RESUMEN
Opioids are powerful analgesics, but also carry significant side effects and abuse potential. Here we describe a modulator of the µ-opioid receptor (MOR1), the transient receptor potential channel subfamily vanilloid member 1 (TRPV1). We show that TRPV1 binds MOR1 and blocks opioid-dependent phosphorylation of MOR1 while leaving G protein signaling intact. Phosphorylation of MOR1 initiates recruitment and activation of the ß-arrestin pathway, which is responsible for numerous opioid-induced adverse effects, including the development of tolerance and respiratory depression. Phosphorylation stands in contrast to G protein signaling, which is responsible for the analgesic effect of opioids. Calcium influx through TRPV1 causes a calcium/calmodulin-dependent translocation of G protein-coupled receptor kinase 5 (GRK5) away from the plasma membrane, thereby blocking its ability to phosphorylate MOR1. Using TRPV1 to block phosphorylation of MOR1 without affecting G protein signaling is a potential strategy to improve the therapeutic profile of opioids.
Asunto(s)
Receptores Opioides mu/metabolismo , Canales Catiónicos TRPV/metabolismo , Membrana Celular/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.
Asunto(s)
Proteínas Cullin/metabolismo , Complejos Multiproteicos/metabolismo , Péptido Hidrolasas/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ácido Fítico/biosíntesis , Secuencia de Aminoácidos , Complejo del Señalosoma COP9 , Dominio Catalítico , Proteínas Cullin/química , Proteínas Cullin/genética , Estabilidad de Enzimas , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Dominios y Motivos de Interacción de Proteínas , Homología de Secuencia de Aminoácido , Electricidad Estática , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPγS), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTPγS binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the µ-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Conteo por Cintilación/métodos , Guanosina 5'-O-(3-Tiotrifosfato)/análisis , Células HEK293 , Humanos , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/metabolismo , Transducción de Señal , Radioisótopos de Azufre/análisisRESUMEN
Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development.
Asunto(s)
Trastornos Congénitos de Glicosilación/genética , Modelos Animales de Enfermedad , Hidroliasas/genética , Sistema Nervioso/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Glicosilación , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Mutación Missense , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Pez CebraRESUMEN
In humans, mutations in electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETFDH) lead to MADD/glutaric aciduria type II, an autosomal recessively inherited disorder characterized by a broad spectrum of devastating neurological, systemic and metabolic symptoms. We show that a zebrafish mutant in ETFDH, xavier, and fibroblast cells from MADD patients demonstrate similar mitochondrial and metabolic abnormalities, including reduced oxidative phosphorylation, increased aerobic glycolysis, and upregulation of the PPARG-ERK pathway. This metabolic dysfunction is associated with aberrant neural proliferation in xav, in addition to other neural phenotypes and paralysis. Strikingly, a PPARG antagonist attenuates aberrant neural proliferation and alleviates paralysis in xav, while PPARG agonists increase neural proliferation in wild type embryos. These results show that mitochondrial dysfunction, leading to an increase in aerobic glycolysis, affects neurogenesis through the PPARG-ERK pathway, a potential target for therapeutic intervention.