RESUMEN
The recruitment of signaling proteins into activated receptor tyrosine kinases (RTKs) to produce rapid, high-fidelity downstream response is exposed to the ambiguity of random diffusion to the target site. Liquid-liquid phase separation (LLPS) overcomes this by providing elevated, localized concentrations of the required proteins while impeding competitor ligands. Here, we show a subset of phosphorylation-dependent RTK-mediated LLPS states. We then investigate the formation of phase-separated droplets comprising a ternary complex including the RTK, (FGFR2); the phosphatase, SHP2; and the phospholipase, PLCγ1, which assembles in response to receptor phosphorylation. SHP2 and activated PLCγ1 interact through their tandem SH2 domains via a previously undescribed interface. The complex of FGFR2 and SHP2 combines kinase and phosphatase activities to control the phosphorylation state of the assembly while providing a scaffold for active PLCγ1 to facilitate access to its plasma membrane substrate. Thus, LLPS modulates RTK signaling, with potential consequences for therapeutic intervention.
Asunto(s)
Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Transducción de Señal , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival and Akt-mediated glycolysis and triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent antitumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Neoplasias/enzimología , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Genes p53 , Glucólisis/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Trasplante Heterólogo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a 'numbers game', where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid-liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras , Transducción de Señal , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Probabilidad , Sistemas de Liberación de MedicamentosRESUMEN
Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.
Asunto(s)
ADN Bacteriano/inmunología , ADN/inmunología , Inmunidad Innata/inmunología , Interleucinas/inmunología , Células Th17/inmunología , Receptor Toll-Like 9/inmunología , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Ratones , Psoriasis/inmunología , Receptores de Interleucina/inmunología , Receptores de Interleucina/metabolismoRESUMEN
Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.
Asunto(s)
Proteína Adaptadora GRB2/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Dimerización , Células HEK293 , Humanos , Modelos Moleculares , Fosforilación , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/químicaRESUMEN
Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis.
Asunto(s)
Estudio de Asociación del Genoma Completo , Melanoma/genética , Mutagénesis , Rayos Ultravioleta , Secuencia de Aminoácidos , Células Cultivadas , Exoma , Humanos , Melanocitos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas B-raf/genética , Alineación de Secuencia , Proteína de Unión al GTP rac1/genéticaRESUMEN
Loss of glycogen myophosphorylase (PYGM) expression results in an inability to break down muscle glycogen, leading to McArdle disease-an autosomal recessive metabolic disorder characterized by exercise intolerance and muscle cramps. While previously considered relatively benign, this condition has recently been associated with pattern dystrophy in the retina, accompanied by variable sight impairment, secondary to retinal pigment epithelial (RPE) cell involvement. However, the pathomechanism of this condition remains unclear. In this study, we generated a PYGM-null induced pluripotent stem cell line and differentiated it into mature RPE to examine structural and functional defects, along with metabolite release into apical and basal media. Mutant RPE exhibited normal photoreceptor outer segment phagocytosis but displayed elevated glycogen levels, reduced transepithelial resistance, and increased cytokine secretion across the epithelial layer compared to isogenic WT controls. Additionally, decreased expression of the visual cycle component, RDH11, encoding 11-cis-retinol dehydrogenase, was observed in PYGM-null RPE. While glycolytic flux and oxidative phosphorylation levels in PYGM-null RPE were near normal, the basal oxygen consumption rate was increased. Oxygen consumption rate in response to physiological levels of lactate was significantly greater in WT than PYGM-null RPE. Inefficient lactate utilization by mutant RPE resulted in higher glucose dependence and increased glucose uptake from the apical medium in the presence of lactate, suggesting a reduced capacity to spare glucose for photoreceptor use. Metabolic tracing confirmed slower 13C-lactate utilization by PYGM-null RPE. These findings have key implications for retinal health since they likely underlie the vision impairment in individuals with McArdle disease.
RESUMEN
As an environment-dependent pleiotropic gene regulator in Gram-negative bacteria, the H-NS protein is crucial for adaptation and toxicity control of human pathogens such as Salmonella, Vibrio cholerae or enterohaemorrhagic Escherichia coli. Changes in temperature affect the capacity of H-NS to form multimers that condense DNA and restrict gene expression. However, the molecular mechanism through which H-NS senses temperature and other physiochemical parameters remains unclear and controversial. Combining structural, biophysical and computational analyses, we show that human body temperature promotes unfolding of the central dimerization domain, breaking up H-NS multimers. This unfolding event enables an autoinhibitory compact H-NS conformation that blocks DNA binding. Our integrative approach provides the molecular basis for H-NS-mediated environment-sensing and may open new avenues for the control of pathogenic multi-drug resistant bacteria.
Asunto(s)
Proteínas Bacterianas/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Desplegamiento Proteico , Proteínas Bacterianas/genética , ADN Bacteriano/química , Proteínas de Unión al ADN/genética , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/patogenicidad , Interacción Gen-Ambiente , Humanos , Dominios Proteicos , Multimerización de Proteína/genética , Salmonella/genética , Salmonella/patogenicidad , Temperatura , Vibrio cholerae/genética , Vibrio cholerae/patogenicidadRESUMEN
The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.
Asunto(s)
Cadherinas/fisiología , Flagelos/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Precursores de Proteínas/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/fisiología , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Relacionadas con las Cadherinas , Embrión de Pollo , Clatrina/fisiología , Ratones , Fosforilación , Transporte de Proteínas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/análisisRESUMEN
The aim of rational drug design is to develop small molecules using a quantitative approach to optimize affinity. This should enhance the development of chemical compounds that would specifically, selectively, reversibly, and with high affinity interact with a target protein. It is not yet possible to develop such compounds using computational (i.e., in silico) approach and instead the lead molecules are discovered in high-throughput screening searches of large compound libraries. The main reason why in silico methods are not capable to deliver is our poor understanding of the compound structure-thermodynamics and structure-kinetics correlations. There is a need for databases of intrinsic binding parameters (e.g., the change upon binding in standard Gibbs energy (ΔGint), enthalpy (ΔHint), entropy (ΔSint), volume (ΔVintr), heat capacity (ΔCp,int), association rate (ka,int), and dissociation rate (kd,int)) between a series of closely related proteins and a chemically diverse, but pharmacophoric group-guided library of compounds together with the co-crystal structures that could help explain the structure-energetics correlations and rationally design novel compounds. Assembly of these data will facilitate attempts to provide correlations and train data for modeling of compound binding. Here, we report large datasets of the intrinsic thermodynamic and kinetic data including over 400 primary sulfonamide compound binding to a family of 12 catalytically active human carbonic anhydrases (CA). Thermodynamic parameters have been determined by the fluorescent thermal shift assay, isothermal titration calorimetry, and by the stopped-flow assay of the inhibition of enzymatic activity. Kinetic measurements were performed using surface plasmon resonance. Intrinsic thermodynamic and kinetic parameters of binding were determined by dissecting the binding-linked protonation reactions of the protein and sulfonamide. The compound structure-thermodynamics and kinetics correlations reported here helped to discover compounds that exhibited picomolar affinities, hour-long residence times, and million-fold selectivities over non-target CA isoforms. Drug-lead compounds are suggested for anticancer target CA IX and CA XII, antiglaucoma CA IV, antiobesity CA VA and CA VB, and other isoforms. Together with 85 X-ray crystallographic structures of 60 compounds bound to six CA isoforms, the database should be of help to continue developing the principles of rational target-based drug design.
Asunto(s)
Antineoplásicos/química , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Simulación por Computador , Bicarbonatos/química , Dominio Catalítico , Estabilidad de Enzimas , Humanos , Cinética , Estructura Molecular , Isoformas de Proteínas/química , Sulfonamidas/química , TermodinámicaRESUMEN
Although essential for T cell function, the identity of the T cell receptor "inside-out" pathway for lymphocyte function-associated antigen 1 (LFA-1) adhesion has proved elusive. Here, we define the "inside-out" pathway mediated by N-terminal SKAP1 (SKAP-55) domain binding to the C-terminal SARAH domain of RapL. TcR induced Rap1-RapL complex formation and LFA-1 binding failed to occur in Skap1(-/-) primary T cells. SKAP1 generated a SKAP1-RapL-Rap1 complex that bound to LFA-1, whereas a RapL mutation (L224A) that abrogated SKAP1 binding without affecting MST1 disrupted component colocalization in vesicles as well as T cell-dendritic cell (DC) conjugation. RapL expression also "slowed" T cell motility in D011.10 transgenic T cells in lymph nodes (LNs), an effect reversed by the L224A mutation with reduced dwell times between T cells and DCs. Overall, our findings define a TCR "inside-out" pathway via N-SKAP1-C-RapL that regulates T cell adhesion, motility, and arrest times with DCs in LNs.
Asunto(s)
Ganglios Linfáticos/inmunología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Reguladoras de la Apoptosis , Adhesión Celular , Movimiento Celular , Células Cultivadas , Células Dendríticas/inmunología , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/inmunología , Mutación , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Unión Proteica , Transporte de Proteínas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Proteínas de Unión al GTP rap1/inmunologíaRESUMEN
Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo II/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , GMP Cíclico/metabolismo , Regulación Alostérica , Animales , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400â mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum â¼40-70â mM NaCl and a reverse bell-shaped dependence of interprotein affinity. Meanwhile, intrinsic SiR activity, as measured in a methyl viologen-dependent assay, exhibited saturation above 100â mM NaCl. Thus, two assays suggested that interprotein interaction is crucial in controlling Fd-dependent SiR activity. Calorimetric analyses showed the monotonic decrease in interprotein affinity on increasing NaCl concentrations, distinguished from a reverse bell-shaped interprotein affinity observed from Fd-dependent SiR activity assay. Furthermore, Fd:SiR complex formation and interprotein affinity were thermodynamically adjusted by both enthalpy and entropy through electrostatic and non-electrostatic interactions. A residue-based NMR investigation on the addition of SiR to 15N-labeled Fd at the various NaCl concentrations also demonstrated that a combination of electrostatic and non-electrostatic forces stabilized the complex with similar interfaces and modulated the binding affinity and mode. Our findings elucidate that non-electrostatic forces are also essential for the formation and modulation of the Fd:SiR complex. We suggest that a complex configuration optimized for maximum enzymatic activity near physiological salt conditions is achieved by structural rearrangement through controlled non-covalent interprotein interactions.
Asunto(s)
Ferredoxinas/metabolismo , Sulfito Reductasa (Ferredoxina)/metabolismo , Calorimetría , Dicroismo Circular , Transporte de Electrón/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Oxidación-Reducción/efectos de los fármacos , Cloruro de Sodio/farmacología , TermodinámicaRESUMEN
LiaR is a 'master regulator' of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR(D191N) increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/química , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Daptomicina/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Secuencia de Consenso , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Bacteriana , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Modelos Moleculares , Mutación , Operón , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
Noise caused by stochastic fluctuations in genetic circuits (transcription and translation) is now appreciated as a central aspect of cell function and phenotypic behavior. Noise has also been detected in signaling networks, but the origin of this noise and how it shapes cellular outcomes remain poorly understood. Here, we argue that noise in signaling networks results from the intrinsic promiscuity of protein-protein interactions (PPIs), and that this noise has shaped cellular signal transduction. Features promoted by the presence of this molecular signaling noise include multimerization and clustering of signaling components, pleiotropic effects of gross changes in protein concentration, and a probabilistic rather than a linear view of signal propagation.
Asunto(s)
Fenómenos Fisiológicos Celulares/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Fenómenos Fisiológicos Celulares/genética , Redes Reguladoras de Genes , Modelos Genéticos , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Proteínas/química , Proteínas/genética , Transducción de Señal/genéticaRESUMEN
The RET proto-oncogene, a tyrosine kinase receptor, is widely known for its essential role in cell survival. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2, a dominant inherited cancer syndrome that affects neuroendocrine organs. However, the mechanisms by which RET promotes cell survival and prevents cell death remain elusive. We demonstrate that in addition to cytoplasmic localization, RET is localized in the nucleus and functions as a tyrosine-threonine dual specificity kinase. Knockdown of RET by shRNA in medullary thyroid cancer-derived cells stimulated expression of activating transcription factor 4 (ATF4), a master transcription factor for stress-induced apoptosis, through activation of its target proapoptotic genes NOXA and PUMA. RET knockdown also increased sensitivity to cisplatin-induced apoptosis. We observed that RET physically interacted with and phosphorylated ATF4 at tyrosine and threonine residues. Indeed, RET kinase activity was required to inhibit the ATF4-dependent activation of the NOXA gene because the site-specific substitution mutations that block threonine phosphorylation increased ATF4 stability and activated its targets NOXA and PUMA. Moreover, chromatin immunoprecipitation assays revealed that ATF4 occupancy increased at the NOXA promoter in TT cells treated with tyrosine kinase inhibitors or the ATF4 inducer eeyarestatin as well as in RET-depleted TT cells. Together these findings reveal RET as a novel dual kinase with nuclear localization and provide mechanisms by which RET represses the proapoptotic genes through direct interaction with and phosphorylation-dependent inactivation of ATF4 during the pathogenesis of medullary thyroid cancer.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Apoptosis , Proteínas Proto-Oncogénicas c-ret/metabolismo , Factor de Transcripción Activador 4/química , Transporte Activo de Núcleo Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Treonina/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Histonas/química , Isoxazoles/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Biotinilación , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Isoxazoles/síntesis química , Isoxazoles/farmacología , Cinética , Ligandos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Docilidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , metaminobenzoatos/síntesis química , metaminobenzoatos/química , metaminobenzoatos/farmacologíaRESUMEN
Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation. Dynamic light scattering and fluorescent microscale thermophoresis of the isolated SAM domain (residues 1-78) revealed evidence of dimers and tetramers. Consistently, deletion of the SAM region eliminated SLP-76 co-precipitation of itself, concurrent with a loss of microcluster formation, nuclear factor of activated T-cells (NFAT) transcription, and interleukin-2 production in Jurkat or primary T-cells. Furthermore, the H5 α helix within the SAM domain contributed to self-association. Retention of H5 in the absence of H1-4 sufficed to support SLP-76 self-association with smaller microclusters that nevertheless enhanced anti-CD3-driven AP1/NFAT transcription and IL-2 production. By contrast, deletion of the H5 α helix impaired self-association and anti-CD3 induced AP1/NFAT transcription. Our data identified for the first time a role for the SAM domain in mediating SLP-76 self-association for T-cell function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Activación de Linfocitos/inmunología , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Complejo CD3/inmunología , Complejo CD3/metabolismo , Células Cultivadas , Dicroismo Circular , Humanos , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Jurkat , Luz , Microscopía Confocal , Modelos Moleculares , Mutación , Factores de Transcripción NFATC/genética , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Dispersión de Radiación , Linfocitos T/metabolismo , Transcripción GenéticaRESUMEN
PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.
Asunto(s)
Células Presentadoras de Antígenos , Antígeno B7-H1 , Comunicación Celular , Proteína 2 Ligando de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1 , Linfocitos T , Animales , Células Presentadoras de Antígenos/química , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígeno B7-1/química , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Comunicación Celular/inmunología , Humanos , Ratones , Modelos Inmunológicos , Resonancia Magnética Nuclear Biomolecular , Proteína 2 Ligando de Muerte Celular Programada 1/química , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/inmunología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Linfocitos T/química , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.