RESUMEN
Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial-both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2'-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.
Asunto(s)
Oligonucleótidos Antisentido/análisis , Oligonucleótidos Fosforotioatos/análisis , Espectrometría de Masa de Ion Secundario/métodos , Células 3T3-L1 , Acetilgalactosamina/administración & dosificación , Acetilgalactosamina/análisis , Animales , Receptor de Asialoglicoproteína/análisis , Cesio , Células HEK293 , Células HeLa , Humanos , Riñón/química , Riñón/ultraestructura , Hígado/química , Hígado/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Miocardio/química , Miocardio/ultraestructura , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacocinética , Seudópodos/química , Seudópodos/ultraestructura , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/biosíntesis , ARN Largo no Codificante/genética , Fracciones Subcelulares/química , Azufre/análisis , Isótopos de Azufre/análisis , Distribución TisularRESUMEN
Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their therapeutic properties. Given the importance of PS backbone for interaction with proteins, we systematically replaced anionic PS-linkages in toxic ASOs with charge-neutral alkylphosphonate linkages. Site-specific incorporation of alkyl phosphonates altered the RNaseH1 cleavage patterns but overall rates of cleavage and activity versus the on-target gene in cells and in mice were only minimally affected. However, replacing even one PS-linkage at position 2 or 3 from the 5'-side of the DNA-gap with alkylphosphonates reduced or eliminated toxicity of several hepatotoxic gapmer ASOs. The reduction in toxicity was accompanied by the absence of nucleolar mislocalization of paraspeckle protein P54nrb, ablation of P21 mRNA elevation and caspase activation in cells, and hepatotoxicity in mice. The generality of these observations was further demonstrated for several ASOs versus multiple gene targets. Our results add to the types of structural modifications that can be used in the gap-region to enhance ASO safety and provide insights into understanding the biochemistry of PS ASO protein interactions.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Oligonucleótidos Antisentido/química , Organofosfonatos/química , Oligonucleótidos Fosforotioatos/química , Células 3T3-L1 , Animales , Caspasas/metabolismo , Línea Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Proteínas de Unión al ADN , Células HeLa , Hepatocitos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Fosforotioatos/administración & dosificación , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.
Asunto(s)
Músculo Esquelético , Miocardio , Oligonucleótidos Antisentido/química , Células 3T3-L1 , Albúminas/metabolismo , Animales , Colesterol/química , Interacciones Hidrofóbicas e Hidrofílicas , Lipoproteínas/metabolismo , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/toxicidad , Palmitatos/química , Ratas Sprague-Dawley , Tocoferoles/químicaRESUMEN
The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.
Asunto(s)
Ácidos Nucleicos/metabolismo , Oligonucleótidos Fosforotioatos/química , Proteínas/metabolismo , HumanosRESUMEN
Antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages are broadly used as research tools and therapeutic agents. Chemically modified PS-ASOs can mediate efficient target reduction by site-specific cleavage of RNA through RNase H1. PS-ASOs are known to be internalized via a number of endocytotic pathways and are released from membrane-enclosed endocytotic organelles, mainly late endosomes (LEs). This study was focused on the details of PS-ASO trafficking through endocytic pathways. It was found that lysobisphosphatidic acid (LBPA) is required for release of PS-ASOs from LEs. PS-ASOs exited early endosomes (EEs) rapidly after internalization and became co-localized with LBPA by 2 hours in LEs. Inside LEs, PS-ASOs and LBPA were co-localized in punctate, dot-like structures, likely intraluminal vesicles (ILVs). Deactivation of LBPA using anti-LBPA antibody significantly decreased PS-ASO activities without affecting total PS-ASO uptake. Reduction of Alix also substantially decreased PS-ASO activities without affecting total PS-ASO uptake. Furthermore, Alix reduction decreased LBPA levels and limited co-localization of LBPA with PS-ASOs at ILVs inside LEs. Thus, the fusion properties of ILVs, which are supported by LBPA, contribute to PS-ASO intracellular release from LEs.
Asunto(s)
Endosomas/metabolismo , Espacio Intracelular/metabolismo , Lisofosfolípidos/metabolismo , Monoglicéridos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Transporte Biológico , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Proteína Niemann-Pick C1 , Oligonucleótidos Antisentido/químicaRESUMEN
Antisense oligonucleotide (ASO) therapeutics show tremendous promise for the treatment of previously intractable human diseases but to exert their effects on cellular RNA processing they must first cross the plasma membrane by endocytosis. The conjugation of ASOs to a receptor ligand can dramatically increase their entry into certain cells and tissues, as demonstrated by the implementation of N-acetylgalactosamine (GalNAc)-conjugated ASOs for Asialoglycoprotein Receptor (ASGR)-mediated uptake into liver hepatocytes. We compared the internalization and activity of GalNAc-conjugated ASOs and their parents in endogenous ASGR-expressing cells and were able to recapitulate hepatocyte ASO uptake and activity in cells engineered to heterologously express the receptor. We found that the minor receptor subunit, ASGR2, is not required for effective in vitro or in vivo uptake of GalNAc-conjugated ASO and that the major subunit, ASGR1, plays a small but significant role in the uptake of unconjugated phosphorothioate ASOs into hepatocytes. Moreover, our data demonstrates there is a large excess capacity of liver ASGR for the effective uptake of GalNAc-ASO conjugates, suggesting broad opportunities to exploit receptors with relatively moderate levels of expression.
Asunto(s)
Acetilgalactosamina , Receptor de Asialoglicoproteína/metabolismo , Hepatocitos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Animales , Transporte Biológico , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/químicaRESUMEN
Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release.
Asunto(s)
Anexina A2/metabolismo , Endocitosis , Endosomas/metabolismo , Oligonucleótidos Antisentido/metabolismo , Anexina A2/deficiencia , Transporte Biológico , Línea Celular , Humanos , Proteínas S100/metabolismo , Sales (Química)RESUMEN
Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins.
Asunto(s)
Linfocitos B/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Ácidos Nucleicos/metabolismo , Oligonucleótidos/farmacología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Ácidos Siálicos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Ácidos Siálicos/síntesis química , Ácidos Siálicos/química , Relación Estructura-ActividadRESUMEN
The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization.
Asunto(s)
Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Piridinas , Aminas/síntesis química , Aminas/química , Aminas/farmacología , Animales , Cristalografía por Rayos X , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Furanos/síntesis química , Furanos/química , Furanos/farmacología , Humanos , Concentración 50 Inhibidora , Ratones , Estructura Molecular , Neoplasias/tratamiento farmacológico , Piridinas/síntesis química , Piridinas/farmacocinética , Piridinas/farmacología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The phosphorothioate (PS) linkage in an essential component of therapeutic oligonucleotides. PS in the DNA region of gapmer antisense oligonucleotides (ASOs) supports RNaseH1 activity and enhances nuclease stability. PS also promotes binding to plasma, cell surface, and intracellular proteins, which facilitates tissue distribution, cellular uptake, and endosomal escape of PS ASOs. We recently showed that site-specific replacement of PS in the DNA gap with methoxylpropyl phosphonate (MOP) linkages can enhance the therapeutic index of gapmer ASOs. In this article, we explored 18 phosphorus- and non-phosphorus-based neutral backbone modifications to determine the structure-activity relationship of neutral linkages for enhancing therapeutic index. Replacing MOP with other alkyl phosphonate and phosphotriester linkages enhanced therapeutic index, but these linkages were susceptible to chemical degradation during oligonucleotide deprotection from solid supports following synthesis. Replacing MOP with non-phosphorus linkages resulted in improved chemical stability, but these linkages were introduced into ASOs as nucleotide dimers, which limits their versatility. Overall, linkages such as isopropyl and isobutyl phosphonates and O-isopropyl and O-tetrahydrofuranosyl phosphotriesters, formacetal, and C3-amide showed improved activity in mice relative to MOP. Our data suggest that site-specific incorporation of any neutral backbone linkage can improve therapeutic index, but the size, hydrophobicity, and RNA-binding affinity of the linkage influence ASO activity.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Animales , Endosomas/metabolismo , Ratones , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Oligonucleótidos Fosforotioatos/genética , Fósforo , Índice TerapéuticoRESUMEN
We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.
Asunto(s)
Oligonucleótidos Antisentido , Receptor de Angiotensina Tipo 1 , Animales , Ratones , Oligonucleótidos Antisentido/farmacología , Receptor de Angiotensina Tipo 1/genética , Transducción de SeñalRESUMEN
Synthetic therapeutic oligonucleotides (STO) represent the third bonafide platform for drug discovery in the pharmaceutical industry after small molecule and protein therapeutics. So far, thirteen STOs have been approved by regulatory agencies and over one hundred of them are in different stages of clinical trials. STOs hybridize to their target RNA or DNA in cells via Watson-Crick base pairing to exert their pharmacological effects. This unique class of therapeutic agents has the potential to target genes and gene products that are considered undruggable by other therapeutic platforms. However, STOs must overcome several extracellular and intracellular obstacles to interact with their biological RNA targets inside cells. These obstacles include degradation by extracellular nucleases, scavenging by the reticuloendothelial system, filtration by the kidney, traversing the capillary endothelium to access the tissue interstitium, cell-surface receptor-mediated endocytic uptake, and escape from endolysosomal compartments to access the nuclear and/or cytoplasmic compartments where their targets reside. In this review, we present the recent advances in this field with a specific focus on antisense oligonucleotides (ASOs) and siRNA therapeutics.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos , Descubrimiento de Drogas , ARN Interferente PequeñoRESUMEN
Despite recent advances, targeted delivery of therapeutic oligonucleotide to extra-hepatic tissues continues to be a challenging endeavor and efficient ligand-receptor systems need to be identified. To determine the feasibility of using neurotensin to improve the productive uptake of antisense oligonucleotides (ASO), we synthesized neurotensin-ASO conjugates and evaluated their cellular uptake and activity in cells and in mice. We performed a comprehensive structure-activity relationship study of the conjugates and determined the influence of ASO charge, ASO length, peptide charge, linker chemistry and ligand identity on receptor binding and internalization. We identified a modified neurotensin peptide capable of improving the cellular uptake and activity of gapmer ASOs in sortilin expressing cells (sixfold) and in spinal cord in mice (twofold). Neurotensin conjugation also improved the potency of morpholino ASO designed to correct splicing of survival motor neuron pre-mRNA in the cortex and striatum after intracerebroventricular injection. Neurotensin-mediated targeted delivery represents a possible approach for enhancing the potency of ASOs with diverse nucleic acid modifications.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Neurotensina/química , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Animales , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Morfolinos/administración & dosificación , Morfolinos/química , Morfolinos/farmacocinética , Oligonucleótidos Antisentido/químicaRESUMEN
Antisense oligonucleotides (ASOs) are chemically synthesized nucleic acid analogs designed to bind to RNA by Watson-Crick base pairing. Following binding to the targeted RNA, the ASO perturbs RNA function by promoting selective degradation of the targeted RNA, altering RNA intermediary metabolism, or disrupting function of the RNA. Most antisense drugs are chemically modified to enhance their pharmacological properties and for passive targeting of the tissues of therapeutic interest. Recent advances in selective tissue targeting have resulted in a newer generation of ASO drugs that are more potent and better tolerated than previous generations, spawning renewed interest in identifying selective ligands that enhance targeted delivery of ASOs to tissues.
Asunto(s)
Sistemas de Liberación de Medicamentos , Oligonucleótidos Antisentido , Animales , Humanos , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/uso terapéuticoRESUMEN
Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.
Asunto(s)
Endocitosis/fisiología , Receptor trkA/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Técnicas de Cultivo de Célula , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/fisiología , Clonación Molecular , Ratones , Datos de Secuencia Molecular , Ratas , Receptor trkA/química , Receptor trkA/genéticaRESUMEN
Oligonucleotide therapeutics have emerged as a third distinct platform for drug discovery within the pharmaceutical industry. Five oligonucleotide-based drugs have been approved by the US FDA and over 100 oligonucleotides drugs are currently at different stages of human trials. Several of these oligonucleotide drugs are modified using the phosphorothioate (PS) backbone modification where one of the nonbridging oxygen atoms of the phosphodiester linkage is replaced with sulfur. In this review, we summarize our knowledge on receptor-mediated uptake of PS antisense oligonucleotides (ASOs) within different cell types of the liver-a privileged organ for the discovery of oligonucleotide-based therapeutics.
Asunto(s)
Endocitosis , Técnicas de Transferencia de Gen , Glicoconjugados/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , ARN Interferente Pequeño/metabolismo , Acetilgalactosamina/metabolismo , Animales , Receptor de Asialoglicoproteína/genética , Receptor de Asialoglicoproteína/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Ingeniería Celular/métodos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células HEK293 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/genética , ARN Interferente Pequeño/genética , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase A/metabolismoRESUMEN
Many protein interactions with G-protein-coupled receptors (GPCRs) appear to influence receptor signaling and functional regulation. There is great interest therefore in methods for the identification of novel or unanticipated GPCR binding proteins. A proven method for identifying such protein interactions is the yeast two-hybrid screen, which involves screening the protein products of a cDNA library with a selected domain derived from a GPCR. Once it is established that a candidate protein produces a specific positive interaction within the yeast two-hybrid system, it is important to demonstrate further that this interaction is likely to occur in vivo. Coimmunoprecipitation, in which proteins of interest are copurified with the receptor under study, is a good way to address this important issue. Together, the yeast two-hybrid screen and coimmunoprecipitation are a useful way to identify and sort through candidate GPCR-interacting proteins prior to analysis in physiological studies.
Asunto(s)
Pruebas de Precipitina/métodos , Mapeo de Interacción de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Técnicas del Sistema de Dos Híbridos , Biblioteca de Genes , Plásmidos , Unión Proteica , Receptores Acoplados a Proteínas G/genética , Transformación Genética , Levaduras/genética , Levaduras/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
OBJECTIVE: Because previous preclinical and clinical studies have implicated the endogenous opioid system in major depression and in the neurochemical action of antidepressants, the authors examined how DNA variation in the mu-opioid receptor gene may influence population variation in response to citalopram treatment. METHOD: A total of 1,953 individuals from the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study were treated with citalopram and genotyped for 53 single nucleotide polymorphisms (SNPs) in a 100-kb region of the OPRM1 gene. The sample consisted of Non-Hispanic Caucasians, Hispanic Caucasians, and African Americans. Population stratification was corrected using 119 ancestry informative markers and principal components analysis. Markers were tested for association with phenotypes for general and specific citalopram response as well as remission. RESULTS: Association between one SNP and specific citalopram response was observed. After Bonferroni correction, the strongest finding was the association between the rs540825 SNP and specific response. The rs540825 polymorphism is a nonsynonymous SNP in the final exon of the mu-opioid receptor-1X isoform of the OPRM1 gene, resulting in a histidine to glutamine change in the intracellular domain of the receptor. When Hispanic and Non-Hispanic Caucasians were analyzed separately, similar results in the population-corrected analyses were detected. CONCLUSIONS: These results suggest that rates of response to antidepressants and consequent remission from major depressive disorder are influenced by variation in the mu-opioid receptor gene as a result of either an effect on placebo response or true pharmacologic response.
Asunto(s)
Antidepresivos de Segunda Generación/uso terapéutico , Citalopram/uso terapéutico , Trastorno Depresivo Mayor/tratamiento farmacológico , Estudios de Asociación Genética , Receptores Opioides mu/genética , Trastorno Depresivo Mayor/genética , Genotipo , Haplotipos/genética , Hispánicos o Latinos/genética , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Escalas de Valoración Psiquiátrica , Resultado del Tratamiento , Población Blanca/genéticaRESUMEN
Mu-type opioid receptors are physiologically important G-protein-coupled receptors that are generally thought to recycle after agonist-induced endocytosis. Here we show that several alternatively spliced receptor variants fail to do so efficiently because of splice-mediated removal of an endocytic sorting sequence that is present specifically in the MOR1 variant. All of the recycling-impaired receptor variants were found to undergo proteolytic down-regulation more rapidly than MOR1, irrespective of moderate differences in endocytic rate, indicating that alternative splicing plays a specific role in distinguishing the trafficking itinerary of receptors after endocytosis. The recycling-impaired MOR1B variant was similar to MOR1 in its ability to mediate opioid-dependent inhibition of adenylyl cyclase, and to undergo opioid-induced desensitization in intact cells. Functional recovery (resensitization) of MOR1B-mediated cellular responsiveness after opioid removal, however, was significantly impaired (4-fold reduction in rate) compared with MOR1. To our knowledge the present results are the first to establish a role of alternative RNA processing in specifying the post-endocytic sorting of G-protein-coupled receptors between divergent and functionally distinct membrane pathways.
Asunto(s)
Empalme Alternativo/fisiología , Membrana Celular/metabolismo , Regulación hacia Abajo/fisiología , Endocitosis/fisiología , Receptores Opioides mu/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Línea Celular , Membrana Celular/genética , Humanos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas/fisiología , Receptores Opioides mu/agonistas , Receptores Opioides mu/genéticaRESUMEN
Ubiquitination of cytoplasmic lysine residues can target G protein-coupled receptors (GPCRs) to proteasomes and has recently been shown to also be required for sorting of certain GPCRs to lysosomes following ligand-induced endocytosis. We addressed the generality of this mechanism by examining regulated proteolysis of the murine delta opioid receptor (DOR) expressed in human embryonic kidney 293 cells, a well characterized model system in which receptors are sorted to lysosomes. Incubation of cells in the presence of the highly specific proteasome inhibitor lactacystin did not detectably affect ligand-induced proteolysis of DOR but significantly delayed ligand-induced proteolysis of epidermal growth factor receptors. Mutation of all cytoplasmic lysine residues in DOR, creating a mutant opioid receptor that is unable to be ubiquitinated, did not detectably inhibit either ligand-induced endocytosis or proteolytic degradation of endocytosed receptors. Furthermore, the lysine-mutated DOR, like its wild type counterpart, colocalized extensively with lysosomes after ligand-induced endocytosis. These results demonstrate that ubiquitination of DOR is not required either for its ligand-induced endocytosis or for postendocytic trafficking to lysosomes.