RESUMEN
Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein his6EGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the in silico binding study, where the analogue of ephrinA, his6EGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The his6EGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.
Asunto(s)
Azurina , Neoplasias de la Próstata , Receptor EphA6 , Masculino , Humanos , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Azurina/genética , Medicina de Precisión , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Efrinas/química , Efrinas/metabolismoRESUMEN
Eph receptors, the largest known family of receptor tyrosine kinases, and ephrin ligands have been implicated in a variety of human cancers. The novel bidirectional signaling events initiated by binding of Eph receptors to their cognate ephrin ligands modulate many cellular processes such as proliferation, metastasis, angiogenesis, invasion, and apoptosis. The relationships between the abundance of a unique subset of Eph receptors and ephrin ligands with associated cellular processes indicate a key role of these molecules in tumorigenesis. The combinatorial expression of these molecules converges on MAP kinase and/or AKT/mTOR signaling pathways. The intracellular target proteins of the initial signal may, however, vary in some cancers. Furthermore, we have also described the commonality of up- and down-regulation of individual receptors and ligands in various cancers. The current state of research in Eph receptors illustrates MAP kinase and mTOR pathways as plausible targets for therapeutic interventions in various cancers.
Asunto(s)
Neoplasias , Receptores de la Familia Eph , Humanos , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Efrinas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ligandos , Neoplasias/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Serina-Treonina Quinasas TORRESUMEN
Erythropoietin-producing hepatoma (Eph) receptors belong to a family of tyrosine kinase receptors that plays a pivotal role in the development of the brain. Eph can be divided broadly into two groups, namely, EphA and EphB, comprising nine and five members, respectively. In recent years, the role of EphA-4 has become increasingly apparent in the onset of Alzheimer's disease (AD). Emerging evidence suggests that EphA-4 results in synaptic dysfunction, which in turn promotes the progression of AD. Moreover, pharmacological or genetic ablation of EphA-4 in the murine model of AD can alleviate the symptoms. The current review summarizes different pathways by which EphA-4 can influence pathogenesis. Since, majority of the studies had reported the protective effect of EphA-4 inhibition during AD, designing therapeutics based on decreasing its enzymatic activity might be necessary for introducing the novel interventions. Therefore, the review described peptide and nanobodies inhibitors of EphA-4 that exhibit the potential to modulate EphA-4 and could be used as lead molecules for the targeted therapy of AD.
Asunto(s)
Enfermedad de Alzheimer , Animales , Humanos , Ratones , Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/metabolismo , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismoRESUMEN
Eph receptors are the largest family of receptor tyrosine kinases and by interactions with ephrin ligands mediate a myriad of processes from embryonic development to adult tissue homeostasis. The interaction of Eph receptors, especially at their transmembrane (TM) domains is key to understanding their mechanism of signal transduction across cellular membranes. We review the structural and functional aspects of EphA1/A2 association and the techniques used to investigate their TM domains: NMR, molecular modelling/dynamics simulations and fluorescence. We also introduce transmembrane peptides, which can be used to alter Eph receptor signaling and we provide a perspective for future studies.
Asunto(s)
Membrana Celular/metabolismo , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos/fisiologíaRESUMEN
EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane (JM) region, pseudokinase and SAM domains. Using small-angle X-ray scattering and cross-linking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosine residues in the JM region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signalling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically targeted to counter oncogenic signalling.
Asunto(s)
Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Transducción de Señal/genética , Motivo alfa Estéril/genética , Dominios Homologos src/genética , Adenosina Trifosfato/metabolismo , Animales , Humanos , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Inhibidores de Proteínas Quinasas/metabolismo , Receptores de la Familia Eph/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera/citología , Tirosina/metabolismoRESUMEN
In two proof-of-concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA-interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss-of-function mutants of the DNA modulating domain of DEK [1]. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidad , Pruebas de Toxicidad/métodos , Animales , Sesgo , Biocatálisis , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/toxicidad , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/toxicidad , Escherichia coli/genética , Humanos , Mutación con Pérdida de Función , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas Oncogénicas/toxicidad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/toxicidad , Dominios Proteicos/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/toxicidad , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Receptores de la Familia Eph/toxicidad , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reproducibilidad de los Resultados , Factores de Tiempo , Pruebas de Toxicidad/normasRESUMEN
Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
Asunto(s)
Moléculas de Adhesión Celular/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Humanos , Ratones , Modelos Moleculares , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/antagonistas & inhibidores , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Spodoptera , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
Eph receptors, the largest subfamily of receptor tyrosine kinases, are linked with proliferative disease, such as cancer, as a result of their deregulated expression or mutation. Unlike other tyrosine kinases that have been clinically targeted, the development of therapeutics against Eph receptors remains at a relatively early stage. The major reason is the limited understanding on the Eph receptor regulatory mechanisms at a molecular level. The complexity in understanding Eph signalling in cells arises due to following reasons: (1) Eph receptors comprise 14 members, two of which are pseudokinases, EphA10 and EphB6, with relatively uncharacterised function; (2) activation of Eph receptors results in dimerisation, oligomerisation and formation of clustered signalling centres at the plasma membrane, which can comprise different combinations of Eph receptors, leading to diverse downstream signalling outputs; (3) the non-catalytic functions of Eph receptors have been overlooked. This review provides a structural perspective of the intricate molecular mechanisms that drive Eph receptor signalling, and investigates the contribution of intra- and inter-molecular interactions between Eph receptors intracellular domains and their major binding partners. We focus on the non-catalytic functions of Eph receptors with relevance to cancer, which are further substantiated by exploring the role of the two pseudokinase Eph receptors, EphA10 and EphB6. Throughout this review, we carefully analyse and reconcile the existing/conflicting data in the field, to allow researchers to further the current understanding of Eph receptor signalling.
Asunto(s)
Receptores de la Familia Eph/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Catálisis , Humanos , Ligandos , Receptores de la Familia Eph/química , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Animals have a greater diversity of signalling pathways than their unicellular relatives, consistent with the evolution and expansion of these pathways occurring in parallel with the origin of animal multicellularity. However, the genomes of sponges and ctenophores - non-bilaterian basal animals - typically encode no, or far fewer, recognisable signalling ligands compared to bilaterians and cnidarians. For instance, the largest subclass of receptor tyrosine kinases (RTKs) in bilaterians, the Eph receptors (Ephs), are present in sponges and ctenophores, but their cognate ligands, the ephrins, have not yet been detected. RESULTS: Here, we use an iterative HMM analysis to identify for the first time membrane-bound ephrins in sponges and ctenophores. We also expand the number of Eph-receptor subtypes identified in these animals and in cnidarians. Both sequence and structural analyses are consistent with the Eph ligand binding domain (LBD) and the ephrin receptor binding domain (RBD) having evolved via the co-option of ancient galactose-binding (discoidin-domain)-like and monodomain cupredoxin domains, respectively. Although we did not detect a complete Eph-ephrin signalling pathway in closely-related unicellular holozoans or in other non-metazoan eukaryotes, truncated proteins with Eph receptor LBDs and ephrin RBDs are present in some choanoflagellates. Together, these results indicate that Eph-ephrin signalling was present in the last common ancestor of extant metazoans, and perhaps even in the last common ancestor of animals and choanoflagellates. Either scenario pushes the origin of Eph-ephrin signalling back much earlier than previously reported. CONCLUSIONS: We propose that the Eph-LBD and ephrin-RBD, which were ancestrally localised in the cytosol, became linked to the extracellular parts of two cell surface proteins before the divergence of sponges and ctenophores from the rest of the animal kingdom. The ephrin-RBD lost the ancestral capacity to bind copper, and the Eph-LBD became linked to an ancient RTK. The identification of divergent ephrin ligands in sponges and ctenophores suggests that these ligands evolve faster than their cognate receptors. As this may be a general phenomena, we propose that the sequence-structure approach used in this study may be usefully applied to other signalling systems where no, or a small number of, ligands have been identified.
Asunto(s)
Ctenóforos/metabolismo , Efrinas/metabolismo , Poríferos/metabolismo , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Evolución Molecular , Humanos , Ligandos , Filogenia , Unión Proteica , Dominios Proteicos , Receptores de la Familia Eph/químicaRESUMEN
Ephrin (Eph) receptor tyrosine kinases have evolutionarily diverged from other tyrosine kinases to respond to specific activation and regulatory signals that require close coupling of kinase catalytic and regulatory functions. However, the evolutionary basis for such functional coupling is not fully understood. We employed an evolutionary systems approach involving statistical mining of large sequence and structural data sets to define the hallmarks of Eph kinase evolution and functional specialization. We found that some of the most distinguishing Eph-specific residues structurally tether the flanking juxtamembrane and sterile α motif (SAM) linker regions to the kinase domain, and substitutions of these residues in EphA3 resulted in faster kinase activation. We report for the first time that the SAM domain linker is functionally coupled to the juxtamembrane through co-conserved residues in the kinase domain and that together these residues provide a structural framework for coupling catalytic and regulatory functions. The unique organization of Eph-specific tethering networks and the identification of other Eph-specific sequence features of unknown functions provide new hypotheses for future functional studies and new clues to disease mutations altering Eph kinase-specific functions.
Asunto(s)
Receptores de la Familia Eph/química , Motivo alfa Estéril , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Activación Enzimática , Evolución Molecular , Humanos , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , Receptores de la Familia Eph/genética , Alineación de SecuenciaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus associated with Kaposi's sarcoma and two B-cell malignancies. The rhesus monkey rhadinovirus (RRV) is a virus of nonhuman primates that is closely related to KSHV. Eph family receptor tyrosine kinases (Ephs) are cellular receptors for the gH/gL glycoprotein complexes of both KSHV and RRV. Through sequence analysis and mutational screens, we identified conserved residues in the N-terminal domain of KSHV and RRV glycoprotein H that are critical for Eph-binding in vitro. Homology-based structural predictions of the KSHV and RRV gH/gL complexes based on the Epstein-Barr-Virus gH/gL crystal structure located these amino acids in a beta-hairpin on gH, which is likely stabilized by gL and is optimally positioned for protein-protein interactions. Guided by these predictions, we generated recombinant RRV and KSHV strains mutated in the conserved motif as well as an RRV gL null mutant. Inhibition experiments using these mutants confirmed that disruption of the identified Eph-interaction motif or of gL expression resulted in complete detargeting from Ephs. However, all mutants were infectious on all cell types tested, exhibiting normal attachment but a reduction in infectivity of up to one log order of magnitude. While Eph-binding-negative RRV mutants were replication-competent on fibroblasts, their infectivity was comparatively more reduced on endothelial cells with a substantial subpopulation of endothelial cells remaining resistant to infection. Together, this provides evidence for a cell type-specific use of Ephs by RRV. Furthermore, our results demonstrate that gL is dispensable for infection by RRV. Its deletion caused a reduction in infectivity similar to that observed after mutation of Eph-binding residues in gH. Our findings would be compatible with an ability of KSHV and RRV to use other, less efficient entry mediators in lieu of Ephs, although these host factors may not be uniformly expressed by all cells.
Asunto(s)
Herpesvirus Humano 8/metabolismo , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Células A549 , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Macaca mulatta , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Receptores de la Familia Eph/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genéticaRESUMEN
Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization-condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.
Asunto(s)
Efrinas/metabolismo , Multimerización de Proteína , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Células HEK293 , Humanos , Polimerizacion , Receptores de la Familia Eph/químicaRESUMEN
The Eph receptor-ephrin system is an emerging target for the development of novel anti-angiogenic therapies. Research programs aimed at developing small-molecule antagonists of the Eph receptors are still in their initial stage as available compounds suffer from pharmacological drawbacks, limiting their application in vitro and in vivo. In the present work, we report the design, synthesis and evaluation of structure-activity relationships of a class of Δ(5)-cholenoyl-amino acid conjugates as Eph-ephrin antagonists. As a major achievement of our exploration, we identified N-(3ß-hydroxy-Δ(5)-cholen-24-oyl)-L-tryptophan (UniPR1331) as the first small molecule antagonist of the Eph-ephrin system effective as an anti-angiogenic agent in endothelial cells, bioavailable in mice by the oral route and devoid of biological activity on G protein-coupled and nuclear receptors targeted by bile acid derivatives.
Asunto(s)
Aminoácidos/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Efrinas/antagonistas & inhibidores , Receptores de la Familia Eph/antagonistas & inhibidores , Aminoácidos/síntesis química , Aminoácidos/química , Inhibidores de la Angiogénesis/síntesis química , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Efrinas/química , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Receptores de la Familia Eph/química , Relación Estructura-ActividadRESUMEN
The Eph receptors are the largest sub-family of Receptor Tyrosine Kinases (RTK). They, together with their ephrin ligands, play central roles in cell-cell communication during development, and also in the maintenance of a normal adult physiology. Their malfunction, therefore, can contribute to various human diseases. Since the structures of the Eph receptors and ephrins are by now well characterized, there has been extensive recent work to develop ways to manipulate their action in order to achieve therapeutic benefits. Although few reagents have progressed to clinical trials thus far, it is evident that the Eph receptors are valid targets for therapeutic drugs. In this review we first summarize studies on the three-dimensional structures of Eph receptors. We then give an overview on small molecule inhibitors and activators using Ephs as targets. We put a special focus on the latest developments in the field of monoclonal antibodies and antibody fragments for inhibiting or activating the Eph/ephrin signaling.
Asunto(s)
Descubrimiento de Drogas , Efrinas/metabolismo , Receptores de la Familia Eph/metabolismo , Anticuerpos de Cadena Única/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Sitios de Unión , Humanos , Modelos Moleculares , Conformación Proteica , Receptores de la Familia Eph/agonistas , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/química , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
UNLABELLED: The ~ 800 kDa laminin heterotrimer forms a distinctive cross-shaped structure that further self-assembles into networks within the extracellular matrix. The domains at the laminin chain termini, which engage in network formation and cell-surface interaction, are well understood both structurally and functionally. By contrast, the structures and roles of additional domains embedded within the limbs of the laminin cross have remained obscure. Here, we report the X-ray crystal structure, determined to 1.2 Å resolution, of the human laminin α2 subunit L4b domain, site of an inframe deletion mutation associated with mild congenital muscular dystrophy. The α2 L4b domain is an irregular ß-sandwich with many short and broken strands linked by extended loops. The most similar known structures are the carbohydrate-binding domains of bacterial cellulases, the ephrin-binding domain of ephrin receptors, and MAM adhesion domains in various other eukaryotic cell-surface proteins. This similarity to mammalian adhesion modules, which was not predicted on the basis of amino acid sequence alone due to lack of detectable homology, suggests that laminin internal domains evolved from a progenitor adhesion molecule and may retain a role in cell adhesion in the context of the laminin trimer. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/) under codes 4YEP and 4YEQ.
Asunto(s)
Laminina/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Matriz Extracelular/metabolismo , Glicoproteínas/química , Humanos , Laminina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de la Familia Eph/química , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Eph receptors and their ligands, ephrins, represent the largest group of the receptor tyrosine kinase (RTK) family, and they mediate numerous developmental processes in a variety of organisms. Ephrins are membrane-bound proteins that are mainly divided into two classes: A class ephrins, which are linked to the membrane by a glycosylphosphatidylinositol (GPI) linkage, and B class ephrins, which are transmembrane ligands. Based on their domain structures and affinities for ligand binding, the Eph receptors are also divided into two groups. Trans-dimerization of Eph receptors with their membrane-tethered ligands regulates cell-cell interactions and initiates bidirectional signaling pathways. These pathways are intimately involved in regulating cytoskeleton dynamics, cell migration, and alterations in cellular dynamics and shapes. The EphBs and ephrinBs are specifically localized and modified to promote higher-order clustering and initiate of bidirectional signaling. In this review, we present an in-depth overview of the structure, mechanisms, cell signaling, and functions of EphB/ephrinB in cell adhesion and migration.
Asunto(s)
Adhesión Celular , Movimiento Celular , Efrinas/metabolismo , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Animales , Citoesqueleto/fisiología , Desarrollo Embrionario , Efrinas/química , Receptores de la Familia Eph/químicaRESUMEN
Eph receptor tyrosine kinases control cell-cell interactions during normal and oncogenic development, and are implicated in a range of processes including angiogenesis, stem cell maintenance and metastasis. They are thus of great interest as targets for cancer therapy. EphA3, originally isolated from leukemic and melanoma cells, is presently one of the most promising therapeutic targets, with multiple tumor-promoting roles in a variety of cancer types. This review focuses on EphA3, its functions in controlling cellular behavior, both in normal and pathological development, and most particularly in cancer.
Asunto(s)
Neoplasias/metabolismo , Receptores de la Familia Eph/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/química , Receptores de la Familia Eph/genéticaRESUMEN
The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.
Asunto(s)
Receptor TIE-2/fisiología , Receptores de la Familia Eph/fisiología , Transducción de Señal , Angiopoyetinas/química , Angiopoyetinas/metabolismo , Sitios de Unión , Activación Enzimática , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína , Receptor TIE-2/química , Receptor TIE-2/metabolismo , Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismoRESUMEN
Drug designing targeting protein-protein interactions is challenging. Because structural elucidation and computational analysis have revealed the importance of hot spot residues in stabilizing these interactions, there have been on-going efforts to develop drugs which bind the hot spots and out-compete the native protein partners. The question arises as to what are the key 'druggable' properties of hot spots in protein-protein interactions and whether these mimic the general hot spot definition. Identification of orthosteric (at the protein- protein interaction site) and allosteric (elsewhere) druggable hot spots is expected to help in discovering compounds that can more effectively modulate protein-protein interactions. For example, are there any other significant features beyond their location in pockets in the interface? The interactions of protein-protein hot spots are coupled with conformational dynamics of protein complexes. Currently increasing efforts focus on the allosteric drug discovery. Allosteric drugs bind away from the native binding site and can modulate the native interactions. We propose that identification of allosteric hot spots could similarly help in more effective allosteric drug discovery. While detection of allosteric hot spots is challenging, targeting drugs to these residues has the potential of greatly increasing the hot spot and protein druggability.
Asunto(s)
Sitio Alostérico , Biología Computacional , Diseño de Fármacos , Proteínas/química , Regulación Alostérica , Humanos , Ligandos , Modelos Moleculares , Péptidos/química , Péptidos/farmacología , Unión Proteica , Receptores de la Familia Eph/química , Proteínas ras/químicaRESUMEN
Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.