RESUMEN
Proper tuning of ß-catenin activity in osteoblasts is required for bone homeostasis, because both increased and decreased ß-catenin activity have pathologic consequences. In the classical pathway for ß-catenin activation, stimulation with WNT ligands suppresses constitutive phosphorylation of ß-catenin by glycogen synthase kinase 3ß, preventing ß-catenin ubiquitination and proteasomal degradation. Here, we have found that mitogen-activated protein kinase kinase kinase 2 (MAP3K2 or MEKK2) mediates an alternative pathway for ß-catenin activation in osteoblasts that is distinct from the canonical WNT pathway. FGF2 activates MEKK2 to phosphorylate ß-catenin at serine 675, promoting recruitment of the deubiquitinating enzyme, ubiquitin-specific peptidase 15 (USP15). USP15 in turn prevents the basal turnover of ß-catenin by inhibiting its ubiquitin-dependent proteasomal degradation, thereby enhancing WNT signaling. Analysis of MEKK2-deficient mice and genetic interaction studies between Mekk2- and ß-catenin-null alleles confirm that this pathway is an important physiologic regulator of bone mass in vivo. Thus, an FGF2/MEKK2 pathway mediates an alternative nonclassical pathway for ß-catenin activation, and this pathway is a key regulator of bone formation by osteoblasts.
Asunto(s)
Desarrollo Óseo , MAP Quinasa Quinasa Quinasa 2/metabolismo , beta Catenina/metabolismo , Animales , Ratones , Tamaño de los Órganos , Osteoblastos/citología , FosforilaciónRESUMEN
E2-25K/Hip2 is an unusual ubiquitin-conjugating enzyme that interacts with the frameshift mutant of ubiquitin B (UBB(+1)) and has been identified as a crucial factor regulating amyloid-ß neurotoxicity. To study the structural basis of the neurotoxicity mediated by the E2-25K-UBB(+1) interaction, we determined the three-dimensional structures of UBB(+1), E2-25K and the E2-25K/ubiquitin, and E2-25K/UBB(+1) complex. The structures revealed that ubiquitin or UBB(+1) is bound to E2-25K via the enzyme MGF motif and residues in α9 of the enzyme. Polyubiquitylation assays together with analyses of various E2-25K mutants showed that disrupting UBB(+1) binding markedly diminishes synthesis of neurotoxic UBB(+1)-anchored polyubiquitin. These results suggest that the interaction between E2-25K and UBB(+1) is critical for the synthesis and accumulation of UBB(+1)-anchored polyubiquitin, which results in proteasomal inhibition and neuronal cell death.
Asunto(s)
Complejos Multiproteicos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular Tumoral , Supervivencia Celular , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitina/química , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.
Asunto(s)
Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Receptor Patched-1/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Proteínas Hedgehog/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Osteoblastos/citología , Receptor Patched-1/genética , Transducción de SeñalRESUMEN
Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1fl/fl;Dmp1-Cre) and Mekk2-/- each displaying skeletal defects, Nf1fl/fl;Mekk2-/-;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Imidazoles/farmacología , MAP Quinasa Quinasa Quinasa 2/metabolismo , Neurofibromatosis 1/etiología , Piridazinas/farmacología , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , MAP Quinasa Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa Quinasa 2/genética , Masculino , Ratones Transgénicos , Neurofibromatosis 1/tratamiento farmacológico , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Osteoblastos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Cráneo/citologíaRESUMEN
It has been demonstrated that ubiquitin-conjugated proteins were accumulated by ectopically-expressed S5a as well as the ubiquitin-interacting motifs of S5a (S5a-UIMs). In this study, we further found that free S5a-UIMs stabilized only a subset of proteasomal substrates including p53, c-Fos, c-Jun, and p27 but not beta-catenin, p15, and ornithine decarboxylase. Both S5a-UIMs and epoxomicin inhibited the proliferation of A549 lung cancer cells but arrest at the different stages of cell cycle. Together, our results suggest a potential role of S5a-UIMs as an upstream proteasomal inhibitor by blocking the subset of substrates from delivery to the 26S proteasome.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Ubiquitina/metabolismo , Secuencias de Aminoácidos/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Humanos , Oligopéptidos/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas de Unión al ARN , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
Recent studies have identified a specialized subset of CD31hiendomucinhi (CD31hiEMCNhi) vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3-/- mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.
Asunto(s)
Resorción Ósea/patología , Huesos/patología , Endotelio/patología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Resorción Ósea/diagnóstico por imagen , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Endotelio/efectos de los fármacos , Curación de Fractura/efectos de los fármacos , Humanos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteogénesis/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Osteoporosis Posmenopáusica/patología , Ovariectomía , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Sialoglicoproteínas/metabolismo , Proteínas RoundaboutRESUMEN
The c-Jun N-terminal kinases (JNKs) are ancient and evolutionarily conserved regulators of proliferation, differentiation, and cell death responses. Currently, in vitro studies offer conflicting data about whether the JNK pathway augments or represses osteoblast differentiation, and the contribution of the JNK pathway to regulation of bone mass in vivo remains unclear. Here we show that Jnk1-/- mice display severe osteopenia due to impaired bone formation, whereas Jnk2-/- mice display a mild osteopenia only evident in long bones. In order to both confirm that these effects were osteoblast intrinsic and assess whether redundancy with JNK1 masks a potential contribution of JNK2, mice with a conditional deletion of both JNK1 and JNK2 floxed conditional alleles in osteoblasts (Jnk1-2osx ) were bred. These mice displayed a similar degree of osteopenia to Jnk1-/- mice due to decreased bone formation. In vitro, Jnk1-/- osteoblasts display a selective defect in the late stages of osteoblast differentiation with impaired mineralization activity. Downstream of JNK1, phosphorylation of JUN is impaired in Jnk1-/- osteoblasts. Transcriptome analysis showed that JNK1 is required for upregulation of several osteoblast-derived proangiogenic factors such as IGF2 and VEGFa. Accordingly, JNK1 deletion results in a significant reduction skeletal vasculature in mice. Taken together, this study establishes that JNK1 is a key mediator of osteoblast function in vivo and in vitro. © 2017 American Society for Bone and Mineral Research.
Asunto(s)
Enfermedades Óseas Metabólicas/enzimología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Osteoblastos/enzimología , Osteogénesis , Animales , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/patología , Factor II del Crecimiento Similar a la Insulina/genética , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/genética , Osteoblastos/patología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Physiological bone remodeling requires that bone formation by osteoblasts be tightly coupled to bone resorption by osteoclasts. However, relatively little is understood about how this coupling is regulated. Here, we demonstrate that modulation of NF-κB signaling in osteoclasts via a novel activity of charged multivesicular body protein 5 (CHMP5) is a key determinant of systemic rates of bone turnover. A conditional deletion of CHMP5 in osteoclasts leads to increased bone resorption by osteoclasts coupled with exuberant bone formation by osteoblasts, resembling an early onset, polyostotic form of human Paget's disease of bone (PDB). These phenotypes are reversed by haploinsufficiency for Rank, as well as by antiresorptive treatments, including alendronate, zolendronate, and OPG-Fc. Accordingly, CHMP5-deficient osteoclasts display increased RANKL-induced NF-κB activation and osteoclast differentiation. Biochemical analysis demonstrated that CHMP5 cooperates with the PDB genetic risk factor valosin-containing protein (VCP/p97) to stabilize the inhibitor of NF-κBα (IκBα), down-regulating ubiquitination of IκBα via the deubiquitinating enzyme USP15. Thus, CHMP5 tunes NF-κB signaling downstream of RANK in osteoclasts to dampen osteoclast differentiation, osteoblast coupling and bone turnover rates, and disruption of CHMP5 activity results in a PDB-like skeletal disorder.
Asunto(s)
Desarrollo Óseo/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , FN-kappa B/metabolismo , Osteoclastos/metabolismo , Transducción de Señal/fisiología , Adenosina Trifosfatasas/metabolismo , Animales , Secuencia de Bases , Desarrollo Óseo/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Luciferasas , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Inhibidor NF-kappaB alfa , Osteoblastos/citología , Ligando RANK/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Ubiquitinación , Proteína que Contiene ValosinaRESUMEN
In this study using non-reduced/reduced 2-dimensional electrophoresis (NR/R-2DE), we clearly demonstrated that E3-independent ubiquitination by Ube2K produced not only unanchored but also Ube2K-linked polyubiquitins through thioester and isopeptide bonds. E3-independent assembly of polyubiquitins on the catalytic cysteine of Ube2K strongly supports the possibility of 'en bloc transfer' for polyubiquitination. From the same analyses of E3-independent ubiquitination products by other E2s, we also found that different lengths of polyubiquitins were linked to different E2s through thioester bond; longer chains by Cdc34 like Ube2K, short chains by Ube2g2, and mono-ubiquitin by UbcH10. Our results suggest that E2s possess the different intrinsic catalytic activities for polyubiquitination.
Asunto(s)
Biocatálisis , Electroforesis en Gel Bidimensional/métodos , Poliubiquitina/aislamiento & purificación , Poliubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Dominio Catalítico , Cisteína , ÉsteresRESUMEN
Accumulating evidence suggests that intracellular amyloid-beta (Abeta) peptide triggers the early pathological events in Alzheimer's disease (AD). However, little is known about the consequence of cytosolic Abeta. In this study, we ectopically expressed Abeta42 in the cytoplasm of SH-SY5Y neuroblastoma cells by expressing a fusion protein of GFP-tagged ubiquitin and Abeta42 (GFPUb-Abeta42). Although GFPUb and Abeta42 are stochastically produced with the same molar ratio in the cytoplasm, Abeta42 was completely degraded in more than 50% of the GFPUb-expressing cells. However, if Abeta42 was not degraded in their cytoplasm, then Abeta42-expressing cells underwent apoptosis. The number of Abeta42-expressing cells is significantly increased by the inhibition of proteasome with MG132. Cytosolic Abeta42 which has escaped degradation inhibits proteasome and thereby may accelerate the accumulation of Abeta42 and its detrimental effects. Our findings suggest that cells have the potential to degrade Abeta42 in their cytoplasm but if Abeta42 appears in the cytoplasm due to its incomplete degradation, it accumulates and may trigger the fatal cascade of pathology of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apoptosis , Citosol/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Amyloid-beta peptide 42 (Abeta42) mediates neuronal degeneration in Alzheimer's disease (AD). We sought to produce recombinant Abeta42 as an ubiquitin extension. A synthetic oligonucleotide encoding Abeta42 was constructed and cloned as an extended polypeptide of hexahistidine-tagged ubiquitin (H(6)Ub) using the pET vector. Isopropyl-beta-D-thiogalactopyranoside induction of transformed Escherichia coli resulted in the production of large amounts of insoluble H(6)Ub-Abeta42 fusion protein. H(6)Ub-Abeta42 was solubilized in 8 M urea and applied to a nickel-nitrilotriacetic acid affinity column for purification. Column washing removed the urea and soluble H(6)Ub-Abeta42 was eluted, indicating that covalently attached ubiquitin prevented Abeta42 from aggregating. Abeta42 was cleaved from H(6)Ub using recombinant yeast ubiquitin hydrolase-1 (YUH-1) and purified using reverse-phase chromatography. The recombinant Abeta42 prepared in this study has the same toxic effect on human neuroblastoma SH-SY5Y cells comparing with chemically synthesized, commercial one. The peptide yield was more than 4 mg/L culture, indicating this ubiquitin fusion technique is an attractive method for production of aggregation-prone peptides such as Abeta42.
Asunto(s)
Péptidos beta-Amiloides/biosíntesis , Fragmentos de Péptidos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Ubiquitina/genética , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/aislamiento & purificación , Secuencia de Bases , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Ubiquitina/aislamiento & purificación , Ubiquitina/metabolismoRESUMEN
A Gram-negative, non-motile, non-spore-forming, moderately halophilic rod (strain SM-1T) was isolated from salt marsh around the junction of the Youngsan River and the Yellow Sea in Korea and subjected to a polyphasic taxonomic study. This organism grew optimally at 37 degrees C and was able to grow at 10 and 45 degrees C. It grew optimally in the presence of 2-3% (w/v) NaCl. The major fatty acids in strain SM-1T were iso-C15:0 and C16:0. Strain SM-1T and Microbulbifer hydrolyticus DSM 11525T were characterized by having ubiquinone-8 as the predominant respiratory lipoquinone. The DNA G+C content of strain SM-1T was 59 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain SM-1T formed a coherent cluster with M. hydrolyticus; this relationship was supported by a bootstrap resampling value of 100%. The level of 16S rDNA identity between strain SM-1T and the type strain of M. hydrolyticus was 98.6%. The mean level of DNA-DNA relatedness between strain SM-1T and the type strain of M. hydrolyticus was 20.6%. Therefore, on the basis of phenotypic properties, phylogeny and genomic data, strain SM-1T should be placed in the genus Microbulbifer as a member of a novel species, for which the name Microbulbifer salipaludis sp. nov. is proposed. The type strain of the novel species is strain SM-1T (=KCCM 41586T =JCM 11542T).
Asunto(s)
Alteromonadaceae/clasificación , Alteromonadaceae/aislamiento & purificación , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Composición de Base , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/genética , Ácidos Grasos/análisis , Agua Dulce/microbiología , Corea (Geográfico) , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Agua de Mar/microbiologíaRESUMEN
A Gram-negative, motile, non-spore-forming and moderately halophilic rod-shaped strain, SW-45T, was isolated from sea water of the East Sea in Korea. The organism grew optimally at 30-37 degrees C and grew at 4 and 46 degrees C. It grew in the presence of 0.5-18% (v/w) NaCl, with an optimum of 2-7% NaCl. Strain SW-45T was chemotaxonomically characterized by having ubiquinone-9 (Q-9) as the major respiratory lipoquinone and C16 : 0, C18 : 1omega9c and C16 : 1omega9c as the predominant fatty acids. The DNA G + C content was found to be 55 mol%. Phylogenetic analysis based on 16S rDNA sequences showed that strain SW-45T forms a coherent cluster with the clade comprising the two Marinobacter species. 16S rDNA sequence similarities between strain SW-45T and the Marinobacter species was 94.9% to Marinobacter hydrocarbonoclasticus DSM 8798T and 95.3% to Marinobacter aquaeolei DSM 11845T. Levels of DNA-DNA relatedness between strain SW-45T and the type strains of M. hydrocarbonoclasticus and M. aquaeolei were respectively 4.3 and 5.5%. On the basis of phenotypic properties, phylogeny and genomic data, strain SW-45T (=KCCM 41591T =JCM 11547T) should be placed in the genus Marinobacter as a member of a novel species, for which the name Marinobacter litoralis sp. nov. is proposed. As part of this study, the major respiratory lipoquinone of M. hydrocarbonoclasticus and M. aquaeolei was also found to be Q-9.