RESUMEN
Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells.
Asunto(s)
Infecciones por VIH/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Línea Celular , VIH-2 , HumanosRESUMEN
BACKGROUND: TASOR, a component of the HUSH repressor epigenetic complex, and SAMHD1, a cellular triphosphohydrolase (dNTPase), are both anti-HIV proteins antagonized by HIV-2/SIVsmm Viral protein X. As a result, the same viral protein is able to relieve two different blocks along the viral life cell cycle, one at the level of reverse transcription, by degrading SAMHD1, the other one at the level of proviral expression, by degrading TASOR. Phosphorylation of SAMHD1 at T592 has been shown to downregulate its antiviral activity. The discovery that T819 in TASOR was lying within a SAMHD1 T592-like motif led us to ask whether TASOR is phosphorylated on this residue and whether this post-translational modification could regulate its repressive activity. RESULTS: Using a specific anti-phospho-antibody, we found that TASOR is phosphorylated at T819, especially in cells arrested in early mitosis by nocodazole. We provide evidence that the phosphorylation is conducted by a Cyclin/CDK1 complex, like that of SAMHD1 at T592. While we could not detect TASOR in quiescent CD4 + T cells, TASOR and its phosphorylated form are present in activated primary CD4 + T lymphocytes. In addition, TASOR phosphorylation appears to be independent from TASOR repressive activity. Indeed, on the one hand, nocodazole barely reactivates HIV-1 in the J-Lat A1 HIV-1 latency model despite TASOR T819 phosphorylation. On the other hand, etoposide, a second cell cycle arresting drug, reactivates latent HIV-1, without concomitant TASOR phosphorylation. Furthermore, overexpression of wt TASOR or T819A or T819E similarly represses gene expression driven by an HIV-1-derived LTR promoter. Finally, while TASOR is degraded by HIV-2 Vpx, TASOR phosphorylation is prevented by HIV-1 Vpr, likely as a consequence of HIV-1 Vpr-mediated-G2 arrest. CONCLUSIONS: Altogether, we show that TASOR phosphorylation occurs in vivo on T819. This event does not appear to correlate with TASOR-mediated HIV-1 silencing. We speculate that TASOR phosphorylation is related to a role of TASOR during cell cycle progression.
Asunto(s)
Infecciones por VIH , VIH-1 , Proteínas de Unión al GTP Monoméricas , Humanos , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , VIH-1/fisiología , Fosforilación , Treonina , Nocodazol/metabolismo , Latencia del Virus , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.
Asunto(s)
Colestasis Intrahepática , Colestasis , Subfamilia B de Transportador de Casetes de Unión a ATP , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Simulación del Acoplamiento Molecular , FosfatidilcolinasRESUMEN
ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Hepatocitos/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transporte Biológico Activo , Membrana Celular/genética , Células HEK293 , Células HeLa , Humanos , Fosfatidilcolinas/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
Because proteins are the main mediators of most cellular processes they are also prime therapeutic targets. Identifying physical links among proteins and between drugs and their protein targets is essential in order to understand the mechanisms through which both proteins themselves and the molecules they are targeted with act. Thus, there is a strong need for sensitive methods that enable mapping out these biomolecular interactions. Here we present a robust and sensitive approach to screen proteome-scale collections of proteins for binding to proteins or small molecules using the well validated MAPPIT (Mammalian Protein-Protein Interaction Trap) and MASPIT (Mammalian Small Molecule-Protein Interaction Trap) assays. Using high-density reverse transfected cell microarrays, a close to proteome-wide collection of human ORF clones can be screened for interactors at high throughput. The versatility of the platform is demonstrated through several examples. With MAPPIT, we screened a 15k ORF library for binding partners of RNF41, an E3 ubiquitin protein ligase implicated in receptor sorting, identifying known and novel interacting proteins. The potential related to the fact that MAPPIT operates in living human cells is illustrated in a screen where the protein collection is scanned for interactions with the glucocorticoid receptor (GR) in its unliganded versus dexamethasone-induced activated state. Several proteins were identified the interaction of which is modulated upon ligand binding to the GR, including a number of previously reported GR interactors. Finally, the screening technology also enables detecting small molecule target proteins, which in many drug discovery programs represents an important hurdle. We show the efficiency of MASPIT-based target profiling through screening with tamoxifen, a first-line breast cancer drug, and reversine, an investigational drug with interesting dedifferentiation and antitumor activity. In both cases, cell microarray screens yielded known and new potential drug targets highlighting the utility of the technology beyond fundamental biology.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Análisis de Matrices Tisulares/métodos , Células HEK293 , Humanos , Bibliotecas de Moléculas Pequeñas/metabolismo , Tamoxifeno/metabolismoRESUMEN
ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Simulación del Acoplamiento Molecular , Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Colestasis Intrahepática/genética , Colestasis Intrahepática/metabolismo , Transporte de Proteínas , Ensayos Analíticos de Alto Rendimiento/métodos , Células HEK293RESUMEN
The pleiotropic cytokine hormone leptin, by activating its receptor OB-R, plays a major role in many biological processes, including energy homeostasis, immune function, and cell survival and proliferation. Abnormal leptin action is associated with obesity, autoimmune diseases, and cancer. The pharmacological characterization of OB-R and the development of synthetic OB-R ligands are still in their infancy because currently available binding assays are not compatible with ligand saturation binding experiments and high-throughput screening (HTS) approaches. We have developed here a novel homogeneous time-resolved fluorescence-based binding assay that overcomes these limitations. In this assay, fluorescently labeled leptin or leptin antagonist binds to the SNAP-tagged OB-R covalently labeled with terbium cryptate (Tb). Successful binding is monitored by measuring the energy transfer between the Tb energy donor and the fluorescently labeled leptin energy acceptor. Ligand binding saturation experiments revealed high-affinity dissociation constants in the subnanomolar range with an excellent signal-to-noise ratio. The assay performed in a 384-well format shows high specificity and reproducibility, making it perfectly compatible with HTS applications to identify new OB-R agonists or antagonists. In addition, fluorescently labeled leptin and SNAP-tagged OB-R will be valuable tools for monitoring leptin and OB-R trafficking in cells and tissues.
Asunto(s)
Fluorescencia , Receptores de Leptina/metabolismo , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Sitios de Unión/efectos de los fármacos , Células Cultivadas , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Antagonistas de Hormonas/química , Antagonistas de Hormonas/metabolismo , Antagonistas de Hormonas/farmacología , Humanos , Leptina/antagonistas & inhibidores , Leptina/química , Leptina/metabolismo , Ligandos , Unión Proteica , Receptores de Leptina/análisis , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R.
Asunto(s)
Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Receptores de Leptina/metabolismo , Animales , Proteínas Portadoras/genética , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/genética , Lisosomas/metabolismo , Mutación , Transporte de Proteínas/fisiología , Ratas , Receptores de Leptina/genética , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
CONTEXT: The genomic organization of the LEPR gene is complex and generates three independent transcripts whose respective functions are still poorly understood. METHODS/RESULTS: We describe here a 7-year old patient with a homozygous 80 kb deletion in the chromosomal 1p31.3 region with early onset obesity, mental retardation and epilepsy. The deleted region comprises the proximal promoter and exons 1 and 2 of the LEPR gene and exons 5 to 19 of the DNAJC6 gene. The deletion leads to the deficiency of all canonical OB-R isoforms but maintains the B219 OB-R short isoforms controlled by the preserved second LEPR promoter. The DNAJC6 gene encodes auxilin-1, a protein required for clathrin-dependent recycling of synaptic vesicles in neurons that is possibly at the origin of the mental retardation and epilepsy phenotype. The obese phenotype and the absence of signaling-competent OB-R are consistent with previously reported individuals with OB-R deficiency. The deletion eliminates an additional transcript of the LEPR gene that encodes endospanin-1, a protein that has been genetically and biochemically linked to OB-R function. CONCLUSIONS: Our study confirms the phenotype of individuals with OB-R deficiency and postulates the effects of auxilin-1 deficiency (mental retardation/epilepsy) and endospanin-1 deficiency (OB-R specific functions) in humans.
Asunto(s)
Cromosomas Humanos Par 1/genética , Epilepsia/genética , Proteínas del Choque Térmico HSP40/genética , Discapacidad Intelectual/genética , Obesidad/genética , Receptores de Leptina/genética , Eliminación de Secuencia , Adolescente , Adulto , Edad de Inicio , Niño , Preescolar , Femenino , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
The Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin recruits the histone methyl-transferase SETDB1 to spread H3K9me3 repressive marks across genes and transgenes in an integration site-dependent manner. The deposition of these repressive marks leads to heterochromatin formation and inhibits gene expression, but the underlying mechanism is not fully understood. Here, we show that TASOR silencing or HIV-2 Vpx expression, which induces TASOR degradation, increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. Furthermore, using a yeast 2-hybrid screen, we identify new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 synergistically repress HIV expression from its LTR. Similar to the RNA-induced transcriptional silencing complex found in fission yeast, we show that TASOR interacts with the RNA exosome and RNA Polymerase II, predominantly under its elongating state. Finally, we show that TASOR facilitates the association of RNA degradation proteins with RNA polymerase II and is detected at transcriptional centers. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral expression.
Asunto(s)
Represión Epigenética , VIH-2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estabilidad del ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensamble y Desensamble de Cromatina , Expresión Génica , Silenciador del Gen , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , Células HeLa , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Humanos , Fosfoproteínas , Provirus/genética , ARN Polimerasa II/metabolismo , SchizosaccharomycesRESUMEN
ABCB4, is an adenosine triphosphate-binding cassette (ABC) transporter localized at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine secretion into bile. Gene variations of ABCB4 cause different types of liver diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3). The molecular mechanisms underlying the trafficking of ABCB4 to and from the canalicular membrane are still unknown. We identified the serine/threonine kinase Myotonic dystrophy kinase-related Cdc42-binding kinase isoform α (MRCKα) as a novel partner of ABCB4. The role of MRCKα was explored, either by expression of dominant negative mutant or by gene silencing using the specific RNAi and CRISPR-cas9 strategy in cell models. The expression of a dominant-negative mutant of MRCKα and MRCKα inhibition by chelerythrine both caused a significant increase in ABCB4 steady-state expression in primary human hepatocytes and HEK-293 cells. RNA interference and CRISPR-Cas9 knockout of MRCKα also caused a significant increase in the amount of ABCB4 protein expression. We demonstrated that the effect of MRCKα was mediated by its downstream effector, the myosin II regulatory light chain (MRLC), which was shown to also bind ABCB4. Our findings provide evidence that MRCKα and MRLC bind to ABCB4 and regulate its cell surface expression.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Colestasis Intrahepática , Colestasis , Proteína Quinasa de Distrofia Miotónica , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Células HEK293 , Humanos , Cadenas Ligeras de Miosina , Miosina Tipo II , Proteína Quinasa de Distrofia Miotónica/metabolismoRESUMEN
Obesity is a major public health problem and is often associated with type 2 diabetes mellitus, cardiovascular disease, and metabolic syndrome. Leptin is the crucial adipostatic hormone that controls food intake and body weight through the activation of specific leptin receptors (OB-R) in the hypothalamic arcuate nucleus (ARC). However, in most obese patients, high circulating levels of leptin fail to bring about weight loss. The prevention of this "leptin resistance" is a major goal for obesity research. We report here a successful prevention of diet-induced obesity (DIO) by silencing a negative regulator of OB-R function, the OB-R gene-related protein (OB-RGRP), whose transcript is genetically linked to the OB-R transcript. We provide in vitro evidence that OB-RGRP controls OB-R function by negatively regulating its cell surface expression. In the DIO mouse model, obesity was prevented by silencing OB-RGRP through stereotactic injection of a lentiviral vector encoding a shRNA directed against OB-RGRP in the ARC. This work demonstrates that OB-RGRP is a potential target for obesity treatment. Indeed, regulators of the receptor could be more appropriate targets than the receptor itself. This finding could serve as the basis for an approach to identifying potential new therapeutic targets for a variety of diseases, including obesity.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Leptina/metabolismo , Obesidad/prevención & control , Receptores de Leptina/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dieta , Grasas de la Dieta/administración & dosificación , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Oligonucleótidos Antisentido/genética , Receptores de Leptina/antagonistas & inhibidores , Receptores de Leptina/genética , Transducción de SeñalRESUMEN
Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Retículo Endoplásmico/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Roscovitina/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente , Humanos , Estructura Molecular , Proteínas Mutantes , Inhibidores de Proteínas Quinasas/química , Transporte de Proteínas/efectos de los fármacos , Roscovitina/análogos & derivados , Roscovitina/químicaRESUMEN
Human ABC (ATP Binding Cassette) transporters form a superfamily of forty-eight transmembrane proteins, which transport their substrates across biological membranes against important concentration gradients, in an energy-dependent manner. Gene variations in approximately half of these transporters have been identified in subjects with rare and often severe genetic diseases, highlighting the importance of their biological function. For missense variations leading to defects in ABC transporters, the current challenge is to identify new molecules with therapeutic potential able to rescue the induced molecular deficiency. In this review, we first address the progress provided by emerging pharmacotherapies in cystic fibrosis, the most frequent monogenic disease caused by variations of an ABC transporter, i.e. ABCC7/CFTR. Then, we enlarge the topic to the other ABC transporters, more notably to canalicular ABC transporters, the variations of which cause rare hepatobiliary diseases, and we discuss the first promising attempts aiming to correct molecular defects of these proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Sistemas de Liberación de Medicamentos/tendencias , Terapia Genética/tendencias , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Fibrosis Quística/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Variación Genética/genética , HumanosRESUMEN
The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.
Asunto(s)
Proteínas Portadoras/metabolismo , Obesidad/metabolismo , Receptores de Leptina/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Peso Corporal/fisiología , Proteínas Portadoras/fisiología , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Leptina/metabolismo , Leptina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Receptores de Leptina/fisiología , Factor de Transcripción STAT3/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The leptin receptor, OBR, is involved in the regulation of whole-body energy homeostasis. Most obese people are resistant to leptin and do not respond to the hormone. The prevention and reversal of leptin resistance is one of the major current goals of obesity research. We showed previously that increased OBR cell surface expression concomitantly increases cellular leptin signaling and prevents obesity development in mice. Improvement of OBR cell surface expression can thus be considered as an interesting anti-obesity therapeutic strategy. To identify compounds that increase the surface expression of OBR, we developed a cell-based, phenotypic assay to perform a high-content screen (HCS) against a library of 50,000 chemical compounds. We identified 67 compounds that increased OBR cell surface expression with AC50 values in the low micromolar range and no effect on total OBR expression and cellular toxicity. Compounds were classified into 16 chemical clusters, of which 4 potentiated leptin-promoted signaling through the JAK2/STAT3 pathway. In conclusion, development of a robust phenotypic screening approach resulted in the discovery of four new scaffolds that demonstrate the desired biological activity and could constitute an original therapeutic solution against obesity and associated disorders.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Obesidad/metabolismo , Fenotipo , Receptores de Leptina/metabolismo , Línea Celular , Descubrimiento de Drogas/métodos , Expresión Génica , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Obesidad/tratamiento farmacológico , Obesidad/genética , Receptores de Leptina/genética , Proteínas Recombinantes de Fusión , Bibliotecas de Moléculas PequeñasRESUMEN
We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced beta-arrestin (betaARR) intramolecular rearrangements and recruitment to G protein-coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing betaARR assays and offer new options for the development of future BRET assays.