RESUMEN
Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP7 and IP8, synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.
Asunto(s)
Fosfatos de Inositol , Fosfotransferasas (Aceptor del Grupo Fosfato) , Transducción de Señal , Humanos , Fosfatos de Inositol/metabolismo , Animales , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Homeostasis , Metabolismo Energético , Adenosina Trifosfato/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genéticaRESUMEN
Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for â¼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.
Asunto(s)
Caenorhabditis elegans , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica/métodos , Técnicas de Silenciamiento del Gen , FenotipoRESUMEN
Decades of research have identified genetic factors and biochemical pathways involved in neurodegenerative diseases (NDDs). We present evidence for the following eight hallmarks of NDD: pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death. We describe the hallmarks, their biomarkers, and their interactions as a framework to study NDDs using a holistic approach. The framework can serve as a basis for defining pathogenic mechanisms, categorizing different NDDs based on their primary hallmarks, stratifying patients within a specific NDD, and designing multi-targeted, personalized therapies to effectively halt NDDs.
Asunto(s)
Enfermedades Neurodegenerativas , Humanos , Enfermedades Neurodegenerativas/patología , Proteostasis , Agregación Patológica de Proteínas/metabolismo , Muerte Celular , Citoesqueleto/metabolismoRESUMEN
Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
Asunto(s)
Glucagón , Receptores de Glucagón , Membrana Celular/metabolismo , Glucagón/metabolismo , Receptores de Glucagón/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
How cells become specialized, or "mature," is important for cell and developmental biology. While maturity is usually deemed a terminal fate, it may be more helpful to consider maturation not as a switch but as a dynamic continuum of adaptive phenotypic states set by genetic and environment programing. The hallmarks of maturity comprise changes in anatomy (form, gene circuitry, and interconnectivity) and physiology (function, rhythms, and proliferation) that confer adaptive behavior. We discuss efforts to harness their chemical (nutrients, oxygen, and growth factors) and physical (mechanical, spatial, and electrical) triggers in vitro and in vivo and how maturation strategies may support disease research and regenerative medicine.
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Diferenciación Celular , Animales , Investigación Biomédica , Proliferación Celular , Humanos , Modelos BiológicosRESUMEN
Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.
Asunto(s)
Biocombustibles/análisis , Desarrollo Sostenible , Vías Biosintéticas , Ciclo del Carbono , Humanos , Lignina/metabolismo , ResiduosRESUMEN
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Receptor de Androstano Constitutivo/metabolismo , Lipólisis , Receptores Acoplados a Proteínas G/metabolismo , Termogénesis , Adipocitos/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Frío , Grasas de la Dieta/farmacología , Humanos , Ratones Endogámicos C57BL , Fenotipo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Sistema Nervioso Simpático/metabolismo , Transcripción GenéticaRESUMEN
The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.
Asunto(s)
Química Farmacéutica/métodos , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Técnicas de Sonda Molecular , Terapia Molecular Dirigida/métodos , Transferencia de Energía por Resonancia de Bioluminiscencia , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Genes Reporteros , Humanos , Cinética , Imagen Óptica/métodos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Delgadez/genética , Tejido Adiposo/metabolismo , Adulto , Animales , Línea Celular , Estudios de Cohortes , Drosophila/genética , Estonia , Femenino , Humanos , Leptina/genética , Lipólisis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Interferencia de ARN/fisiología , Adulto JovenRESUMEN
Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.
Asunto(s)
Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Aciltransferasas/metabolismo , Sitios de Unión , Dominio Catalítico , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Ácidos Grasos/biosíntesis , Ácidos Grasos/química , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Animales , Sitios de Unión , Células CHO , Agonistas de Receptores de Cannabinoides/síntesis química , Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/síntesis química , Antagonistas de Receptores de Cannabinoides/farmacología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Animales , Archaea/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Filogenia , Conformación ProteicaRESUMEN
Local translation meets protein turnover and plasticity demands at synapses, however, the location of its energy supply is unknown. We found that local translation in neurons is powered by mitochondria and not by glycolysis. Super-resolution microscopy revealed that dendritic mitochondria exist as stable compartments of single or multiple filaments. To test if these mitochondrial compartments can serve as local energy supply for synaptic translation, we stimulated individual synapses to induce morphological plasticity and visualized newly synthesized proteins. Depletion of local mitochondrial compartments abolished both the plasticity and the stimulus-induced synaptic translation. These mitochondrial compartments serve as spatially confined energy reserves, as local depletion of a mitochondrial compartment did not affect synaptic translation at remote spines. The length and stability of dendritic mitochondrial compartments and the spatial functional domain were altered by cytoskeletal disruption. These results indicate that cytoskeletally tethered local energy compartments exist in dendrites to fuel local translation during synaptic plasticity.
Asunto(s)
Mitocondrias/metabolismo , Neuronas/metabolismo , Biosíntesis de Proteínas/fisiología , Animales , Citoesqueleto/metabolismo , Dendritas/metabolismo , Espinas Dendríticas/metabolismo , Femenino , Masculino , Mitocondrias/fisiología , Plasticidad Neuronal/fisiología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sinapsis/metabolismoRESUMEN
Current machine learning techniques enable robust association of biological signals with measured phenotypes, but these approaches are incapable of identifying causal relationships. Here, we develop an integrated "white-box" biochemical screening, network modeling, and machine learning approach for revealing causal mechanisms and apply this approach to understanding antibiotic efficacy. We counter-screen diverse metabolites against bactericidal antibiotics in Escherichia coli and simulate their corresponding metabolic states using a genome-scale metabolic network model. Regression of the measured screening data on model simulations reveals that purine biosynthesis participates in antibiotic lethality, which we validate experimentally. We show that antibiotic-induced adenine limitation increases ATP demand, which elevates central carbon metabolism activity and oxygen consumption, enhancing the killing effects of antibiotics. This work demonstrates how prospective network modeling can couple with machine learning to identify complex causal mechanisms underlying drug efficacy.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Adenina/metabolismo , Biología Computacional/métodos , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/metabolismo , Aprendizaje Automático , Redes y Vías Metabólicas/inmunología , Modelos Teóricos , Purinas/metabolismoRESUMEN
Chromatin is a mighty consumer of cellular energy generated by metabolism. Metabolic status is efficiently coordinated with transcription and translation, which also feed back to regulate metabolism. Conversely, suppression of energy utilization by chromatin processes may serve to preserve energy resources for cell survival. Most of the reactions involved in chromatin modification require metabolites as their cofactors or coenzymes. Therefore, the metabolic status of the cell can influence the spectra of posttranslational histone modifications and the structure, density and location of nucleosomes, impacting epigenetic processes. Thus, transcription, translation, and DNA/RNA biogenesis adapt to cellular metabolism. In addition to dysfunctions of metabolic enzymes, imbalances between metabolism and chromatin activities trigger metabolic disease and life span alteration. Here, we review the synthesis of the metabolites and the relationships between metabolism and chromatin function. Furthermore, we discuss how the chromatin response feeds back to metabolic regulation in biological processes.
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Cromatina/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Cromatina/genética , Ensamble y Desensamble de Cromatina , Metabolismo Energético , Epigénesis Genética , Código de Histonas , Humanos , Longevidad/genética , Longevidad/fisiología , Modelos BiológicosRESUMEN
As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/química , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and ßγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Metabolismo Energético , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/genéticaRESUMEN
The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.
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Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Ingestión de Alimentos , Secretina/metabolismo , Termogénesis , Adipocitos Marrones/citología , Tejido Adiposo Pardo/citología , Animales , Células HEK293 , Humanos , Lipólisis , Ratones , Ratones Noqueados , Ratones Obesos , Secretina/genéticaRESUMEN
The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Metabolismo Energético , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adipocitos/patología , Tejido Adiposo/patología , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Línea Celular Transformada , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteínas Serina-Treonina Quinasas/genética , Quinasa de Factor Nuclear kappa BRESUMEN
The bacterial chaperonin GroEL and its cofactor, GroES, form a nano-cage for a single molecule of substrate protein (SP) to fold in isolation. GroEL and GroES undergo an ATP-regulated interaction cycle to close and open the folding cage. GroEL consists of two heptameric rings stacked back to back. Here, we show that GroEL undergoes transient ring separation, resulting in ring exchange between complexes. Ring separation occurs upon ATP-binding to the trans ring of the asymmetric GroEL:7ADP:GroES complex in the presence or absence of SP and is a consequence of inter-ring negative allostery. We find that a GroEL mutant unable to perform ring separation is folding active but populates symmetric GroEL:GroES2 complexes, where both GroEL rings function simultaneously rather than sequentially. As a consequence, SP binding and release from the folding chamber is inefficient, and E. coli growth is impaired. We suggest that transient ring separation is an integral part of the chaperonin mechanism.