RESUMEN
Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.
Asunto(s)
Antinematodos , Tylenchoidea , Animales , Humanos , Antinematodos/química , Antinematodos/metabolismo , Antinematodos/farmacología , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/metabolismo , Tylenchoidea/efectos de los fármacos , Tylenchoidea/metabolismo , Tiazoles/química , Tiazoles/metabolismo , Tiazoles/farmacología , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/parasitología , Enfermedades de las Plantas , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.
Asunto(s)
Receptores ErbB/metabolismo , Mapas de Interacción de Proteínas , Transducción de Señal , Familia-src Quinasas/metabolismo , Animales , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/agonistas , Receptores ErbB/genética , Células HEK293 , Humanos , Ratones , Mutación , Fosforilación , Mapeo de Interacción de Proteínas , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Transfección , Técnicas del Sistema de Dos Híbridos , Familia-src Quinasas/genéticaRESUMEN
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride and bicarbonate channel in secretory epithelia with a critical role in maintaining fluid homeostasis. Mutations in CFTR are associated with Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasians. While remarkable treatment advances have been made recently in the form of modulator drugs directly rescuing CFTR dysfunction, there is still considerable scope for improvement of therapeutic effectiveness. Here, we report the application of a high-throughput screening variant of the Mammalian Membrane Two-Hybrid (MaMTH-HTS) to map the protein-protein interactions of wild-type (wt) and mutant CFTR (F508del), in an effort to better understand CF cellular effects and identify new drug targets for patient-specific treatments. Combined with functional validation in multiple disease models, we have uncovered candidate proteins with potential roles in CFTR function/CF pathophysiology, including Fibrinogen Like 2 (FGL2), which we demonstrate in patient-derived intestinal organoids has a significant effect on CFTR functional expression.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Animales , Membrana Celular/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrinógeno/genética , Fibrinógeno/metabolismo , Fibrinógeno/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Mamíferos , MutaciónRESUMEN
Receptor tyrosine kinases (RTKs) are recognized as targets of precision medicine in human cancer upon their gene amplification or constitutive activation, resulting in increased downstream signal complexity including heterotypic crosstalk with other RTKs. The Met RTK exhibits such reciprocal crosstalk with several members of the human EGFR (HER) family of RTKs when amplified in cancer cells. We show that Met signaling converges on HER3-tyrosine phosphorylation across a panel of seven MET-amplified cancer cell lines and that HER3 is required for cancer cell expansion and oncogenic capacity in vitro and in vivo. Gene expression analysis of HER3-depleted cells identified MPZL3, encoding a single-pass transmembrane protein, as HER3-dependent effector in multiple MET-amplified cancer cell lines. MPZL3 interacts with HER3 and MPZL3 loss phenocopies HER3 loss in MET-amplified cells, while MPZL3 overexpression can partially rescue proliferation upon HER3 depletion. Together, these data support an oncogenic role for a HER3-MPZL3 axis in MET-amplified cancers.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptor ErbB-3/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos NOD , Inestabilidad de Microsatélites , Fosforilación , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/genética , Transducción de Señal/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Trasplante HeterólogoRESUMEN
Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular , Línea Celular Tumoral , ADN Nucleotidiltransferasas/genética , Descubrimiento de Drogas , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Genes Reporteros , Humanos , Luciferasas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Fosforilación/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Estaurosporina/análogos & derivados , Estaurosporina/farmacologíaRESUMEN
SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Productos del Gen nef/fisiología , VIH-1/fisiología , Tejido Linfoide/virología , Proteínas de la Membrana/metabolismo , Replicación Viral/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Colobus/virología , Células HEK293 , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Virus de la Inmunodeficiencia de los Simios/genéticaRESUMEN
Melatonin, a neuro-hormone released by the pineal gland, has multiple effects in the central nervous system including the regulation of dopamine (DA) levels, but how melatonin accomplishes this task is not clear. Here, we show that melatonin MT1 and MT2 receptors co-immunoprecipitate with the DA transporter (DAT) in mouse striatal synaptosomes. Increased DA re-uptake and decreased amphetamine-induced locomotor activity were observed in the striatum of mice with targeted deletion of MT1 or MT2 receptors. In vitro experiments confirmed the interactions and recapitulated the inhibitory effect of melatonin receptors on DA re-uptake. Melatonin receptors retained DAT in the endoplasmic reticulum in its immature non-glycosylated form. In conclusion, we reveal one of the first molecular complexes between G protein-coupled receptors (MT1 and MT2) and transporters (DAT) in which melatonin receptors regulate the availability of DAT at the plasma membrane, thus limiting the striatal DA re-uptake capacity in mice.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/metabolismo , Animales , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Unión Proteica , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT2/genética , Sinaptosomas/metabolismoRESUMEN
Protein-protein interactions (PPIs) are useful for understanding signaling cascades, predicting protein function, associating proteins with disease and fathoming drug mechanism of action. Currently, only â¼ 10% of human PPIs may be known, and about one-third of human proteins have no known interactions. We introduce FpClass, a data mining-based method for proteome-wide PPI prediction. At an estimated false discovery rate of 60%, we predicted 250,498 PPIs among 10,531 human proteins; 10,647 PPIs involved 1,089 proteins without known interactions. We experimentally tested 233 high- and medium-confidence predictions and validated 137 interactions, including seven novel putative interactors of the tumor suppressor p53. Compared to previous PPI prediction methods, FpClass achieved better agreement with experimentally detected PPIs. We provide an online database of annotated PPI predictions (http://ophid.utoronto.ca/fpclass/) and the prediction software (http://www.cs.utoronto.ca/~juris/data/fpclass/).
Asunto(s)
Biología Computacional/métodos , Simulación por Computador , Minería de Datos/métodos , Mapeo de Interacción de Proteínas/métodos , Humanos , Proteoma , Programas Informáticos , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , Humanos , Receptor de Adenosina A2A/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Quitina Sintasa/metabolismo , Detergentes , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Unión Proteica , Mapeo de Interacción de Proteínas , Proteoma/análisis , Proteoma/química , Proteoma/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Cell signaling, one of key processes in both normal cellular function and disease, is coordinated by numerous interactions between membrane proteins that change in response to stimuli. We present a split ubiquitin-based method for detection of integral membrane protein-protein interactions (PPIs) in human cells, termed mammalian-membrane two-hybrid assay (MaMTH). We show that this technology detects stimulus (hormone or agonist)-dependent and phosphorylation-dependent PPIs. MaMTH can detect changes in PPIs conferred by mutations such as those in oncogenic ErbB receptor variants or by treatment with drugs such as the tyrosine kinase inhibitor erlotinib. Using MaMTH as a screening assay, we identified CRKII as an interactor of oncogenic EGFR(L858R) and showed that CRKII promotes persistent activation of aberrant signaling in non-small cell lung cancer cells. MaMTH is a powerful tool for investigating the dynamic interactomes of human integral membrane proteins.
Asunto(s)
Membrana Celular/metabolismo , Mapeo de Interacción de Proteínas/métodos , Técnicas del Sistema de Dos Híbridos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Supervivencia Celular , Citosol/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Mutación , Fosforilación , Fosfotirosina/química , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Biología de Sistemas/métodos , Factores de Transcripción/química , Ubiquitina/químicaRESUMEN
High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.
Asunto(s)
Centrosoma/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Cromosomas Humanos/metabolismo , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations.
Asunto(s)
Investigación Biomédica/métodos , Proteómica/métodos , Transducción de Señal , Análisis por Matrices de Proteínas , Proteoma/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (â¼12% and â¼39%, respectively) by chelating calcium (EGTA, 50 µM; BAPTA-AM, 25 µM), whereas increasing intracellular calcium (thapsigargin, 1.5 µM) led to increased nucleoside uptake (â¼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 µM) significantly increased nucleoside uptake (â¼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 µM), or CaM antagonist, W7 (50 µM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/química , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sitios de Unión , Calcio/química , Células HEK293 , Humanos , Unión Proteica , Receptores de N-Metil-D-Aspartato/químicaRESUMEN
Over the past two decades, the field of systems biology, which encompasses the numerous, widely popular "OMICs" approaches, has driven many significant advances in biomedical research, enabling researchers to generate huge datasets at multiple levels of biological organization. Despite such successes, some scientists still think that "OMICs"-based research introduces a lot of chaos into the biomedical field and claim that the resultant data are often not reproducible and do not reveal deep mechanistic aspects of biological processes. In this editorial, I argue the following: first, that "OMICs" technologies have improved significantly to yield much better datasets; and second, that follow-up studies on components identified in "OMICs" analyses have yielded many valuable biological insights.
Asunto(s)
Conjuntos de Datos como Asunto , Genómica/tendencias , Biología de Sistemas/tendencias , Animales , Investigación Biomédica , Genómica/métodos , Humanos , Biología de Sistemas/métodosRESUMEN
Studying protein interaction networks of all proteins in an organism ("interactomes") remains one of the major challenges in modern biomedicine. Such information is crucial to understanding cellular pathways and developing effective therapies for the treatment of human diseases. Over the past two decades, diverse biochemical, genetic, and cell biological methods have been developed to map interactomes. In this review, we highlight basic principles of interactome mapping. Specifically, we discuss the strengths and weaknesses of individual assays, how to select a method appropriate for the problem being studied, and provide general guidelines for carrying out the necessary follow-up analyses. In addition, we discuss computational methods to predict, map, and visualize interactomes, and provide a summary of some of the most important interactome resources. We hope that this review serves as both a useful overview of the field and a guide to help more scientists actively employ these powerful approaches in their research.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Animales , Biología Computacional/métodos , Humanos , Mamíferos/metabolismo , Mapas de Interacción de ProteínasRESUMEN
A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol O-Aciltransferasa/metabolismo , Esteroles/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Esterificación/fisiología , Lipoproteínas/genética , Lipoproteínas/metabolismo , Microdominios de Membrana/genética , Ratones , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esterol O-Aciltransferasa/genéticaRESUMEN
In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT1 and MT2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT1 , but not with MT2 , we identified several presynaptic proteins, suggesting a potential role of MT1 in neurotransmission. Presynaptic localization of MT1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT1 physically interacts with the voltage-gated calcium channel Cav 2.2 and inhibits Cav 2.2-promoted Ca(2+) entry in an agonist-independent manner. In conclusion, we show that MT1 is part of the presynaptic protein network and negatively regulates Cav 2.2 activity, providing a first hint for potential synaptic functions of MT1.
Asunto(s)
Encéfalo/metabolismo , Canales de Calcio Tipo N/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Receptor de Melatonina MT1/metabolismo , Canales de Calcio Tipo N/genética , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/genética , Receptor de Melatonina MT1/genéticaRESUMEN
GPR37 is an orphan G protein-coupled receptor mostly enriched in brain areas such as the cerebellum, striatum, and hippocampus. Identified as a substrate of parkin, GPR37 has been suggested to play a role in Parkinson's disease. Distributed throughout the brain, the function of GPR37, however, remains unknown. We now provide the first mapping of GPR37 within the hippocampus, where GPR37 is widely expressed and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts, and axon terminals. GPR37 per se does not appear to play a role in learning and memory, since knocking out GPR37 (GPR37-KO) did not alter the performance in different hippocampal-related memory tasks. This is in agreement with slice electrophysiology experiments showing no differences both in short-term plasticity paired-pulse facilitation and long-term potentiation between WT and GPR37-KO mice. However, we report a potential functional interaction between GPR37 and adenosine A2A receptors (A2 A R) in the hippocampus, with A2 A R modulating the GPR37-associated phenotype. Thus, the absence of GPR37 appeared to sensitize mice to hippocampal A2 A R-mediated signaling, as observed by the effect of the A2 A R antagonist SCH58261 increasing synaptic depotentiation, reducing novel object recognition memory and reverting the anxiolytic effect of GPR37 deletion. Collectively, these findings afford insight into the localization and role of the orphan GPR37 within the hippocampus with potential involvement in A2 A R function (i.e., A2 A R sensitization). GPR37 is an orphan G protein-coupled receptor widely expressed in the hippocampus and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts and axon terminals. This orphan receptor per se does not appear to directly control the learning and memory processes; however knocking-out GPR37 triggers anxiolytic-like effects and sensitizes mice to hippocampal A2A R-mediated signalling.