RESUMEN
CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryoelectron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.
Asunto(s)
Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/fisiología , Ribonucleasas/fisiología , Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Microscopía por Crioelectrón/métodos , Endonucleasas/metabolismo , Células HEK293 , Humanos , Conformación Molecular , ARN/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/ultraestructura , Ribonucleasas/metabolismo , Ribonucleasas/ultraestructuraRESUMEN
Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/ß5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.
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Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Remodelación Ósea , Fibronectinas/metabolismo , Integrina alfaV/metabolismo , Osteocitos/metabolismo , Osteólisis/metabolismo , Adipocitos/patología , Animales , Línea Celular Tumoral , Femenino , Fibronectinas/genética , Células HEK293 , Humanos , Integrina alfaV/genética , Ratones , Osteocitos/patología , Osteólisis/genéticaRESUMEN
We report the discovery of a fluorescent small molecule probe. This probe exhibits an emission increase in the presence of the oncoprotein MYC that can be attenuated by a competing inhibitor. Hydrogen-deuterium exchange mass spectrometry analysis, rationalized by induced-fit docking, suggests it binds to the "coiled-coil" region of the leucine zipper domain. Point mutations of this site produced functional MYC constructs resistant to inhibition in an oncogenic transformation assay by compounds that displace the probe. Utilizing this probe, we have developed a high-throughput assay to identify MYC inhibitor scaffolds. Screening of a diversity library (N = 1408, 384-well) and a library of pharmacologically active compounds (N = 1280, 1536-well) yielded molecules with greater drug-like properties than the probe. One lead is a potent inhibitor of oncogenic transformation and is specific for MYC relative to resistant mutants and transformation-inducing oncogenes. This method is simple, inexpensive, and does not require protein modification, DNA binding, or the dimer partner MAX. This assay presents an opportunity for MYC inhibition researchers to discover unique scaffolds.
Asunto(s)
Desarrollo de Medicamentos , Colorantes Fluorescentes/farmacología , Ensayos Analíticos de Alto Rendimiento , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Estructura Molecular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-ActividadRESUMEN
AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the ß-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including ß2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1ß2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1ß1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Activadores de Enzimas/química , Proteínas Quinasas Activadas por AMP/metabolismo , Dominio Catalítico , Células Hep G2 , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.
Asunto(s)
Enzimas Inmovilizadas/química , Pepsina A/química , Proteolisis , Animales , Biocatálisis , Deuterio/química , Medición de Intercambio de Deuterio , Espectrometría de Masas , Sarraceniaceae/enzimología , Especificidad por Sustrato , PorcinosRESUMEN
AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/metabolismo , Nucleótidos/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Adenina/química , Sitios de Unión , Humanos , Modelos Moleculares , Nucleótidos/químicaRESUMEN
Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.
RESUMEN
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Análisis de Varianza , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Deuterio/análisis , Medición de Intercambio de Deuterio/instrumentación , Hidrógeno/análisis , Ligandos , Espectrometría de Masas/instrumentación , Péptidos/análisis , Proteínas/química , Receptores de Calcitriol/química , Reproducibilidad de los ResultadosRESUMEN
Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.
Asunto(s)
Proteínas de la Matriz Extracelular , Sinapsis , Humanos , Proteínas Portadoras/metabolismo , Microscopía por Crioelectrón , Proteínas de la Matriz Extracelular/metabolismo , Células Fotorreceptoras/metabolismo , Sinapsis/metabolismoRESUMEN
GPR158 is an orphan G proteincoupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryoelectron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/química , Proteínas RGS/química , Receptores Acoplados a Proteínas G/química , Sitios de Unión , Microscopía por Crioelectrón , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Humanos , Ligandos , Modelos Moleculares , Fosfolípidos/química , Unión Proteica , Conformación Proteica , Conformación Proteica en Hélice alfa , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfocitos T , Linfocitos B , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , InmunoterapiaRESUMEN
Circularized nandiscs (cNDs) exhibit superb monodispersity and have the potential to transform functional and structural studies of membrane proteins. In particular, cNDs can stabilize large patches of lipid bilayers for the reconstitution of complex membrane biochemical reactions, enabling the capture of crucial intermediates involved in synaptic transmission and viral entry. However, previous methods for building cNDs require multiple steps and suffer from low yields. We herein introduce a simple, one-step approach to ease the construction of cNDs using the SpyCatcher-SpyTag technology. This approach increases the yield of cNDs by over 10-fold and is able to rapidly generates cNDs with diameters ranging from 11 to over 100 nm. We demonstrate the utility of these cNDs for mechanistic interrogations of vesicle fusion and protein-lipid interactions that are unattainable using small nanodiscs. Together, the remarkable performance of SpyCatcher-SpyTag in nanodisc circularization paves the way for the use of cNDs in membrane biochemistry and structural biology.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteínas de la Membrana/genética , Nanoestructuras/química , Péptidos/genética , Ingeniería de Proteínas/métodos , Ingeniería Celular/métodos , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Humanos , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Nanoestructuras/ultraestructura , Oxadiazoles/química , Tamaño de la Partícula , Péptidos/química , Péptidos/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Coloración y Etiquetado/métodosRESUMEN
The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.
Asunto(s)
Proteasas ATP-Dependientes/ultraestructura , Proteínas Mitocondriales/ultraestructura , Proteasas ATP-Dependientes/antagonistas & inhibidores , Proteasas ATP-Dependientes/genética , Proteasas ATP-Dependientes/metabolismo , Adenosina Trifosfato/metabolismo , Envejecimiento/metabolismo , Bortezomib/farmacología , Dominio Catalítico/efectos de los fármacos , Microscopía por Crioelectrón , Pruebas de Enzimas , Humanos , Hidrólisis , Mitocondrias/metabolismo , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Oxidación-Reducción , Unión Proteica/efectos de los fármacos , Dominios Proteicos/genética , Proteolisis , Proteostasis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to 'sense' the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.
Asunto(s)
Coactivador 3 de Receptor Nuclear/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Elementos de Respuesta , Animales , Sitios de Unión , ADN/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Espectrometría de Masas/métodos , Ratones Endogámicos BALB C , Coactivador 3 de Receptor Nuclear/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Conformación Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Despite a myriad of available pharmacotherapies for the treatment of type 2 diabetes (T2D), challenges still exist in achieving glycemic control. Several novel glucose-lowering strategies are currently under clinical investigation, highlighting the need for more robust treatments. Previously, we have shown that suppressing peroxisome proliferator-activated receptor gamma coactivator 1-alpha activity with a small molecule (SR18292, 16) can reduce glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Despite structural similarities in 16 to known ß-blockers, detailed structure-activity relationship studies described herein have led to the identification of analogues lacking ß-adrenergic activity that still maintain the ability to suppress glucagon-induced glucose release from hepatocytes and ameliorate hyperglycemia in diabetic mouse models. Hence, these compounds exert their biological effects in a mechanism that does not include adrenergic signaling. These probe molecules may lead to a new therapeutic approach to treat T2D either as a single agent or in combination therapy.
Asunto(s)
Glucagón/antagonistas & inhibidores , Gluconeogénesis/efectos de los fármacos , Hipoglucemiantes/farmacología , Indoles/farmacología , Propanoles/farmacología , Adipocitos Marrones/efectos de los fármacos , Adipocitos Marrones/metabolismo , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hipoglucemiantes/química , Indoles/química , Lipólisis/efectos de los fármacos , Glucógeno Hepático/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR gamma/efectos de los fármacos , Propanoles/química , Receptores Adrenérgicos beta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Sistema Nervioso Simpático/efectos de los fármacosRESUMEN
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/inmunología , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Humanos , Fragmentos Fab de Inmunoglobulinas , Modelos Moleculares , Fosforilación , Conformación Proteica , Dominios Proteicos , Ingeniería de ProteínasRESUMEN
Obesity and rheumatic disease are mechanistically linked via chronic inflammation. The orphan receptor TREM-1 (triggering receptor expressed on myeloid cells-1) is a potent amplifier of proinflammatory and noninfectious immune responses. Here, we show that the pan modulator SR1903 effectively blocks TREM-1 activation. SR1903 emerged from a chemical series of potent RORγ inverse agonists, although unlike close structural analogues, it has modest agonist activity on LXR and weak repressive activity (inverse agonism) of PPARγ, three receptors that play essential roles in inflammation and metabolism. The anti-inflammatory and antidiabetic efficacy of this unique modulator in collagen-induced arthritis and diet-induced obesity mouse models is demonstrated. Interestingly, in the context of obesity, SR1903 aided in the maintenance of the thymic homeostasis unlike selective RORγ inverse agonists. SR1903 was well-tolerated following chronic administration, and combined, these data suggest that it may represent a viable strategy for treatment of both metabolic and inflammatory disease. More importantly, the ability of SR1903 to block LPS signaling suggests the potential utility of this unique polypharmacological modulator for treatment of innate immune response disorders.
Asunto(s)
Compuestos de Bifenilo/farmacología , Inflamación/metabolismo , Piperazinas/farmacología , Polifarmacología , Propanoles/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Animales , Artritis Experimental/tratamiento farmacológico , Compuestos de Bifenilo/uso terapéutico , Dieta , Modelos Animales de Enfermedad , Agonismo Inverso de Drogas , Ligandos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Obesidad/tratamiento farmacológico , Obesidad/etiología , PPAR gamma/agonistas , PPAR gamma/metabolismo , Piperazinas/uso terapéutico , Propanoles/uso terapéutico , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Receptor Activador Expresado en Células Mieloides 1/metabolismoRESUMEN
Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it's unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous-but not synthetic-ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.
Asunto(s)
Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Células HEK293 , Humanos , Ligandos , Modelos Moleculares , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Unión Proteica , Electricidad EstáticaRESUMEN
The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.