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1.
Proc Natl Acad Sci U S A ; 117(12): 6784-6791, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152109

RESUMO

Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.


Assuntos
Caderinas/química , Caderinas/metabolismo , Microscopia Crioeletrônica/métodos , Enterovirus/química , Enterovirus/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Virais/metabolismo , Enterovirus/classificação , Infecções por Enterovirus/virologia , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica
2.
Nat Commun ; 11(1): 472, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980653

RESUMO

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.


Assuntos
Junções Aderentes/fisiologia , Junções Aderentes/efeitos da radiação , Actomiosina/química , Animais , Antígenos CD/química , Fenômenos Biomecânicos , Caderinas/química , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/efeitos da radiação , Células Epiteliais/ultraestrutura , Humanos , Luz , Microscopia de Força Atômica , Fenômenos Ópticos , Processos Fotoquímicos , Xenopus laevis/embriologia , alfa Catenina/química
3.
BMC Cancer ; 20(1): 62, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992226

RESUMO

BACKGROUND: Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1α, stemness genes and EMT markers in glioma cells. Here, we reveal NFкB (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells. METHODS: In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0 kb FAT1 promoter (- 3220 bp to + 848 bp w.r.t. TSS + 1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells. RESULTS: Expression levels of NFкB (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n = 16), REMBRANDT GBM-database (n = 214) and TCGA GBM-database (n = 153). In addition to glioma, positive correlation between NFкB (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0 kb FAT1-promoter-construct [- 3220 bp/+ 848 bp, transcription start site (TSS) + 1, having 17 NFкB (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [- 200 bp/+ 848 bp, with 3-NFкB (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NFкB (RelA)-activators [severe-hypoxia/TNF-α/ectopic-NFкB (RelA) + IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NFкB (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NFкB (RelA)-motif at - 90 bp/- 80 bp [pGL3F1δ1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90 bp/- 80 bp and ChIP assay for endogenous-NFкB (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NFкB (RelA). CONCLUSION: Since FAT1 and NFкB (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1α/EMT/stemness in tumors, targeting the NFкB (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.


Assuntos
Neoplasias Encefálicas/metabolismo , Caderinas/genética , Glioma/metabolismo , Fator de Transcrição RelA/metabolismo , Sítios de Ligação , Neoplasias Encefálicas/genética , Caderinas/química , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Simulação por Computador , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Transdução de Sinais
4.
J Hum Genet ; 65(4): 397-410, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31911611

RESUMO

Molecular diagnosis of rare inherited palmoplantar keratoderma (PPK) is still challenging. We investigated at the clinical and genetic level a consanguineous Tunisian family presenting an autosomal dominant atypical form of transgrediens and progrediens PPK to better characterize this ultrarare disease and to identify its molecular etiology. Whole-exome sequencing (WES), filtering strategies, and bioinformatics analysis have been achieved. Clinical investigation and follow up over 13 years of this Tunisian family with three siblings formerly diagnosed as an autosomal recessive form of Mal de Melela-like conducted us to reconsider its initial phenotype. Indeed, the three patients presented clinical features that overlap both Mal de Meleda and progressive symmetric erythrokeratoderma (PSEK). The mode of inheritance was also reconsidered, since the mother, initially classified as unaffected, exhibited a similar expression of the disease. WES analysis showed the absence of potentially functional rare variants in known PPKs or PSEK-related genes. Results revealed a novel heterozygous nonsynonymous variant in cadherin-12 gene (CDH12, NM_004061, c.1655C > A, p.Thr552Asn) in all affected family members. This variant is absent in dbSNP and in 50 in-house control exomes. In addition, in silico analysis of the mutated 3D domain structure predicted that this variant would result in cadherin-12 protein destabilization and thermal instability. Functional annotation and biological network construction data provide further supporting evidence for the potential role of CDH12 in the maintenance of skin integrity. Taken together, these results suggest that CDH12 gene is a potential candidate gene for an atypical presentation of an autosomal dominant form of transgrediens and progrediens PPK.


Assuntos
Caderinas , Transtornos Cromossômicos , Eritroceratodermia Variável , Genes Dominantes , Mutação de Sentido Incorreto , Adulto , Idoso , Caderinas/química , Caderinas/genética , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Simulação por Computador , Eritroceratodermia Variável/genética , Eritroceratodermia Variável/patologia , Feminino , Humanos , Masculino , Domínios Proteicos , Pele/patologia , Sequenciamento Completo do Exoma
5.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 12): 750-757, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31797817

RESUMO

The cryo-electron microscopy (cryo-EM) structure of the complex between the trimeric human adenovirus B serotype 3 fibre knob and human desmoglein 2 fragments containing cadherin domains EC2 and EC3 has been published, showing 3:1 and 3:2 complexes. Here, the crystal structure determined at 4.5 Šresolution is presented with one EC2-EC3 desmoglein fragment bound per fibre knob monomer in the asymmetric unit, leading to an apparent 3:3 stoichiometry. However, in concentrated solution the 3:2 complex is predominant, as shown by small-angle X-ray scattering (SAXS), while cryo-EM at lower concentrations showed a majority of the 3:1 complex. Substitution of the calcium ions bound to the desmoglein domains by terbium ions allowed confirmation of the X-ray model using their anomalous scattering and shows that at least one binding site per cluster of calcium ions is intact and exchangeable and, combined with SAXS data, that the cadherin domains are folded even in the distal part that is invisible in the cryo-EM reconstruction.


Assuntos
Adenovírus Humanos/metabolismo , Caderinas/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Desmogleína 2/química , Desmogleína 2/metabolismo , Adenovírus Humanos/classificação , Sequência de Aminoácidos , Caderinas/química , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Sorogrupo
6.
Proc Natl Acad Sci U S A ; 116(43): 21545-21555, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31591245

RESUMO

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•ß-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and ß-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of ß-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.


Assuntos
Caderinas/química , Caderinas/metabolismo , Mecanotransdução Celular , alfa Catenina/química , alfa Catenina/metabolismo , beta Catenina/química , beta Catenina/metabolismo , Junções Aderentes/química , Junções Aderentes/genética , Junções Aderentes/metabolismo , Motivos de Aminoácidos , Caderinas/genética , Humanos , Conformação Molecular , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , alfa Catenina/genética , beta Catenina/genética
7.
Commun Biol ; 2: 354, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583286

RESUMO

Cadherins form a large family of calcium-dependent adhesive proteins involved in morphogenesis, cell differentiation, and neuronal connectivity. Non-clustered δ1 protocadherins form a cadherin subgroup of proteins with seven extracellular cadherin (EC) repeats and cytoplasmic domains distinct from those of classical cadherins. Non-clustered δ1 protocadherins mediate homophilic adhesion and have been implicated in various diseases including asthma, autism, and cancer. Here we present X-ray crystal structures of human Protocadherin-1 (PCDH1), a δ1-protocadherin member essential for New World Hantavirus infection that is typically expressed in the brain, airway epithelium, skin keratinocytes, and lungs. The structures suggest a binding mode that involves antiparallel overlap of repeats EC1 to EC4. Mutagenesis combined with binding assays and biochemical experiments validated this mode of adhesion. Overall, these results reveal the molecular mechanism underlying adhesiveness of PCDH1 and δ1-protocadherins, also shedding light on PCDH1's role in maintaining airway epithelial integrity, the loss of which causes respiratory diseases.


Assuntos
Caderinas/metabolismo , Transtornos Respiratórios/etiologia , Transtornos Respiratórios/metabolismo , Caderinas/química , Adesão Celular , Dissulfetos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Transtornos Respiratórios/patologia
8.
Proc Natl Acad Sci U S A ; 116(36): 17825-17830, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31431536

RESUMO

Clustered protocadherins, a large family of paralogous proteins that play important roles in neuronal development, provide an important case study of interaction specificity in a large eukaryotic protein family. A mammalian genome has more than 50 clustered protocadherin isoforms, which have remarkable homophilic specificity for interactions between cellular surfaces. A large antiparallel dimer interface formed by the first 4 extracellular cadherin (EC) domains controls this interaction. To understand how specificity is achieved between the numerous paralogs, we used a combination of structural and computational approaches. Molecular dynamics simulations revealed that individual EC interactions are weak and undergo binding and unbinding events, but together they form a stable complex through polyvalency. Strongly evolutionarily coupled residue pairs interacted more frequently in our simulations, suggesting that sequence coevolution can inform the frequency of interaction and biochemical nature of a residue interaction. With these simulations and sequence coevolution, we generated a statistical model of interaction energy for the clustered protocadherin family that measures the contributions of all amino acid pairs at the interface. Our interaction energy model assesses specificity for all possible pairs of isoforms, recapitulating known pairings and predicting the effects of experimental changes in isoform specificity that are consistent with literature results. Our results show that sequence coevolution can be used to understand specificity determinants in a protein family and prioritize interface amino acid substitutions to reprogram specific protein-protein interactions.


Assuntos
Caderinas/química , Caderinas/metabolismo , Caderinas/genética , Evolução Molecular , Variação Genética , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Relação Estrutura-Atividade
9.
Mol Med Rep ; 20(3): 2609-2616, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322239

RESUMO

The aim of the present study was to elucidate the role of the non­syndromic autosomal recessive deafness 12 allelic variant of cadherin 23 (CDH23) in Chinese patients with non­syndromic hearing loss. The present study focused on a Chinese family with hearing loss in which there were two siblings with autosomal, recessive deafness, ranging from severe to profound hearing loss over all frequencies. DNA sequencing was used to assess the genetic factors in the disease etiology. The data revealed a compound heterozygous mutation of CDH23 in both patients. Genetic CDH23 variants are known to be responsible for non­syndromic hearing loss, and CDH23 variants frequently occur in various populations, including Japanese and Republic of Korean. Results from the present study, indicated a significant contribution of CDH23 variants to the non­syndromic hearing loss in Chinese patients.


Assuntos
Caderinas/genética , Perda Auditiva Neurossensorial/genética , Adolescente , Sequência de Aminoácidos , Grupo com Ancestrais do Continente Asiático/genética , Caderinas/química , Feminino , Perda Auditiva/genética , Heterozigoto , Humanos , Masculino , Modelos Moleculares , Mutação , Linhagem , Alinhamento de Sequência , Irmãos , Adulto Jovem
10.
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
11.
PLoS Comput Biol ; 15(6): e1007041, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31158220

RESUMO

Cadherins are homophilic cell-cell adhesion molecules whose aberrant expression has often been shown to correlate with different stages of tumor progression. In this work, we investigate the interaction of two peptidomimetic ligands with the extracellular portion of human E-cadherin using a combination of NMR and computational techniques. Both ligands have been previously developed as mimics of the tetrapeptide sequence Asp1-Trp2-Val3-Ile4 of the cadherin adhesion arm, and have been shown to inhibit E-cadherin-mediated adhesion in epithelial ovarian cancer cells with millimolar potency. To sample a set of possible interactions of these ligands with the E-cadherin extracellular portion, STD-NMR experiments in the presence of two slightly different constructs, the wild type E-cadherin-EC1-EC2 fragment and the truncated E-cadherin-(Val3)-EC1-EC2 fragment, were carried out at three temperatures. Depending on the protein construct, a different binding epitope of the ligand and also a different temperature effect on STD signals were observed, both suggesting an involvement of the Asp1-Trp2 protein sequence among all the possible binding events. To interpret the experimental results at the atomic level and to probe the role of the cadherin adhesion arm in the dynamic interaction with the peptidomimetic ligand, a computational protocol based on docking calculations and molecular dynamics simulations was applied. In agreement with NMR data, the simulations at different temperatures unveil high variability/dynamism in ligand-cadherin binding, thus explaining the differences in ligand binding epitopes. In particular, the modulation of the signals seems to be dependent on the protein flexibility, especially at the level of the adhesive arm, which appears to participate in the interaction with the ligand. Overall, these results will help the design of novel cadherin inhibitors that might prevent the swap dimer formation by targeting both the Trp2 binding pocket and the adhesive arm residues.


Assuntos
Caderinas , Biologia Computacional/métodos , Espectroscopia de Ressonância Magnética/métodos , Peptidomiméticos , Caderinas/química , Caderinas/metabolismo , Humanos , Ligantes , Simulação de Dinâmica Molecular , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Ligação Proteica
12.
Int J Mol Sci ; 20(11)2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31195621

RESUMO

E-cadherin is the core component of epithelial adherens junctions, essential for tissue development, differentiation, and maintenance. It is also fundamental for tissue barrier formation, a critical function of epithelial tissues. The colon or large intestine is lined by an epithelial monolayer that encompasses an E-cadherin-dependent barrier, critical for the homeostasis of the organ. Compromised barriers of the colonic epithelium lead to inflammation, fibrosis, and are commonly observed in colorectal cancer. In addition to its architectural role, E-cadherin is also considered a tumor suppressor in the colon, primarily a result of its opposing function to Wnt signaling, the predominant driver of colon tumorigenesis. Beyond these well-established traditional roles, several studies have portrayed an evolving role of E-cadherin as a signaling epicenter that regulates cell behavior in response to intra- and extra-cellular cues. Intriguingly, these recent findings also reveal tumor-promoting functions of E-cadherin in colon tumorigenesis and new interacting partners, opening future avenues of investigation. In this Review, we focus on these emerging aspects of E-cadherin signaling, and we discuss their implications in colon biology and disease.


Assuntos
Caderinas/química , Caderinas/metabolismo , Colo/metabolismo , Doenças do Colo/metabolismo , Homeostase , Transdução de Sinais , Animais , Colo/microbiologia , Doenças do Colo/microbiologia , Microbioma Gastrointestinal , Humanos
13.
Proc Natl Acad Sci U S A ; 116(22): 11048-11056, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31072932

RESUMO

Hair cells, the sensory receptors of the inner ear, respond to mechanical forces originating from sounds and accelerations. An essential feature of each hair cell is an array of filamentous tip links, consisting of the proteins protocadherin 15 (PCDH15) and cadherin 23 (CDH23), whose tension is thought to directly gate the cell's transduction channels. These links are considered far too stiff to represent the gating springs that convert hair bundle displacement into forces capable of opening the channels, and no mechanism has been suggested through which tip-link stiffness could be varied to accommodate hair cells of distinct frequency sensitivity in different receptor organs and animals. Consequently, the gating spring's identity and mechanism of operation remain central questions in sensory neuroscience. Using a high-precision optical trap, we show that an individual monomer of PCDH15 acts as an entropic spring that is much softer than its enthalpic stiffness alone would suggest. This low stiffness implies that the protein is a significant part of the gating spring that controls a hair cell's transduction channels. The tip link's entropic nature then allows for stiffness control through modulation of its tension. We find that a PCDH15 molecule is unstable under tension and exhibits a rich variety of reversible unfolding events that are augmented when the Ca2+ concentration is reduced to physiological levels. Therefore, tip link tension and Ca2+ concentration are likely parameters through which nature tunes a gating spring's mechanical properties.


Assuntos
Caderinas/química , Caderinas/metabolismo , Elasticidade/fisiologia , Células Ciliadas Auditivas/fisiologia , Animais , Orelha Interna/fisiologia , Células HEK293 , Humanos , Camundongos , Pinças Ópticas
14.
Nature ; 569(7755): 280-283, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30971825

RESUMO

Neurite self-recognition and avoidance are fundamental properties of all nervous systems1. These processes facilitate dendritic arborization2,3, prevent formation of autapses4 and allow free interaction among non-self neurons1,2,4,5. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, ß- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities1,2,4-13. Avoidance is observed between neurons that express identical protocadherin repertoires2,5, and single-isoform differences are sufficient to prevent self-recognition10. Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers10,14-20. Although these interactions have previously been characterized in isolation15,17-20, structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains11.


Assuntos
Caderinas/metabolismo , Caderinas/ultraestrutura , Microscopia Crioeletrônica , Neurônios/química , Neurônios/metabolismo , Animais , Caderinas/química , Caderinas/genética , Cristalografia por Raios X , Lipossomos/química , Lipossomos/metabolismo , Camundongos , Modelos Moleculares , Neurônios/ultraestrutura , Domínios Proteicos , Multimerização Proteica
15.
J Biol Chem ; 294(20): 7990-8000, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30936204

RESUMO

Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery. Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer. H. pylori expresses a series of virulence factors in the host, among which high-temperature requirement A (HpHtrA) is a newly identified serine protease secreted by H. pylori. HpHtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctions, allowing H. pylori to access the intercellular space. Here we report the first crystal structure of HpHtrA at 3.0 Å resolution. The structure revealed a new type of HtrA protease trimer stabilized by unique N-terminal domain swapping distinct from other known HtrA homologs. We further observed that truncation of the N terminus completely abrogates HpHtrA trimer formation as well as protease activity. In the presence of unfolded substrate, HpHtrA assembled into cage-like 12-mers or 24-mers. Combining crystallographic, biochemical, and mutagenic data, we propose a mechanistic model of how HpHtrA recognizes and cleaves the well-folded E-cadherin substrate. Our study provides a fundamental basis for the development of anti-H. pylori agents by using a previously uncharacterized HtrA protease as a target.


Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/enzimologia , Modelos Biológicos , Serina Endopeptidases/química , Fatores de Virulência/química , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caderinas/química , Caderinas/genética , Caderinas/metabolismo , Cristalografia por Raios X , Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , Humanos , Domínios Proteicos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Acta Biomater ; 90: 412-423, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30951897

RESUMO

Intracellular transport is fundamental for neuronal function and development and is dependent on the formation of stable actin filaments. N-cadherin, a cell-cell adhesion protein, is actively involved in neuronal growth and actin cytoskeleton organization. Various groups have explored how neurons behaved on substrates engineered to present N-cadherin; however, few efforts have been made to examine how these surfaces modulate neuronal intracellular transport. To address this issue, we assembled a substrate to which recombinant N-cadherin molecules are physiosorbed using graphene oxide (GO) or reduced graphene oxide (rGO). N-cadherin physisorbed on GO and rGO led to a substantial enhancement of intracellular mass transport along neurites relative to N-cadherin on glass, due to increased neuronal adhesion, neurite extensions, dendritic arborization and glial cell adhesion. This study will be broadly useful for recreating active neural tissues in vitro and for improving our understanding of the development, homeostasis, and physiology of neurons. STATEMENT OF SIGNIFICANCE: Intracellular transport of proteins and chemical cues is extremely important for culturing neurons in vitro, as they replenish materials within and facilitate communication between neurons. Various studies have shown that intracellular transport is dependent on the formation of stable actin filaments. However, the extent to which cadherin-mediated cell-cell adhesion modulates intracellular transport is not heavily explored. In this study, N-cadherin was adsorbed onto graphene oxide-based substrates to understand the role of cadherin at a molecular level and the intracellular transport within cells was examined using spatial light interference microscopy. As such, the results of this study will serve to better understand and harness the role of cell-cell adhesion in neuron development and regeneration.


Assuntos
Caderinas , Grafite , Proteínas do Tecido Nervoso , Neuritos/metabolismo , Neurogênese/efeitos dos fármacos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Caderinas/química , Caderinas/farmacologia , Adesão Celular/efeitos dos fármacos , Grafite/química , Grafite/farmacologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/farmacologia , Ratos , Ratos Long-Evans
18.
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
19.
Metallomics ; 11(5): 914-924, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-30848261

RESUMO

Lanthanides are rare-earth metals with a broad range of applications in biological research and medicine. In addition to their unique magnetic and spectroscopic properties, lanthanides are also effective mimics of calcium and can stimulate or inhibit the function of calcium-binding proteins. Cadherins are a large family of calcium-binding proteins that facilitate cell adhesion and play key roles in embryo development, tissue homeostasis and tumour metastasis. However, whether lanthanides can bind cadherins and functionally replace calcium binding has not been comprehensively explored. In this study, we investigated the effect of lanthanide binding on cadherin structure and function using terbium, which is a commonly used lanthanide for protein spectroscopy and a proposed anti-cancer agent. We demonstrate that terbium can compete with calcium for binding to calcium-binding sites in cadherins. Terbium binding to cadherins abolished their cell adhesive activity and rendered cadherins sensitive to proteolysis by trypsin. Molecular dynamics simulations indicate that replacement of calcium by terbium results in structural rearrangements and increases the flexibility of the cadherin ectodomain. These changes in structure and dynamics are likely to underlie the inability of lanthanide-bound cadherins to support cell adhesion. Taken together, our findings further knowledge on lanthanide interactions with calcium-binding proteins and provide new insight into the influence of metal chemistry on cadherin structure, dynamics and function.


Assuntos
Caderinas/metabolismo , Cálcio/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Animais , Células CHO , Caderinas/química , Adesão Celular , Agregação Celular , Cricetulus , Humanos , Íons , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteólise , Térbio/metabolismo , Tripsina/metabolismo
20.
Electrophoresis ; 40(9): 1273-1281, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30702765

RESUMO

During SDS-PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues-long, sequence-homologous, structurally-analogous, extracellular domains of human E- and N-cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively-charged and positively-charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater-than-expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS-protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.


Assuntos
Caderinas/química , Eletroforese em Gel de Poliacrilamida/métodos , Domínios Proteicos , Proteínas/análise , Eletricidade Estática , Ácidos , Humanos , Peso Molecular , Erro Experimental , Dodecilsulfato de Sódio
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