RESUMEN
Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and a/b-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. Significance Statement Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in HDL particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study we show that recently discovered small molecule activators can rescue function in LCAT deficient variants when the mutations occur in the lid and cap domains of the enzyme.
RESUMEN
Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a disease-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with WT CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.
Asunto(s)
Caveolina 1 , Caveolinas , Enfermedad , Humanos , Caveolas/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Subunidades de Proteína/metabolismo , Enfermedad/genéticaRESUMEN
Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome. These patients harbor germline and somatic mutations in Dicer that lead to defective microRNA processing and activity. While most mutations occur within Dicer's catalytic RNase III domains, alterations within the Platform-PAZ (Piwi-Argonaute-Zwille) domain also cause loss of microRNA production. Using a combination of in vitro biochemical and cellular studies, we characterized the effect of disease-relevant Platform-PAZ-associated mutations on the processing of a well-studied oncogenic microRNA, pre-microRNA-21. We then compared these results to those of a representative from another Dicer substrate class, the small nucleolar RNA, snord37. From this analysis, we provide evidence that mutations within the Platform-PAZ domain result in differential impacts on RNA binding and processing, adding new insights into the complexities of Dicer processing of small RNA substrates.
Asunto(s)
MicroARNs , ARN Nucleolar Pequeño , Niño , Humanos , ARN Nucleolar Pequeño/genética , Ribonucleasa III/química , MicroARNs/química , Mutación , ARN Helicasas DEAD-box/genéticaRESUMEN
Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits IκB kinase ε (IKKε) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes the inhibitory effects. IKKε is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKKε and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.
Asunto(s)
Aminopiridinas/farmacología , Ácidos Carboxílicos/farmacología , Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Línea Celular , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/efectos de los fármacos , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.
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Apolipoproteína A-I/fisiología , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Dominio Catalítico , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Activación Enzimática , Humanos , Lipoproteínas HDL/metabolismo , Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Conformación ProteicaRESUMEN
The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R7 and R8 A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC50 values as low as 210â¯nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R7 cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.
Asunto(s)
Quinasa I-kappa B/antagonistas & inhibidores , Obesidad/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Células 3T3-L1 , Aminación , Animales , Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/química , Fármacos Antiobesidad/farmacología , Fármacos Antiobesidad/uso terapéutico , Cromanos/síntesis química , Cromanos/química , Cromanos/farmacología , Cromanos/uso terapéutico , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Obesidad/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Piridinas/síntesis química , Piridinas/uso terapéuticoRESUMEN
Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis. VacA assembles into oligomeric complexes in solution and forms anion-selective channels in cell membranes. Cryo-electron microscopy (cryo-EM) analyses of VacA oligomers in solution provided insights into VacA oligomerization but failed to reveal the structure of the hydrophobic N-terminal region predicted to be a pore-forming domain. In this study, we incubated VacA with liposomes and used single particle cryo-EM to analyze detergent-extracted VacA oligomers. A 3D structure of detergent-solubilized VacA hexamers revealed the presence of six α-helices extending from the center of the oligomers, a feature not observed in previous studies of water-soluble VacA oligomers. Cryo-electron tomography analysis and 2D averages of VacA associated with liposomes confirmed that central regions of the membrane-associated VacA oligomers can insert into the lipid bilayer. However, insertion is heterogenous, with some membrane-associated oligomers appearing only partially inserted and others sitting on top of the bilayer. These studies indicate that VacA undergoes a conformational change when contacting the membrane and reveal an α-helical region positioned to extend into the membrane. Although the reported VacA 3D structure does not represent a selective anion channel, our combined single particle 3D analysis, cryo-electron tomography, and modeling allow us to propose a model for the structural organization of the VacA N-terminus in the context of a hexamer as it inserts into the membrane.
Asunto(s)
Proteínas Bacterianas , Helicobacter pylori , Toxinas Biológicas , Canales Aniónicos Dependientes del Voltaje , Humanos , Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Detergentes , Helicobacter pylori/química , Liposomas/química , Toxinas Biológicas/química , Canales Aniónicos Dependientes del Voltaje/química , Multimerización de ProteínaRESUMEN
Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6. The VPS9 domain is a guanine nucleotide exchange factor for Rab31, a Rab5 subfamily GTPase implicated in trans-Golgi network (TGN)-to-endosome trafficking. Overexpression of Rab31 blocks insulin-stimulated Glut4 translocation, whereas knockdown of Rab31 potentiates insulin-stimulated Glut4 translocation and glucose uptake. Gapex-5 is predominantly cytosolic in untreated cells; its overexpression promotes intracellular retention of Glut4 in adipocytes. Insulin recruits the CIP4/Gapex-5 complex to the plasma membrane, thus reducing Rab31 activity and permitting Glut4 vesicles to translocate to the cell surface, where Glut4 docks and fuses to transport glucose into the cell.
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Adipocitos/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/química , Insulina/metabolismo , Ratones , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Microscopía por Crioelectrón/métodos , Legionella pneumophila/química , Proteínas Bacterianas/química , Conformación Proteica , Especificidad de la Especie , Sistemas de Secreción Tipo IV/químicaRESUMEN
Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.
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Epítopos/química , Proteínas de la Membrana/química , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Cristalografía por Rayos X , Epítopos/inmunología , Humanos , Proteínas de la Membrana/inmunología , Microscopía Electrónica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity. However, it remains challenging to develop vaccine nanoparticles that can preserve and present conformational epitopes of protein antigens for induction of neutralizing antibody responses. Here, we have designed a new lipid-based nanoparticle vaccine platform (NVP) that presents viral proteins (HIV-1 and SARS-CoV-2 antigens) in a conformational manner for induction of antigen-specific antibody responses. We show that NVP was readily taken up by dendritic cells (DCs) and promoted DC maturation and antigen presentation. NVP loaded with BG505.SOSIP.664 (SOSIP) or SARS-CoV-2 receptor-binding domain (RBD) was readily recognized by neutralizing antibodies, indicating the conformational display of antigens on the surfaces of NVP. Rabbits immunized with SOSIP-NVP elicited strong neutralizing antibody responses against HIV-1. Furthermore, mice immunized with RBD-NVP induced robust and long-lasting antibody responses against RBD from SARS-CoV-2. These results suggest that NVP is a promising platform technology for vaccination against infectious pathogens.
Asunto(s)
Vacunas contra el SIDA/química , Vacunas contra la COVID-19/química , Inmunidad Humoral/efectos de los fármacos , Lípidos/química , Nanopartículas , Vacunas Virales/química , Vacunas contra el SIDA/administración & dosificación , Adyuvantes Inmunológicos , Animales , Presentación de Antígeno , Reacciones Antígeno-Anticuerpo , Vacunas contra la COVID-19/administración & dosificación , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , VIH-1 , Humanos , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Conejos , SARS-CoV-2 , Vacunas Virales/administración & dosificaciónRESUMEN
Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes.
Asunto(s)
Proteínas Portadoras/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Apoptosis , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Homólogo 1 de la Proteína Discs Large , Glucosa/metabolismo , Guanilato-Quinasas , Insulina/farmacología , Fusión de Membrana , Microdominios de Membrana/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Estructura Terciaria de Proteína , Transporte de Proteínas , Interferencia de ARN , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rhoRESUMEN
BACKGROUND AND PURPOSE: The δ-opioid receptor is an emerging target for the management of chronic pain and depression. Biased signalling, the preferential activation of one signalling pathway over another downstream of δ-receptors, may generate better therapeutic profiles. BMS 986187 is a positive allosteric modulator of δ-receptors. Here, we ask if BMS 986187 can directly activate the receptor from an allosteric site, without an orthosteric ligand, and if a signalling bias is generated. EXPERIMENTAL APPROACH: We used several clonal cell lines expressing δ-receptors, to assess effects of BMS 986187 on events downstream of δ-receptors by measuring G-protein activation, ß-arrestin 2 recruitment, receptor phosphorylation, loss of surface receptor expression, ERK1/ERK2 phosphorylation, and receptor desensitization. KEY RESULTS: BMS 986187 is a G protein biased allosteric agonist, relative to ß-arrestin 2 recruitment. Despite showing direct and potent G protein activation, BMS 986187 has a low potency to recruit ß-arrestin 2. This appears to reflect the inability of BMS 986187 to elicit any significant receptor phosphorylation, consistent with low receptor internalization and a slower onset of desensitization, compared with the full agonist SNC80. CONCLUSIONS AND IMPLICATIONS: This is the first evidence of biased agonism mediated through direct binding to an allosteric site on an opioid receptor, without a ligand at the orthosteric site. Our data suggest that agonists targeting δ-receptors, or indeed any GPCR, through allosteric sites may be a novel way to promote signalling bias and thereby potentially produce a more specific pharmacology than can be observed by activation via the orthosteric site.
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Proteínas de Unión al GTP/agonistas , Receptores Opioides delta/metabolismo , Xantonas/farmacología , Sitio Alostérico , Animales , Células CHO , Cricetulus , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Masculino , RatonesRESUMEN
Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.
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HDL-Colesterol/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Dominio Catalítico , Activadores de Enzimas/química , Activadores de Enzimas/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Células HEK293 , Humanos , Lípidos de la Membrana/metabolismo , Mutación/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Conformación Proteica , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Previous studies have suggested that activation of the Rho family member GTPase TC10 is necessary but not sufficient for the stimulation of glucose transport by insulin. We show here that endogenous TC10alpha is rapidly activated in response to insulin in 3T3L1 adipocytes in a phosphatidylinositol 3-kinase-independent manner, whereas platelet-derived growth factor was without effect. Knockdown of TC10alpha but not TC10beta by RNA interference inhibited insulin-stimulated glucose uptake as well as the translocation of the insulin-sensitive glucose transporter GLUT4 from intracellular sites to the plasma membrane. In contrast, loss of TC10alpha had no effect on the stimulation of Akt by insulin. Additionally, knockdown of TC10alpha inhibited insulin-stimulated translocation of its effector CIP4. These data indicate that TC10alpha is specifically required for insulin-stimulated glucose uptake in adipocytes.
Asunto(s)
Adipocitos/metabolismo , Glucosa/farmacocinética , Hipoglucemiantes/farmacología , Insulina/farmacología , Proteínas de Unión al GTP rho/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Animales , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/genéticaRESUMEN
Insulin stimulates glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane. The exocyst complex tethers GLUT4-containing vesicles to the plasma membrane, a process that requires the binding of the G protein (heterotrimeric guanine nucleotide-binding protein) RalA to the exocyst complex. We report that upon activation of RalA, the protein kinase TBK1 phosphorylated the exocyst subunit Exo84. Knockdown of TBK1 blocked insulin-stimulated glucose uptake and GLUT4 translocation; knockout of TBK1 in adipocytes blocked insulin-stimulated glucose uptake; and ectopic overexpression of a kinase-inactive mutant of TBK1 reduced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The phosphorylation of Exo84 by TBK1 reduced its affinity for RalA and enabled its release from the exocyst. Overexpression of a kinase-inactive mutant of TBK1 blocked the dissociation of the TBK1/RalA/exocyst complex, and treatment of 3T3-L1 adipocytes with specific inhibitors of TBK1 reduced the rate of complex dissociation. Introduction of phosphorylation-mimicking or nonphosphorylatable mutant forms of Exo84 blocked insulin-stimulated GLUT4 translocation. Thus, these data indicate that TBK1 controls GLUT4 vesicle engagement and disengagement from the exocyst, suggesting that exocyst components not only constitute a tethering complex for the GLUT4 vesicle but also act as "gatekeepers" controlling vesicle fusion at the plasma membrane.
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Adipocitos/efectos de los fármacos , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Transportador de Glucosa de Tipo 4/genética , Hipoglucemiantes/farmacología , Immunoblotting , Ratones , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Insulin stimulates glucose uptake in insulin-responsive tissues by means of the translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane. Two pathways are required, one involving activation of a phosphatidylinositol 3-kinase (PI 3-kinase) and downstream protein kinases, and one involving activation of the Rho-family GTPase TC10. TC10 activation by insulin is catalyzed by the exchange factor C3G, which is translocated to lipid rafts along with its binding partner CrkII as a consequence of Cbl tyrosine phosphorylation by the insulin receptor. This activation of TC10 is dependent on localization of TC10 in the lipid raft subdomains of the plasma membrane. We describe experimental approaches using the insulin-responsive cell line 3T3-L1 adipocytes to study the role of TC10 in insulin-stimulated glucose transport.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Proteínas de Unión al GTP rho/fisiología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Androstadienos/farmacología , Animales , Diferenciación Celular , Centrifugación por Gradiente de Densidad , Electroporación , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Magnesio/farmacología , Ratones , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Recombinantes de Fusión , Wortmanina , Proteínas de Unión al GTP rho/genéticaRESUMEN
The search for effective treatments for obesity and its comorbidities is of prime importance. We previously identified IKK-ε and TBK1 as promising therapeutic targets for the treatment of obesity and associated insulin resistance. Here we show that acute inhibition of IKK-ε and TBK1 with amlexanox treatment increases cAMP levels in subcutaneous adipose depots of obese mice, promoting the synthesis and secretion of the cytokine IL-6 from adipocytes and preadipocytes, but not from macrophages. IL-6, in turn, stimulates the phosphorylation of hepatic Stat3 to suppress expression of genes involved in gluconeogenesis, in the process improving glucose handling in obese mice. Preliminary data in a small cohort of obese patients show a similar association. These data support an important role for a subcutaneous adipose tissue-liver axis in mediating the acute metabolic benefits of amlexanox on glucose metabolism, and point to a new therapeutic pathway for type 2 diabetes.
Asunto(s)
Gluconeogénesis , Hígado/metabolismo , Transducción de Señal , Grasa Subcutánea/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Adulto , Anciano , Aminopiridinas/farmacología , Animales , AMP Cíclico/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Gluconeogénesis/efectos de los fármacos , Glucosa-6-Fosfatasa/metabolismo , Humanos , Inflamación/patología , Resistencia a la Insulina , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores Adrenérgicos beta/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Grasa Subcutánea/efectos de los fármacos , Adulto Joven , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Glycogen and lipids are major storage forms of energy that are tightly regulated by hormones and metabolic signals. We demonstrate that feeding mice a high-fat diet (HFD) increases hepatic glycogen due to increased expression of the glycogenic scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels were augmented in mice and cells after activation of the mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target SREBP1. Deletion of the PTG gene in mice prevented HFD-induced hepatic glycogen accumulation. Of note, PTG deletion also blocked hepatic steatosis in HFD-fed mice and reduced the expression of numerous lipogenic genes. Additionally, PTG deletion reduced fasting glucose and insulin levels in obese mice while improving insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk was due to decreased mTORC1 and SREBP activity in PTG knockout mice or knockdown cells, suggesting a positive feedback loop in which once accumulated, glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to lipogenesis. Together, these data reveal a previously unappreciated broad role for glycogen in the control of energy homeostasis.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/deficiencia , Metabolismo de los Lípidos , Glucógeno Hepático/metabolismo , Obesidad/metabolismo , Animales , Dieta Alta en Grasa , Metabolismo Energético/fisiología , Retroalimentación , Glucógeno/metabolismo , Células HEK293 , Humanos , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Lipogénesis , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate ß-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored ß-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C). Conversely, overexpression of the kinases reduced induction of Ucp1, lipolysis, cAMP levels, and phosphorylation of hormone sensitive lipase in response to isoproterenol or forskolin. Noncanonical IKKs reduce catecholamine sensitivity by phosphorylating and activating the major adipocyte phosphodiesterase PDE3B. In vivo inhibition of these kinases by treatment of obese mice with the drug amlexanox reversed obesity-induced catecholamine resistance, and restored PKA signaling in response to injection of a ß-3 adrenergic agonist. These studies suggest that by reducing production of cAMP in adipocytes, IKKε and TBK1 may contribute to the repression of energy expenditure during obesity. DOI: http://dx.doi.org/10.7554/eLife.01119.001.