/química , Caderinas/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Microscopia Confocal , Mutagênese , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Transfecção
20.
Biomaterials ; 145: 33-43, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28843065

RESUMO

N-cadherin, a transmembrane protein and major component of adherens junction, mediates cell-cell interactions and intracellular signaling that are important to the regulation of cell behaviors and organ development. Previous studies have identified mimetic peptides that possess similar bioactivity as that of N-cadherin, which promotes chondrogenesis of human mesenchymal stem cells (hMSCs); however, the molecular mechanism remains unknown. In this study, we combined the N-cadherin mimetic peptide (HAVDI) with the self-assembling KLD-12 peptide: the resultant peptide is capable of self-assembling into hydrogels functionalized with N-cadherin peptide in phosphate-buffered saline (PBS) at 37 °C. Encapsulation of hMSCs in these hydrogels showed enhanced expression of chondrogenic marker genes and deposition of cartilage specific extracellular matrix rich in proteoglycan and Type II Collagen compared to control hydrogels, with a scrambled-sequence peptide after 14 days of chondrogenic culture. Furthermore, western blot showed a significantly higher expression of active glycogen synthase kinase-3ß (GSK-3ß), which phosphorylates ß-catenin and facilitates ubiquitin-mediated degradation, as well as a lower expression of ß-catenin and LEF1 in the N-cadherin peptide hydrogels versus controls. Immunofluorescence staining revealed significantly less nuclear localization of ß-catenin in N-cadherin mimetic peptide hydrogels. Our findings suggest that N-cadherin peptide hydrogels suppress canonical Wnt signaling in hMSCs by reducing ß-catenin nuclear translocation and the associated transcriptional activity of ß-catenin/LEF-1/TCF complex, thereby enhancing the chondrogenesis of hMSCs. Our biomimetic self-assembled peptide hydrogels can serve as a tailorable and versatile three-dimensional culture platform to investigate the effect of biofunctionalization on stem cell behavior.


Assuntos
Materiais Biomiméticos/farmacologia , Caderinas/química , Condrogênese/efeitos dos fármacos , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Peptídeos/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Sequência de Aminoácidos , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Condrogênese/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Modelos Biológicos , Peptídeos/química , beta Catenina/metabolismo
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