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1.
J Med Chem ; 66(3): 2143-2160, 2023 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-36719862

RESUMEN

The discovery of monokinase-selective inhibitors for patients is challenging because the 500+ kinases encoded by the human genome share highly conserved catalytic domains. Until now, no selective inhibitors unique for a single transforming growth factor ß (TGFß) family transmembrane receptor kinase, including bone morphogenetic protein receptor type 2 (BMPR2), have been reported. This dearth of receptor-specific kinase inhibitors hinders therapeutic options for skeletal defects and cancer as a result of an overactivated BMP signaling pathway. By screening 4.17 billion "unbiased" and "kinase-biased" DNA-encoded chemical library molecules, we identified hits CDD-1115 and CDD-1431, respectively, that were low-nanomolar selective kinase inhibitors of BMPR2. Structure-activity relationship studies addressed metabolic lability and high-molecular-weight issues, resulting in potent and BMPR2-selective inhibitor analogs CDD-1281 (IC50 = 1.2 nM) and CDD-1653 (IC50 = 2.8 nM), respectively. Our work demonstrates that DNA-encoded chemistry technology (DEC-Tec) is reliable for identifying novel first-in-class, highly potent, and selective kinase inhibitors.


Asunto(s)
ADN , Transducción de Señal , Humanos , Biblioteca de Genes , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo
2.
Nat Commun ; 13(1): 2395, 2022 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-35504921

RESUMEN

Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and ß4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Proteínas Morfogenéticas Óseas , Proteínas Morfogenéticas Óseas/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Hipertensión Pulmonar Primaria Familiar , Humanos , Hipertensión Arterial Pulmonar , Transducción de Señal
3.
Nat Commun ; 12(1): 4950, 2021 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-34400635

RESUMEN

Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs.


Asunto(s)
Receptores de Activinas Tipo I/química , Receptores de Activinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Transducción de Señal/fisiología , Receptores de Activinas Tipo I/genética , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Proteínas Morfogenéticas Óseas/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Humanos , Ligandos , Modelos Moleculares , Mutación , Fosforilación , Unión Proteica , Dominios Proteicos , Hipertensión Arterial Pulmonar , Dispersión del Ángulo Pequeño , Transducción de Señal/genética , Proteínas Smad/metabolismo , Difracción de Rayos X
4.
Cell Rep ; 35(12): 109276, 2021 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-34161760

RESUMEN

Developing neurons initially form excessive neurites and then remodel them based on molecular cues and neuronal activity. Developing mitral cells in the olfactory bulb initially extend multiple primary dendrites. They then stabilize single primary dendrites while eliminating others. However, the mechanisms underlying selective dendrite remodeling remain elusive. Using CRISPR-Cas9-based knockout screening combined with in utero electroporation, we identify BMPR-2 as a key regulator for selective dendrite stabilization. Bmpr2 knockout and its rescue experiments show that BMPR-2 inhibits LIMK without ligands and thereby permits dendrite destabilization. In contrast, the overexpression of antagonists and agonists indicates that ligand-bound BMPR-2 stabilizes dendrites, most likely by releasing LIMK. Using genetic and FRET imaging experiments, we demonstrate that free LIMK is activated by NMDARs via Rac1, facilitating dendrite stabilization through F-actin formation. Thus, the selective stabilization of primary dendrites is ensured by concomitant inputs of BMP ligands and neuronal activity.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Dendritas/metabolismo , Bulbo Olfatorio/citología , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Proteínas Morfogenéticas Óseas/metabolismo , Ácido Glutámico/metabolismo , Ligandos , Quinasas Lim/metabolismo , Ratones Endogámicos C57BL , Dominios Proteicos , Receptores de N-Metil-D-Aspartato/metabolismo , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo
5.
J Mol Recognit ; 34(4): e2881, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33137847

RESUMEN

Transforming growth factor-ß1 (TGF-ß1) signaling pathway has been implicated in the fibroblast activation of hypertrophic scarring (HS). Previously, we proposed a new biotherapeutic strategy to combat HS by disrupting the intermolecular interaction of TGF-ß1 with its cognate type-II receptor (TßR-II). Here, we further demonstrate that the binding site of TGF-ß1 to TßR-II is not overlapped with the conformational wrist epitope and linear knuckle epitope that are traditionally recognized as the functional binding sites of bone morphogenetic protein-2 (BMP-2) to its type-II receptor (BMPR-II), which can thus be regarded as a new functional site we called elbow epitope. Structural, energetic, and dynamic investigations reveal that the elbow epitope consists of two sequentially discontinuous, spatially vicinal segments Loop30-34 and Turn90-95 ; they cannot work effectively to independently interact with TßR-II. Rational redesign of the epitope is performed using an integrated in silio-in vitro method based on crystal and modeled structure data. In the procedure, the two epitope segments are split from the interface of TGF-ß1-TßR-II complex and then connected with each other in a head-to-tail manner by adding a flexible poly-(Gly)n linker between them, thus resulting in a series of combined peptides. We found that the peptide affinity reaches maximum at n = 2, which shares a consistent binding mode with the elbow epitope at native complex interface. The linker of either too long (n > 2) or too short (n < 2) cannot properly place the gap space between the two segments, thus impairing the binding compatibility of designed peptides with TßR-II active site.


Asunto(s)
Epítopos/química , Epítopos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/inmunología , Sitios de Unión , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/inmunología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Cicatriz Hipertrófica/terapia , Polarización de Fluorescencia , Humanos , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/química , Receptor Tipo II de Factor de Crecimiento Transformador beta/inmunología , Termodinámica , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/metabolismo
6.
Protein J ; 39(6): 703-710, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33130958

RESUMEN

Bone morphogenic protein-2 (BMP-2) is the most documented member of BMP family and plays a crucial role in bone formation and growth. In this study, we systematically analyze and compare the complex crystal structures and interaction properties of BMP-2 with its cognate receptors BMPR-I/BMPR-II and with its natural antagonist crossveinless-2 (CV-2) using an integrated in silico-in vitro strategy. It is found that the antagonist-binding site is not fully overlapped with the two receptor-binding sites on BMP-2 surface; the antagonist can competitively disrupt BMP-2-BMPR-II interaction using a blocking-out-of-site manner, but has no substantial influence on BMP-2-BMPR-I interaction. Here, the antagonist-binding site is assigned as a new functional epitope armpit to differ from the traditional conformational epitope wrist and linear epitope knuckle at receptor-binding sites. Structural analysis reveals that the armpit comprises three sequentially discontinuous, structurally vicinal peptide segments, separately corresponding to a loop region and two ß-strands crawling on the protein surface. The three segments cannot work independently when splitting from the protein context, but can restore binding capability to CV-2 if they are connected to a single peptide. A systematic combination of different-length polyglycine linkers between these segments obtains a series of designed single peptides, from which several peptides that can potently interact with the armpit-recognition site of CV-2 with high affinity and specificity are identified using energetic analysis and fluorescence assay; they are expected to target BMP-2-CV-2 interaction in a self-inhibitory manner.


Asunto(s)
Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Proteína Morfogenética Ósea 2/química , Proteínas Portadoras/química , Simulación por Computador , Epítopos/química , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Humanos
7.
Sci Rep ; 9(1): 18351, 2019 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-31797984

RESUMEN

Bone morphogenetic proteins (BMPs) are secreted ligands of the transforming growth factor-ß (TGF-ß) family that control embryonic patterning, as well as tissue development and homeostasis. Loss of function mutations in the type II BMP receptor BMPR2 are the leading cause of pulmonary arterial hypertension (PAH), a rare disease of vascular occlusion that leads to high blood pressure in the pulmonary arteries. To understand the structural consequences of these mutations, we determined the crystal structure of the human wild-type BMPR2 kinase domain at 2.35 Å resolution. The structure revealed an active conformation of the catalytic domain that formed canonical interactions with the bound ligand Mg-ADP. Disease-associated missense mutations were mapped throughout the protein structure, but clustered predominantly in the larger kinase C-lobe. Modelling revealed that the mutations will destabilize the protein structure by varying extents consistent with their previously reported functional heterogeneity. The most severe mutations introduced steric clashes in the hydrophobic protein core, whereas those found on the protein surface were less destabilizing and potentially most favorable for therapeutic rescue strategies currently under clinical investigation.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Conformación Proteica , Hipertensión Arterial Pulmonar/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/ultraestructura , Cristalografía por Rayos X , Humanos , Ligandos , Mutación Missense/genética , Dominios Proteicos/genética , Hipertensión Arterial Pulmonar/patología , Transducción de Señal/genética , Relación Estructura-Actividad
8.
Sci Rep ; 7(1): 8778, 2017 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-28821740

RESUMEN

Elaborate regulatory networks of the Bone Morphogenetic Protein (BMP) pathways ensure precise signalling outcome during cell differentiation and tissue homeostasis. Here, we identified IRS4 as a novel regulator of BMP signal transduction and provide molecular insights how it integrates into the signalling pathway. We found that IRS4 interacts with the BMP receptor BMPRII and specifically targets Smad1 for proteasomal degradation consequently leading to repressed BMP/Smad signalling in C2C12 myoblasts while concomitantly activating the PI3K/Akt axis. IRS4 is present in human and primary mouse myoblasts, the expression increases during myogenic differentiation but is downregulated upon final commitment coinciding with Myogenin expression. Functionally, IRS4 promotes myogenesis in C2C12 cells, while IRS4 knockdown inhibits differentiation of myoblasts. We propose that IRS4 is particularly critical in the myoblast stage to serve as a molecular switch between BMP/Smad and Akt signalling and to thereby control cell commitment. These findings provide profound understanding of the role of BMP signalling in early myogenic differentiation and open new ways for targeting the BMP pathway in muscle regeneration.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Proteínas Smad/metabolismo , Animales , Sitios de Unión , Biomarcadores , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/química , Línea Celular , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Sustrato del Receptor de Insulina/química , Ligandos , Ratones , Modelos Biológicos , Desarrollo de Músculos , Mioblastos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-akt/química , Ratas , Proteínas Smad/química , Ubiquitinación
9.
J Biol Chem ; 292(30): 12702-12712, 2017 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-28588028

RESUMEN

Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)- but not TGFß-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knock-out of SMYD2 We further demonstrate that SMYD2 knockdown or knock-out impairs BMP-induced phosphorylation of the signal-transducing protein SMAD1/5 and SMAD1/5 nuclear localization and interaction with SMAD4. The SMYD2 KMT activity was required to facilitate BMP-mediated signal transduction, as treatment with the SMYD2 inhibitor AZ505 suppressed BMP2-induced SMAD1/5 phosphorylation. Furthermore, we present evidence that SMYD2 likely modulates the BMP response through its function in the cytosol. We show that, although SMYD2 interacted with multiple components in the BMP pathway, it specifically methylated the kinase domain of BMP type II receptor BMPR2. Taken together, our findings suggest that SMYD2 may promote BMP signaling by directly methylating BMPR2, which, in turn, stimulates BMPR2 kinase activity and activation of the BMP pathway.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Transducción de Señal , Línea Celular , Células HEK293 , Humanos , Metilación , Dominios Proteicos
10.
J Biol Chem ; 291(7): 3395-410, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26677222

RESUMEN

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-ß family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.


Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Antígenos CD/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Modelos Moleculares , Receptores de Superficie Celular/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/genética , Animales , Antígenos CD/química , Antígenos CD/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Dimerización , Endoglina , Femenino , Factor 2 de Diferenciación de Crecimiento/sangre , Factor 2 de Diferenciación de Crecimiento/aislamiento & purificación , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factores de Diferenciación de Crecimiento/sangre , Factores de Diferenciación de Crecimiento/química , Factores de Diferenciación de Crecimiento/genética , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos BALB C , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Precursores de Proteínas/sangre , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , Organismos Libres de Patógenos Específicos
11.
Hum Mutat ; 36(12): 1113-27, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26387786

RESUMEN

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II-like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next-generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus-specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.


Asunto(s)
Hipertensión Pulmonar/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Asesoramiento Genético , Predisposición Genética a la Enfermedad , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hipertensión Pulmonar/diagnóstico , Hipertensión Pulmonar/metabolismo , Familia de Multigenes , Mutación , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo
12.
Int J Mol Med ; 35(3): 579-86, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25543998

RESUMEN

The aim of the present study was to examine the microRNA (miRNA or miR) expression profiles during the chondrogenic differentiation of human adipose­derived stem cells (hADSCs) and identify the potential mechanisms through which miRNAs may affect the process of chondrogenesis. hADSCs were isolated and cultured. The expression levels of chondrogenic markers was detected by FACS analysis and immunohistochemistry. The miRNA expression profiles were then obtained through a miRNA array and confirmed through northern blot analysis. Putative targets of the miRNAs were predicted and validated through a luciferase reporter assay. The comparison of hADSCs following the induction of chondrogenic differentiation with undifferentiated hADSCs revealed 20 miRNAs that were differentially expressed by at least 2­fold, and these miRNAs included 12 upregulated miRNAs and 8 downregulated miRNAs. Northern blot analysis further confirmed the miRNA expression levels. Of these miRNAs, the expression of miR­490­5p was gradually downregulated following the induction of chondrogenic differentiation. The overexpression of miR­490­5p increased the expression of the chondrogenic markers, collagen, type II, alpha 1 (Col2A1), collagen, type X, alpha 1 (Col10A1) and aggrecan. Furthermore, it was confirmed that miR­490­5p directly targets bone morphogenetic protein receptor type 2 (BMPR2). In conclusion, in this study, we identified a set of miRNAs that may play key roles in the regulation of the chondrogenic differentiation of hADSCs. Our results may provide a basis for the further investigations into the molecular mechanisms of action of miRNAs in hADSC chondrogenesis.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/genética , Condrogénesis/genética , MicroARNs/genética , Células Madre/citología , Células Madre/metabolismo , Transcriptoma , Secuencia de Bases , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/química , Interferencia de ARN , ARN Mensajero/genética , Reproducibilidad de los Resultados
13.
Cell Mol Life Sci ; 71(16): 3165-72, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24337809

RESUMEN

The bone morphogenetic protein (BMP) signaling pathway is essential for normal development and tissue homeostasis. BMP signal transduction occurs when ligands interact with a complex of type 1 and type 2 receptors to activate downstream transcription factors. It is well established that a single BMP receptor may bind multiple BMP ligands with varying affinity, and this has been largely attributed to conformation at the amino acid level. However, all three type 2 BMP receptors (BMPR2, ACVR2A/B) contain consensus N-glycosylation sites in their extracellular domains (ECDs), which could play a role in modulating interaction with ligand. Here, we show a differential pattern of N-glycosylation between BMPR2 and ACVR2A/B. Site-directed mutagenesis reveals that BMPR2 is uniquely glycosylated near its ligand binding domain and at a position that is mutated in patients with heritable pulmonary arterial hypertension. We further demonstrate using a cell-free pulldown assay that N-glycosylation of the BMPR2-ECD enhances its ability to bind BMP2 ligand but has no impact on binding by the closely-related ACVR2B. Our results illuminate a novel aspect of BMP signaling pathway mechanics and demonstrate a functional difference resulting from post-translational modification of type 2 BMP receptors. Additionally, since BMPR2 is required for several aspects of normal development and defects in its function are strongly implicated in human disease, our findings are likely to be relevant in several biological contexts in normal and abnormal human physiology.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Línea Celular , Hipertensión Pulmonar Primaria Familiar , Glicosilación , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína
14.
Cell Signal ; 25(12): 2717-26, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24018044

RESUMEN

TGFß superfamily ligands greatly outnumber their receptors. Thus, receptors are shared between ligands and individual ligands can bind multiple receptors. Bone morphogenetic proteins (BMPs) bind and signal via both BMP type II (BMPR2) and activin type II (ACVR2) receptors. We hypothesized that, in addition to its canonical receptor ACVR2, activin A might similarly bind and signal via BMPR2. First, using surface plasmon resonance, we showed that activin A binds to the BMPR2 extracellular domain (ECD), though with lower affinity compared to the ACVR2-ECD. We confirmed these results in cells, where radiolabeled activin A bound to ACVR2 and BMPR2, but not to other type II receptors (AMHR2 or TGFBR2). Using homology modeling and site-directed mutagenesis, we identified key residues in BMPR2 that mediate its interaction with activin A. The soluble ECDs of ACVR2 or BMPR2 dose-dependently inhibited activin A-, but not TGFß-induced signaling in cells, suggesting that activin binding to BMPR2 could have functional consequences. To address this idea, we altered BMPR2 expression levels in immortalized murine gonadotrope-like cells, LßT2, in which activins potently stimulate follicle-stimulating hormone ß (Fshb) subunit transcription. BMPR2 expression potentiated activin A responses whereas depletion of endogenous BMPR2 with short interfering RNAs attenuated activin A-stimulated Fshb transcription. Additional data suggest, for the first time, that BMPR2 may form functional complexes with the canonical activin type I receptor, activin receptor-like kinase 4. Collectively, our data show that BMPR2, along with ACVR2, functions as a bona fide activin type II receptor in gonadotrope-like cells, thereby broadening our understanding of mechanisms of activin action.


Asunto(s)
Activinas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Gonadotrofos/metabolismo , Transducción de Señal , Receptores de Activinas Tipo I/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células CHO , Línea Celular , Cricetulus , Hormona Folículo Estimulante de Subunidad beta/genética , Células HEK293 , Humanos , Ratones , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Interferencia de ARN , Activación Transcripcional , Regulación hacia Arriba
15.
PLoS One ; 8(8): e72407, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23967299

RESUMEN

Mortality from prostate cancer (PCa) is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor ß (TGFß) superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFß receptor, activin receptor-like kinase 2 (ALK2), and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFß receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA) and bone morphogenetic protein receptor type II (BMPRII). Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.


Asunto(s)
Activinas/metabolismo , Antígenos CD/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptores de Superficie Celular/metabolismo , Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Línea Celular Tumoral , Endoglina , Activación Enzimática , Humanos , Masculino , Invasividad Neoplásica , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Proteína Smad1/metabolismo
16.
Biochemistry ; 51(35): 6968-80, 2012 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-22894880

RESUMEN

One of the most intriguing questions confronting the bone morphogenetic protein family is the mechanism of ligand recognition, because there are more ligands than receptors. Crystal structures of two type II receptors, ActR-II and BMPR-II, are essentially identical, and a loop structure (A-loop) has been suggested to play a role in determining ligand specificity. A solution biophysical study showed mutations of several A-loop residues in these two receptors exert different ligand binding effects. Thus, the issues of mechanism of ligand recognition and specificity remain unresolved. We examined effects of mutations of residues Y40, G47, and S107 in BMPR-II. These residues are not identified as being in contact with the ligand in the BMP-7-BMPR-II complex but are found mutated in genetic diseases. They are likely to be useful in identifying their roles in differentiating the various BMP ligands. Spectroscopic probing revealed little mutation-induced structural change in BMPR-II. Ligand binding studies revealed that Y40 plays a significant role in differentiating three distinct ligands; G47 and S107 affect ligand binding to a lesser extent. The role of the A-loop in ActR-II or BMPR-II is dependent on the host sequence of the receptor extracellular domain (ECD) in which it is embedded, suggesting a host-guest relationship between the A-loop and the rest of the ECD. Computational analysis demonstrated a long-range connectivity between Y40, G47, and S107 and other locations in BMPR-II. An integration of these results on functional energetics and protein structures clearly demonstrates, for the first time, an intradomain communication network within BMPR-II.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Activinas Tipo II/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Alineación de Secuencia , Eliminación de Secuencia , Especificidad por Sustrato
17.
Dev Cell ; 19(4): 547-61, 2010 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-20951346

RESUMEN

During embryogenesis, bone morphogenetic protein (BMP) signaling needs to be finely tuned in a locally restricted manner. Here, we report a cell-intrinsic mode of BMP response control executed by the membrane protein Jiraiya. In the Xenopus embryo, zygotic Jiraiya, expressed exclusively in the neuroectoderm, is essential and sufficient for limiting dorsal neural development, which is dependent on BMP signals. In animal cap assays, Jiraiya selectively and cell-autonomously inhibits BMP signaling, while Jiraiya's knockdown enhances the signaling. In the cell, Jiraiya selectively forms a complex with type II BMP receptor (BMPRII) and downregulates the cell surface localization of functional BMPRII. This functional interaction with Jiraiya depends on the unique tail domain of BMPRII, and, in particular, the conserved EVNNNG motif, the function of which has been unknown. Thus, Jiraiya represents a cell-intrinsic cutoff mechanism for dynamic responsiveness to BMP signals via subtype-selective receptor control.


Asunto(s)
Tipificación del Cuerpo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de la Membrana/metabolismo , Placa Neural/embriología , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Membrana Celular/metabolismo , Secuencia Conservada , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Retículo Endoplásmico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Sistema Nervioso/citología , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Placa Neural/metabolismo , Especificidad de Órganos , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Transducción de Señal/genética , Relación Estructura-Actividad , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética
18.
Mol Biol Cell ; 21(22): 4028-41, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20861306

RESUMEN

Cell surface heparan sulfate (HS) not only binds several major classes of growth factors but also sometimes potentiates their activities--an effect usually termed "coreception." A view that coreception is due to the stabilization of growth factor-receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here, we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and we provide evidence that HS-BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I receptor subunits but instead enhances the subsequent recruitment of type II receptor subunits to BMP-type I receptor complexes. This suggests a view of HS as a catalyst of the formation of signaling complexes, rather than as a stabilizer of growth factor binding.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Heparitina Sulfato/metabolismo , Animales , Sitios de Unión , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Proteínas Morfogenéticas Óseas/farmacología , Línea Celular , Activación Enzimática/efectos de los fármacos , Heparitina Sulfato/farmacología , Immunoblotting , Ligandos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Células PC12 , Fosforilación/efectos de los fármacos , Polisacárido Liasas/farmacología , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Ratas , Proteínas Smad/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
19.
J Biol Chem ; 285(48): 37641-9, 2010 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-20870717

RESUMEN

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Lisosomas/metabolismo , Sarcoma de Kaposi/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células HeLa , Herpesvirus Humano 8/enzimología , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiología , Humanos , Lisosomas/química , Lisosomas/genética , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sarcoma de Kaposi/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteínas Virales/genética , Proteínas Virales/metabolismo
20.
Ann N Y Acad Sci ; 1192: 286-91, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20392248

RESUMEN

Different working modes were proposed to explain how bone morphogenetic protein (BMP) type I and II receptors are involved in Smad phosphorylation cascades. In addition, both parathyroid hormone (PTH) and Wnts are also known to regulate phosphorylation of Smads. Here we established a mouse model in which a C-terminal truncated BMP type II receptor (BMPRII) is expressed specifically in osteoblasts as a dominant negative form in the BMP/Smad signaling pathway. Smad1/5/8 phosphorylation levels were reduced in bone marrow stromal cells from the transgenic mice. The sizes of embryos were smaller, and the mineralization of calvarial bones and lumbar vertebrae were delayed in mice expressing the transgene. In adult transgenic mice, total bone volume was reduced with no significant changes observed in cortical bone. Thus, osteoblast-targeted expression of dominant negative BMPRII leads to inhibited Smad1 phosphorylation, delayed skeletal development, and decreased bone formation in the adult mice. This study provides an in vivo tool to study the role of BMPRII in BMP/Smad signaling and the regulation of this pathway by PTH and Wnts.


Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Ratones Transgénicos , Modelos Animales , Osteoblastos/metabolismo , Animales , Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Citoplasma/metabolismo , Embrión de Mamíferos , Técnicas de Transferencia de Gen , Genes Dominantes , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Hormona Paratiroidea/metabolismo , Hormona Paratiroidea/fisiología , Estructura Terciaria de Proteína/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Proteínas Smad/fisiología , Proteínas Wnt/metabolismo , Proteínas Wnt/fisiología
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