RESUMEN
Kinetochore specification and assembly requires the targeted deposition of specialized nucleosomes containing the histone H3 variant CENP-A at centromeres. However, CENP-A is not sufficient to drive full-kinetochore assembly, and it is not clear how centromeric chromatin is established. Here, we identify CENP-W as a component of the DNA-proximal constitutive centromere-associated network (CCAN) of proteins. We demonstrate that CENP-W forms a DNA-binding complex together with the CCAN component CENP-T. This complex directly associates with nucleosomal DNA and with canonical histone H3, but not with CENP-A, in centromeric regions. CENP-T/CENP-W functions upstream of other CCAN components with the exception of CENP-C, an additional putative DNA-binding protein. Our analysis indicates that CENP-T/CENP-W and CENP-C provide distinct pathways to connect the centromere with outer kinetochore assembly. In total, our results suggest that the CENP-T/CENP-W complex is directly involved in establishment of centromere chromatin structure coordinately with CENP-A.
Asunto(s)
Centrómero , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Cinetocoros/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Proteína A Centromérica , Pollos , Proteínas Cromosómicas no Histona/genética , Células HeLa , Histonas/metabolismo , Humanos , Mutación , Nucleosomas/metabolismoRESUMEN
Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Colitis/enzimología , Colitis/prevención & control , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Animales , Recuento de Células , Quimiocinas/genética , Quimiocinas/metabolismo , Colitis/patología , Colon/patología , Femenino , Células Caliciformes/metabolismo , Células Caliciformes/patología , Células HEK293 , Humanos , Interleucina-10/deficiencia , Interleucina-10/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , FN-kappa B/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas Tirosina Quinasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Quinasa Syk , Dominios Homologos src , Familia-src Quinasas/metabolismoRESUMEN
Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments.
Asunto(s)
Preparaciones Farmacéuticas/química , Cromatografía de Afinidad , Diazometano/química , Ligandos , Microscopía , Preparaciones Farmacéuticas/aislamiento & purificación , Preparaciones Farmacéuticas/efectos de la radiación , Rodaminas/química , Tacrolimus/química , Tacrolimus/aislamiento & purificación , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismo , Rayos UltravioletaRESUMEN
Stomata in the epidermal tissues of leaves are valves through which passes CO(2), and as such they influence the global carbon cycle. The two-dimensional pattern and density of stomata in the leaf epidermis are genetically and environmentally regulated to optimize gas exchange. Two putative intercellular signalling factors, EPF1 and EPF2, function as negative regulators of stomatal development in Arabidopsis, possibly by interacting with the receptor-like protein TMM. One or more positive intercellular signalling factors are assumed to be involved in stomatal development, but their identities are unknown. Here we show that a novel secretory peptide, which we designate as stomagen, is a positive intercellular signalling factor that is conserved among vascular plants. Stomagen is a 45-amino-rich peptide that is generated from a 102-amino-acid precursor protein designated as STOMAGEN. Both an in planta analysis and a semi-in-vitro analysis with recombinant and chemically synthesized stomagen peptides showed that stomagen has stomata-inducing activity in a dose-dependent manner. A genetic analysis showed that TMM is epistatic to STOMAGEN (At4g12970), suggesting that stomatal development is finely regulated by competitive binding of positive and negative regulators to the same receptor. Notably, STOMAGEN is expressed in inner tissues (the mesophyll) of immature leaves but not in the epidermal tissues where stomata develop. This study provides evidence of a mesophyll-derived positive regulator of stomatal density. Our findings provide a conceptual advancement in understanding stomatal development: inner photosynthetic tissues optimize their function by regulating stomatal density in the epidermis for efficient uptake of CO(2).
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Estomas de Plantas/fisiología , Arabidopsis/anatomía & histología , Arabidopsis/citología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Dióxido de Carbono/metabolismo , Proteínas de Unión al ADN/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
To identify the novel factors involved in the postsplicing intron turnover pathway, we carried out immunoprecipitation with known postsplicing factors, hPrp43 and TFIP11. As an interacting factor, we identified C2ORF3 protein by mass spectrometry. We found that C2ORF3 protein is present in the previously characterized Intron Large (IL) complex with an excised lariat intron. In vitro splicing using C2ORF3-depleted nuclear extracts showed significant repression of splicing, suggesting that C2ORF3 protein is required for pre-mRNA splicing through its presumable role in efficient intron turnover. Interestingly, C2ORF3 protein is localized in both the nucleoplasm and nucleoli, which suggests a potential function in rRNA processing.
Asunto(s)
Intrones , Precursores del ARN/metabolismo , Proteínas Represoras/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , Precursores del ARN/genética , Empalme del ARN , Factores de Empalme de ARN , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Regulation of the actin cytoskeleton is crucial for cell morphology and migration. mDia is an actin nucleator that produces unbranched actin filaments downstream of Rho. However, the mechanisms by which mDia activity is regulated in the cell remain unknown. We pulled down Liprin-α as an mDia-binding protein. The binding is mediated through the central region of Liprin-α and through the N-terminal Dia-inhibitory domain (DID) and dimerization domain (DD) of mDia. Liprin-α competes with Dia autoregulatory domain (DAD) for binding to DID, and binds preferably to the open form of mDia. Overexpression of a Liprin-α fragment containing the mDia-binding region decreases localization of mDia to the plasma membrane and attenuates the Rho-mDia-mediated formation of stress fibers in cultured cells. Conversely, depletion of Liprin-α by RNA interference (RNAi) increases the amount of mDia in the membrane fraction and enhances formation of actin stress fibers. Thus, Liprin-α negatively regulates the activity of mDia in the cell by displacing it from the plasma membrane through binding to the DID-DD region.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Proteínas/metabolismo , Fibras de Estrés/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Forminas , Células HeLa , Humanos , Ratones , Transporte de ProteínasRESUMEN
Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AID-interacting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.
Asunto(s)
Cambio de Clase de Inmunoglobulina , Hipermutación Somática de Inmunoglobulina , Factores de Transcripción/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Línea Celular , Citidina Desaminasa/química , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Ratones , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
In the male germline in mammals, chromatoid bodies, a specialized assembly of cytoplasmic ribonucleoprotein (RNP), are structurally evident during meiosis and haploidgenesis, but their developmental origin and regulation remain elusive. The tudor domain containing proteins constitute a conserved class of chromatoid body components. We show that tudor domain containing 7 (Tdrd7), the deficiency of which causes male sterility and age-related cataract (as well as glaucoma), is essential for haploid spermatid development and defines, in concert with Tdrd6, key biogenesis processes of chromatoid bodies. Single and double knockouts of Tdrd7 and Tdrd6 demonstrated that these spermiogenic tudor genes orchestrate developmental programs for ordered remodeling of chromatoid bodies, including the initial establishment, subsequent RNP fusion with ubiquitous processing bodies/GW bodies and later structural maintenance. Tdrd7 suppresses LINE1 retrotransposons independently of piwi-interacting RNA (piRNA) biogenesis wherein Tdrd1 and Tdrd9 operate, indicating that distinct Tdrd pathways act against retrotransposons in the male germline. Tdrd6, in contrast, does not affect retrotransposons but functions at a later stage of spermiogenesis when chromatoid bodies exhibit aggresome-like properties. Our results delineate that chromatoid bodies assemble as an integrated compartment incorporating both germline and ubiquitous features as spermatogenesis proceeds and that the conserved tudor family genes act as master regulators of this unique RNP remodeling, which is genetically linked to the male germline integrity in mammals.
Asunto(s)
Cromatina/metabolismo , Ribonucleoproteínas/metabolismo , Espermatogénesis , Animales , Cromosomas Artificiales Bacterianos , Masculino , Ratones , Ratones Noqueados , Microscopía Inmunoelectrónica , Ribonucleoproteínas/genética , Ribonucleoproteínas/fisiologíaRESUMEN
Transfer RNA (tRNA) modification is essential for proper protein translation, as these modifications play important roles in several biological functions and disease pathophysiologies. AlkB homolog 8 (ALKBH8) is one of the nine mammalian ALKBH family molecules known to regulate selenoprotein translation through the modification of the wobble uridine (U34) in tRNA; however, its specific biological roles remain unclear. In this study, we investigated the role of ALKBH8 using Alkbh8-knockout (Albkh8-/-) mice, which were observed to have reduced 5-methoxycarbonylmethyluridine (mcm5U) and (S)-5-methoxycarbonylhydroxymethyluridine levels; notably, the mcm5U level was partially compensated only in the brain. The results of the novel object recognition test showed reduction in time to explore a novel object in Albkh8-/- mice; increased latency to fall in the rotarod performance test and latency to the immobility period in the forced swim test were also observed. These abnormal behaviors indicate dysfunction of the central nervous system. Furthermore, we observed reduced brain weight and ischemic pathological changes in the cerebral cortex and hippocampus in the form of weak eosin staining in the fiber tracts adjacent to the hippocampal cornu ammonis 1 region and an increase in pyramidal cells in the temporal lobe. Concordantly, we identified the differential expression of oxidative stress-related proteins and metabolites in the cerebral cortex and hippocampus using omics analyses. Finally, neurons and glial cells derived from Albkh8-/- mice show reduced mitochondrial membrane potential. Collectively, these findings indicate that ALKBH8 maintains neural function through an oxidative stress-regulatory mechanism.
RESUMEN
FADD is an adaptor protein that transmits apoptotic signals from death receptors. Additionally, FADD has been shown to play a role in various functions including cell proliferation. However, the physiological role of FADD during embryonic development remains to be delineated. Here, we show the novel roles FADD plays in development and the molecular mechanisms of these roles in Xenopus embryos. By whole-mount in situ hybridization and RT-PCR analysis, we observed that fadd is constantly expressed in early embryos. The upregulation or downregulation of FADD proteins by embryonic manipulation resulted in induction of apoptosis or size changes in the heart during development. Expression of a truncated form of FADD, FADDdd, which lacks pro-apoptotic activity, caused growth retardation of embryos associated with dramatic expressional fluctuations of genes that are regulated by NF-κB. Moreover, we isolated a homolog of mammalian cullin-4 (Cul4), a component of the ubiquitin E3 ligase family, as a FADDdd-interacting molecule in Xenopus embryos. Thus, our study shows that FADD has multiple functions in embryos; it plays a part in the regulation of NF-κB activation and heart formation, in addition to apoptosis. Furthermore, our findings provide new insights into how Cul4-based ligase is related to FADD signaling in embryogenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Antígenos de Diferenciación/fisiología , Apoptosis , Proteína de Dominio de Muerte Asociada a Fas/fisiología , Corazón/embriología , FN-kappa B/metabolismo , Receptores Inmunológicos/fisiología , Proteínas de Xenopus/fisiología , Xenopus/embriología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Blastómeros/enzimología , Blastómeros/metabolismo , Proteínas Cullin/química , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Morfolinos/genética , FN-kappa B/fisiología , Fragmentos de Péptidos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Análisis de Secuencia de ADN , Eliminación de Secuencia , Transducción de Señal , Activación Transcripcional , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.
Asunto(s)
Autoantígenos/biosíntesis , Autoantígenos/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Centrómero/química , Proteína A Centromérica , Pollos , Segregación Cromosómica/genética , Proteínas del Citoesqueleto , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Quinasa I-kappa B/metabolismo , Poliubiquitina/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Citoplasma/metabolismo , Silenciador del Gen , Glicosilación , Células HeLa , Humanos , Cinética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , ARN Interferente Pequeño/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , alfa 1-Antitripsina/químicaRESUMEN
Synaptic loss, which strongly correlates with the decline of cognitive function, is one of the pathological hallmarks of Alzheimer disease. N-cadherin is a cell adhesion molecule essential for synaptic contact and is involved in the intracellular signaling pathway at the synapse. Here we report that the functional disruption of N-cadherin-mediated cell contact activated p38 MAPK in murine primary neurons, followed by neuronal death. We further observed that treatment with Aß(42) decreased cellular N-cadherin expression through NMDA receptors accompanied by increased phosphorylation of both p38 MAPK and Tau in murine primary neurons. Moreover, expression levels of phosphorylated p38 MAPK were negatively correlated with that of N-cadherin in human brains. Proteomic analysis of human brains identified a novel interaction between N-cadherin and JNK-associated leucine zipper protein (JLP), a scaffolding protein involved in the p38 MAPK signaling pathway. We demonstrated that N-cadherin expression had an inhibitory effect on JLP-mediated p38 MAPK signal activation by decreasing the interaction between JLP and p38 MAPK in COS7 cells. Also, this study demonstrated a novel physical and functional association between N-cadherin and p38 MAPK and suggested neuroprotective roles of cadherin-based synaptic contact. The dissociation of N-cadherin-mediated synaptic contact by Aß may underlie the pathological basis of neurodegeneration such as neuronal death, synaptic loss, and Tau phosphorylation in Alzheimer disease brain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer/metabolismo , Antígenos CD/metabolismo , Cadherinas/metabolismo , Degeneración Nerviosa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Animales , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Humanos , Leucina Zippers/fisiología , Masculino , Ratones , Persona de Mediana Edad , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Oligopéptidos/metabolismo , Péptidos Cíclicos/metabolismo , Fosforilación/fisiología , Proteómica , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.
Asunto(s)
Carboxilesterasa/metabolismo , Proteínas Ligadas a GPI/metabolismo , Microdominios de Membrana/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Carboxilesterasa/genética , Carboxilesterasa/aislamiento & purificación , Línea Celular , Humanos , Isoenzimas/metabolismo , Ratones , Mutación Puntual , Isoformas de Proteínas/metabolismo , ConejosRESUMEN
Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.
Asunto(s)
Tejido Elástico/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Microfibrillas/metabolismo , Tropoelastina/metabolismo , Envejecimiento/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Animales , Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Tejido Elástico/ultraestructura , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ratones , Microfibrillas/ultraestructura , Regeneración/fisiología , Piel/metabolismo , Piel/ultraestructuraRESUMEN
FGF23 is a unique member of the fibroblast growth factor (FGF) family because it acts as a hormone that derives from bone and regulates kidney functions, whereas most other family members are thought to regulate various cell functions at a local level. The renotropic activity of circulating FGF23 indicates the possible presence of an FGF23-specific receptor in the kidney. Here we show that a previously undescribed receptor conversion by Klotho, a senescence-related molecule, generates the FGF23 receptor. Using a renal homogenate, we found that Klotho binds to FGF23. Forced expression of Klotho enabled the high-affinity binding of FGF23 to the cell surface and restored the ability of a renal cell line to respond to FGF23 treatment. Moreover, FGF23 incompetence was induced by injecting wild-type mice with an anti-Klotho monoclonal antibody. Thus, Klotho is essential for endogenous FGF23 function. Because Klotho alone seemed to be incapable of intracellular signalling, we searched for other components of the FGF23 receptor and found FGFR1(IIIc), which was directly converted by Klotho into the FGF23 receptor. Thus, the concerted action of Klotho and FGFR1(IIIc) reconstitutes the FGF23 receptor. These findings provide insights into the diversity and specificity of interactions between FGF and FGF receptors.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Glucuronidasa/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Animales , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/genética , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/genética , Humanos , Proteínas Klotho , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Unión Proteica , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Especificidad por SustratoRESUMEN
Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca(2+) in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca(2+)-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC(50) for Ca(2+) of 168 nM, close to the resting Ca(2+) concentration, and that the interaction was disrupted by Ca(2+)-activated calmodulin (CaM) at higher concentrations of Ca(2+). Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca(2+) level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.
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Calcio/metabolismo , Estriol/análogos & derivados , Proteínas Munc18/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmisión Sináptica , Animales , Estriol/metabolismo , Ratones , Proteínas Munc18/genética , Células PC12 , Unión Proteica , RatasRESUMEN
Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.
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Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Pérdida de Tono Postural/fisiología , Receptores Inmunológicos/metabolismo , Estrés Fisiológico/fisiología , Estrés Psicológico/fisiopatología , Animales , Animales Modificados Genéticamente , Western Blotting , Línea Celular , Humanos , Ratones , Microdiálisis , Fosforilación , Receptores Inmunológicos/genética , Estrés Psicológico/metabolismo , NataciónRESUMEN
The transcription factor ATF6 is constitutively synthesized as a type II transmembrane protein embedded in the endoplasmic reticulum (ER). When unfolded proteins accumulate in the ER, ATF6 senses such ER stress via an as yet undetermined mechanism and relocates to the Golgi apparatus where it is cleaved by sequential action of Site-1 and Site-2 proteases, allowing liberated N-terminal fragments to translocate into the nucleus. This ATF6-mediated transcriptional induction of ER-localized molecular chaperones and folding enzymes together with components of ER-associated degradation leads to the maintenance of ER homeostasis in mammals. Here, we demonstrated that the luminal domain of ATF6 alone is sufficient for sensing ER stress and subsequent transportation to the Golgi apparatus. This domain of ATF6 was inserted between the N-terminal signal sequence and C-terminal tandem affinity purification tag. The resulting ATF6(C)-TAP translocated into the ER, where it was glycosylated and disulfide bonded. ATF6(C)-TAP occurred as monomer and dimer, and exhibited a relatively short half-life, similar to full-length ATF6. On application of dithiothreitol- or thapsigargin-induced ER stress, the ER chaperone BiP dissociated from ATF6(C)-TAP, and ATF6(C)-TAP was transported to the Golgi apparatus and then secreted into medium. Calnexin and protein disulfide isomerase were identified as cellular proteins capable of binding to ATF6(C)-TAP in addition to BiP, and subsequent analysis revealed that protein disulfide isomerase was bound to ATF6(C)-TAP with chaperone activity. These findings indicate that ATF6(C)-TAP can be used as a tool to isolate protein(s) that escort ATF6 from the ER to the Golgi apparatus in response to ER stress.