RESUMEN
The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.
Asunto(s)
ARN Helicasas DEAD-box , ARN Bicatenario , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Inmunidad Innata , Estructura Terciaria de Proteína , ARN Viral/genéticaRESUMEN
The RNA sensor MDA5 recruits the signaling adaptor MAVS to initiate type I interferon signaling and downstream antiviral responses, a process that requires K63-linked polyubiquitin chains. Here, we examined the mechanisms whereby K63-polyUb chain regulate MDA5 activation. Only long unanchored K63-polyUbn (n ≥ 8) could mediate tetramerization of the caspase activation and recruitment domains of MDA5 (MDA5CARDs). Cryoelectron microscopy structures of a polyUb13-bound MDA5CARDs tetramer and a polyUb11-bound MDA5CARDs-MAVSCARD assembly revealed a tower-like formation, wherein eight Ubs tethered along the outer rim of the helical shell, bridging MDA5CARDs and MAVSCARD tetramers into proximity. ATP binding and hydrolysis promoted the stabilization of RNA-bound MDA5 prior to MAVS activation via allosteric effects on CARDs-polyUb complex. Abundant ATP prevented basal activation of apo MDA5. Our findings reveal the ordered assembly of a MDA5 signaling complex competent to recruit and activate MAVS and highlight differences with RIG-I in terms of CARD orientation and Ub sensing that suggest different abilities to induce antiviral responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/química , Microscopía por Crioelectrón , Células HEK293 , Humanos , Inmunidad Innata/fisiología , Helicasa Inducida por Interferón IFIH1/química , Helicasa Inducida por Interferón IFIH1/ultraestructura , Poliubiquitina/química , Poliubiquitina/metabolismo , Unión ProteicaRESUMEN
Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cristalografía por Rayos X , Femenino , Humanos , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
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
The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2'-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2'-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2'-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.
RESUMEN
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.
Asunto(s)
Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Descubrimiento de Drogas/economía , Descubrimiento de Drogas/instrumentación , Descubrimiento de Drogas/métodos , Diseño de Equipo , Análisis de Inyección de Flujo/economía , Análisis de Inyección de Flujo/instrumentación , Análisis de Inyección de Flujo/métodos , Humanos , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/economía , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/instrumentación , Ligandos , Proteínas/químicaRESUMEN
Proteins are not rigid bodies under their physiological conditions. Here we discuss a solution-phase structural proteomics technique, hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS), as a means to study protein dynamics, which can complement other structural approaches. We outline the background theory and highlight the utility of HDX-MS measurements in two case studies involving a nuclear receptor and an innate immunity receptor. We also discuss emerging software advances for improving data analysis and three-dimensional visualization.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Proteínas/química , Proteínas/metabolismo , Cinética , Conformación ProteicaRESUMEN
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Análisis de Datos , Concentración de Iones de HidrógenoRESUMEN
Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5'-triphosphate (5'ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2'-O-methylation (Cap1) on the 5' end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5'ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner.
Asunto(s)
Autoinmunidad/genética , Proteína 58 DEAD Box/genética , Inmunidad Innata/genética , Caperuzas de ARN/inmunología , ARN Bicatenario/inmunología , Adenosina Trifosfatasas/metabolismo , Dominio de Reclutamiento y Activación de Caspasas/inmunología , Proteína 58 DEAD Box/química , Proteína 58 DEAD Box/inmunología , Proteína 58 DEAD Box/metabolismo , Medición de Intercambio de Deuterio/métodos , Mutación con Ganancia de Función , Guanosina/análogos & derivados , Guanosina/química , Guanosina/inmunología , Guanosina/metabolismo , Helicasa Inducida por Interferón IFIH1/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Espectrometría de Masas/métodos , Metilación , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Unión Proteica/inmunología , Caperuzas de ARN/química , Caperuzas de ARN/metabolismo , ARN Bicatenario/química , ARN Bicatenario/metabolismo , ARN Viral/inmunología , Receptores Inmunológicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
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.
Asunto(s)
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
Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.
Asunto(s)
Proteínas Portadoras/química , Subunidades beta de la Proteína de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Simulación de Dinámica Molecular , Neuronas/metabolismo , Proteínas RGS/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Espectrometría de Masas , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Multimerización de Proteína , Proteínas RGS/genética , Proteínas RGS/metabolismo , Homología de Secuencia de Aminoácido , Células Sf9 , SpodopteraRESUMEN
Despite the growing evidence suggesting that long noncoding RNAs (lncRNAs) are critical regulators of several biological processes, their functions in the nervous system remain elusive. We have identified an lncRNA, GM12371, in hippocampal neurons that is enriched in the nucleus and necessary for synaptic communication, synapse density, synapse morphology, and dendritic tree complexity. Mechanistically, GM12371 regulates the expression of several genes involved in neuronal development and differentiation, as well as expression of specific lncRNAs and their cognate mRNA targets. Furthermore, we find that cAMP-PKA signaling up-regulates the expression of GM12371 and that its expression is essential for the activity-dependent changes in synaptic transmission in hippocampal neurons. Taken together, our data establish a key role for GM12371 in regulating synapse function.
Asunto(s)
Regulación de la Expresión Génica/genética , ARN Largo no Codificante/genética , Sinapsis/genética , Transcripción Genética/genética , Animales , Diferenciación Celular/genética , Femenino , Hipocampo/fisiología , Ratones , Neuronas/fisiología , Embarazo , Transducción de Señal/genética , Regulación hacia Arriba/genéticaRESUMEN
Targeting peroxisome proliferator-activated receptor γ (PPARγ) by synthetic compounds has been shown to elicit insulin sensitising properties in type 2 diabetics. Treatment with a class of these compounds, the thiazolidinediones (TZDs), has shown adverse side effects such as weight gain, fluid retention, and congestive heart failure. This is due to their full agonist properties on the receptor, where a number of genes are upregulated beyond normal physiological levels. Lessened transactivation of PPARγ by partial agonists has proved beneficial in terms of reducing side effects, while still maintaining insulin sensitising properties. However, some partial agonists have been associated with unfavourable pharmacokinetic profiles due to their acidic moieties, often causing partitioning to the liver. Here we present SR1988, a new partial agonist with favourable non-acid chemical properties. We used a combination of X-ray crystallography and hydrogen/deuterium exchange (HDX) to elucidate the structural basis for reduced activation of PPARγ by SR1988. This structural analysis reveals a mechanism that decreases stabilisation of the AF2 coactivator binding surface by the ligand.
RESUMEN
Microtubules are highly dynamic tubulin polymers that are required for a variety of cellular functions. Despite the importance of a cellular population of tubulin dimers, we have incomplete information about the mechanisms involved in the biogenesis of αß-tubulin heterodimers. In addition to prefoldin and the TCP-1 Ring Complex, five tubulin-specific chaperones, termed cofactors A-E (TBCA-E), and GTP are required for the folding of α- and ß-tubulin subunits and assembly into heterodimers. We recently described the purification of a novel trimer, TBCDâ¢ARL2â¢ß-tubulin. Here, we employed hydrogen/deuterium exchange coupled with mass spectrometry to explore the dynamics of each of the proteins in the trimer. Addition of guanine nucleotides resulted in changes in the solvent accessibility of regions of each protein that led to predictions about each's role in tubulin folding. Initial testing of that model confirmed that it is ARL2, and not ß-tubulin, that exchanges GTP in the trimer. Comparisons of the dynamics of ARL2 monomer to ARL2 in the trimer suggested that its protein interactions were comparable to those of a canonical GTPase with an effector. This was supported by the use of nucleotide-binding assays that revealed an increase in the affinity for GTP by ARL2 in the trimer. We conclude that the TBCDâ¢ARL2â¢ß-tubulin complex represents a functional intermediate in the ß-tubulin folding pathway whose activity is regulated by the cycling of nucleotides on ARL2. The co-purification of guanine nucleotide on the ß-tubulin in the trimer is also shown, with implications to modeling the pathway.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/química , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos/química , Conformación Proteica , Pliegue de Proteína , Transducción de Señal , Tubulina (Proteína)/metabolismoRESUMEN
The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.
Asunto(s)
ADN/química , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , ADN/genética , ADN/metabolismo , Medición de Intercambio de Deuterio , Dimerización , Humanos , Hidrógeno , Ligandos , Espectrometría de Masas , Osteocalcina/genética , Osteocalcina/metabolismo , Unión Proteica , Receptores de Calcitriol/genética , Receptores X Retinoide/química , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMEN
Characterization of interactions between proteins and other molecules is crucial for understanding the mechanisms of action of biological systems and, thus, drug discovery. An increasingly useful approach to mapping these interactions is measurement of hydrogen/deuterium exchange (HDX) using mass spectrometry (HDX-MS), which measures the time-resolved deuterium incorporation of peptides obtained by enzymatic digestion of the protein. Comparison of exchange rates between apo- and ligand-bound conditions results in a mapping of the differential HDX (ΔHDX) of the ligand. Residue-level analysis of these data, however, must account for experimental error, sparseness, and ambiguity due to overlapping peptides. Here, we propose a Bayesian method consisting of a forward model, noise model, prior probabilities, and a Monte Carlo sampling scheme. This method exploits a residue-resolved exponential rate model of HDX-MS data obtained from all peptides simultaneously, and explicitly models experimental error. The result is the best possible estimate of ΔHDX magnitude and significance for each residue given the data. We demonstrate the method by revealing richer structural interpretation of ΔHDX data on two nuclear receptors: vitamin D-receptor (VDR) and retinoic acid receptor gamma (RORγ). The method is implemented in HDX Workbench and as a standalone module of the open source Integrative Modeling Platform.
Asunto(s)
Medición de Intercambio de Deuterio , Espectrometría de Masas , Proteínas/química , Teorema de Bayes , Ligandos , Simulación de Dinámica Molecular , Método de MontecarloRESUMEN
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
The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPARγ), to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.
RESUMEN
Liver receptor homologue-1 (LRH1) is an orphan nuclear receptor that has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the LRH1 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative LRH1 co-regulators. Nuclear fractionation protocol was essential for detection of LRH1 peptides by mass spectrometry (MS), with most peptides being observed in the insoluble fraction (receptor bound to DNA). SERBP1 and ILF3 were identified as LRH1 interacting partners by both Western blot and MS/MS analysis. Receptor knockdown by siRNA showed an increase in SERBP1 expression, while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of LRH1 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, subsequently inhibiting transcription. Given the receptor's role in cancer progression, the study here elucidates additional transcriptional machinery involved in LRH1 signaling and potentially provides new targets for therapeutics development.