RESUMEN
Human serum albumin (HSA) is the most abundant protein in plasma and an important transporter of exogenous small molecules. In this paper, the binary interaction of HSA with gambogic acid (GA), theophylline (TP), and quercetin (QC) and the ternary interaction of the HSA-QC system in the presence of food components were studied. Fluorescence emission and UV-Vis absorption spectra analysis showed that all three compounds bind HSA via a static quenching model. Competition binding site analysis and molecular docking revealed they bind to HSA Site I. The presence of TP or QC preferentially occupied GA's binding site, significantly decreasing the GA-HSA binding constant and affecting the HSA-GA binary system. In vitro cell experiments validated that TP/QC increase GA's free concentration, and both enhance GA's inhibitory effect on HepG2 cells in the presence of plasma proteins. Synchronous fluorescence, 3D fluorescence, and circular dichroism (CD) spectroscopy indicated TP/QC influence GA-induced conformational changes in HSA, with TP having a significantly greater impact than QC. This study offers new insights into GA's interactions with food components and provides dietary recommendations for clinical GA use.
Asunto(s)
Simulación del Acoplamiento Molecular , Quercetina , Albúmina Sérica Humana , Xantonas , Xantonas/química , Xantonas/farmacología , Xantonas/metabolismo , Humanos , Espectrometría de Fluorescencia , Células Hep G2 , Sitios de Unión , Dicroismo Circular , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Unión Competitiva , Quercetina/química , Unión ProteicaRESUMEN
The anti-vascular endothelial growth factor (VEGF) aptamer, t44.27, is a 27-mer RNA that functions as the active component of pegaptanib, an antiangiogenic medicine for neovascular age-related macular degeneration. The t44.27 aptamer is extensively 2'-modified and tightly binds to the heparin-binding domain (HDB) of VEGF165 in a Ca2+-dependent manner. However, the molecular mechanism by which the aptamer selectively recognizes VEGF HDB to antagonize its function is poorly understood. We found that t44.27 binds to VEGF HBD in a two-step manner using a different region in the molecule: a transient interaction using a structured region of t44.27 followed by a tight complex formation with a larger interaction surface. Ca2+ binding stabilizes t44.27 base-pair formation suitable for the initial transient interaction. Meanwhile, the tight complex formation was essential for t44.27 to exert a competitive inhibition of heparin binding to VEGF HBD. These results provide structural insight into how the RNA aptamer specifically interacts with its target molecule to inhibit its activity.
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Inhibidores de la Angiogénesis , Aptámeros de Nucleótidos , Factor A de Crecimiento Endotelial Vascular , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Humanos , Heparina/metabolismo , Heparina/química , Unión Proteica , Unión Competitiva , Calcio/metabolismo , Calcio/química , Sitios de Unión , Conformación de Ácido Nucleico , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/químicaRESUMEN
This study investigates the molecular interactions and physicochemical properties of N6-cyclohexyladenosine (CHA), a neuroprotective adenosine A1 receptor agonist, and 8-(p-sulfophenyl)theophylline (8-SPT), a co-administered non-selective adenosine receptor antagonist with limited blood-brain barrier permeability, and 2-hydroxypropyl-ß-cyclodextrin (HPßCD), which serves as an excipient. The aim is to systematically evaluate how inclusion complexation with HPßCD affects the solubility and compatibility of drugs in combined formulations, using binding constant analysis to guide the investigation. Two analytical approaches were employed to determine the binding affinities of CHA and 8-SPT: capillary electrophoresis (CE) with UV/Vis absorption detection and nuclear magnetic resonance (NMR) spectroscopy. CE method development utilized two distinct buffers: phosphate buffer (pH 2.5) for protonated CHA and acetate buffer (pH 4.6) for 8-SPT. The detection limits were 2.0⯱â¯1.0⯵g/mL (6⯱â¯3⯵M) at 270â¯nm for CHA and 2.0⯱â¯1.0⯵g/mL (6⯱â¯3⯵M) at 210â¯nm for 8-SPT. CE analysis revealed apparent and average binding constants of 136⯱â¯13â¯M-1 for protonated CHA and 121⯱â¯19â¯M-1 for 8-SPT. NMR spectroscopy established the solubility profile of CHA in water and determined an apparent and average binding constant of 308⯱â¯30â¯M-1 for neutral CHA. The relatively small binding constants suggest that 8-SPT does not significantly impact CHA solubility, stability, or availability in combined drug formulations, ensuring sufficient drug availability upon infusion into the bloodstream. These competitive binding constant studies provide valuable guidance for predicting drug solubility and compatibility in combined formulations.
Asunto(s)
2-Hidroxipropil-beta-Ciclodextrina , Adenosina , Teofilina , 2-Hidroxipropil-beta-Ciclodextrina/química , Teofilina/análogos & derivados , Teofilina/química , Teofilina/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Solubilidad , Unión Competitiva , Espectroscopía de Resonancia Magnética/métodos , Excipientes/química , Electroforesis Capilar/métodos , Química Farmacéutica/métodos , Composición de Medicamentos/métodosRESUMEN
A hallmark of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the delayed interferon response. Interferons are typically produced upon host recognition of pathogen- or damage-associated molecular patterns, such as nucleic acids. While the mechanisms by which SARS-CoV-2 evades host recognition of its RNA are well studied, how it evades immune responses to cytosolic DNA-leaked from mitochondria or nuclei during infection-remains poorly understood. Here, we demonstrate that the SARS-CoV-2 nucleocapsid protein directly suppresses DNA sensing by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). Although primarily known for packaging the viral RNA genome, we uncover that the SARS-CoV-2 nucleocapsid protein also binds DNA with high affinity and competitively blocks cGAS activation. Using cell-free biochemical and biophysical approaches, including single-molecule optical tweezers, we show that the nucleocapsid protein binds to DNA at nanomolar concentrations and cocondenses with DNA at micromolar concentrations, thereby impeding stable cGAS-DNA interactions required for signal propagation. Hyperphosphorylation of the nucleocapsid protein diminishes its competitive binding capacity. Our findings reveal an unexpected role of the SARS-CoV-2 nucleocapsid protein in directly suppressing the cGAS-STING pathway, strongly suggesting that this contributes to the delayed interferon response during infection. This study raises the possibility that nucleocapsid proteins of other RNA viruses may also exhibit moonlighting functions by antagonizing host nucleic acid-sensing pathways.
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COVID-19 , Proteínas de la Nucleocápside de Coronavirus , ADN , Nucleotidiltransferasas , Fosfoproteínas , SARS-CoV-2 , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Humanos , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , ADN/metabolismo , COVID-19/virología , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Unión Competitiva , Unión ProteicaRESUMEN
The molecular mechanism by which the essential trace elements (TEs) zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) interact during their uptake, particularly their competition for interaction with the gastrointestinal mucus layer, remains unclear. Thus, in this study we investigated the binding of TEs to gastrointestinal mucins individually and interdependently and analyzed the impact of mucus on their intestinal absorption. Cell-free binding and competition experiments with commercially available mucin and native MUC2 and MUC5AC from porcine gastrointestinal tract showed high TE-binding capacity (Fe>Zn>Cu>Mn) with an average affinity that was highest for Zn and lowest for Mn (Zn>Fe>Cu>Mn). Gastrointestinal mucins contain high-affinity TE-binding sites with physiologically relevant affinities, and TEs mutually affected their interaction with mucins in ratios reflecting the in vivo situation, leading to various cases of displacement or augmented binding. Solely Fe was unaffected by other TEs, apart from Mn that facilitated the Fe-mucin interaction. The relevance of TE-binding by the mucins was underlined by transport studies with two 3D in vitro intestinal models: Caco-2 mono- and Caco-2/HT-29-MTX co-cultures, showing that at least for Fe and Zn, mucus provides a buffering system and increases the availability of the TEs by delivering them to the underlying enterocytes. This study provides the first systematic analysis of the competition between Zn, Fe, Cu, and Mn for binding to gastrointestinal mucins, demonstrating the importance of the mucus layer for TE absorption and that competition for their intestinal uptake already starts at the mucus barrier, with translational relevance for nutrition and (malabsorption) diseases.
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Absorción Intestinal , Mucinas , Oligoelementos , Animales , Humanos , Oligoelementos/metabolismo , Zinc/metabolismo , Manganeso/metabolismo , Cobre/metabolismo , Hierro/metabolismo , Células CACO-2 , Porcinos , Mucinas/metabolismo , Unión Competitiva , Células HT29 , Mucosa Intestinal/metabolismoRESUMEN
Eukaryotic actin filaments bind factors that regulate their assembly and disassembly creating a self-organising system, the actin cytoskeleton. Despite extensive knowledge of signals that modulate actin organisation, significant gaps remain in our understanding of spatiotemporal regulation of de novo filament initiation. Yeast Las17/WASP is essential for actin polymerisation initiation supporting membrane invagination in Saccharomyces cerevisiae endocytosis and therefore its tight regulation is critical. The adaptor protein Sla1 inhibits Las17 but mechanisms underpinning Las17 activation remain elusive. Here we show that Las17 binding of tandem Sla1 SH3 domains is >100-fold stronger than single domains. Furthermore, SH3 domains directly compete with G-actin for binding in the Las17 polyproline region, thus rationalising how SH3 interactions can affect actin polymerisation despite their distance from C-terminal actin-binding and Arp2/3-interacting VCA domains. Our data and proposed model also highlight the likely importance of multiple weak interactions that together ensure spatial and temporal regulation of endocytosis.
Asunto(s)
Actinas , Prolina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteína del Síndrome de Wiskott-Aldrich , Dominios Homologos src , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Actinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Unión Proteica , Endocitosis , Citoesqueleto de Actina/metabolismo , Prolina/metabolismo , Unión Competitiva , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteínas de Microfilamentos/metabolismo , Polimerizacion , Proteínas del CitoesqueletoRESUMEN
DExD- box helicase 21 (DDX21) is overexpressed in colorectal cancer (CRC) and is positively correlated with poor prognosis and the malignant phenotype of CRC. Functional characterization indicated that DDX21 promotes CRC metastasis and angiogenesis both in vitro and in vivo. N-acetyltransferase 10 (NAT10) is a key regulator of the N4-acetylcytidine (ac4C) modification of mRNA, regulating the stabilization of mRNA via ac4C modification. Here, we identified that DDX21 competitive binding with sirtuin 7 (SIRT7), inducing the overexpression of NAT10. Furthermore, DDX21 upregulates NAT10 expression to enhance ac4C modification and the stability of ATAD2, SOX4 and SNX5 mRNAs, which mediate CRC metastasis and angiogenesis. Overall, the present study revealed a mechanism of DDX21/NAT10-mediated mRNA stability in CRC, laying the foundation for the use of DDX21 as a therapeutic target to overcome metastasis and angiogenesis in CRC. DDX21 competitive binding with sirtuin 7 (SIRT7), inducing the overexpression of NAT10. Furthermore, DDX21 upregulates NAT10 expression to enhance ac4C modification and the stability of ATAD2, SOX4 and SNX5 mRNAs, which mediate CRC metastasis and angiogenesis.
Asunto(s)
Acetiltransferasas , Neoplasias Colorrectales , ARN Helicasas DEAD-box , Neovascularización Patológica , Sirtuinas , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Sirtuinas/metabolismo , Sirtuinas/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Patológica/metabolismo , Animales , Acetiltransferasas/metabolismo , Acetiltransferasas/genética , Línea Celular Tumoral , Metástasis de la Neoplasia , Ratones , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Ratones Endogámicos BALB C , Estabilidad del ARN , Unión Competitiva , Unión Proteica , Células HCT116 , AngiogénesisRESUMEN
The estrogen receptor (ER) is a nuclear receptor and one of the most extensively researched targets in the study of endocrine-disrupting chemicals (EDCs). Many biosensors and bioassays for estrogenic EDCs use the ligand-binding domain of human ERα (LBD-hERα) as a biological recognition element. However, the LBD-hERα is poorly stable and difficult to produce as a functional LBD-hERα in the E. coli expression system. In this study, we efficiently expressed the functional LBD-hERα tagged with the cyan fluorescent protein, mTurquoise2 (LBD-hERα-mTq2) by the addition of ethanol (3 %) to E. coli suspension during protein expression (> 40 times more compared to without ethanol). We found that ethanol not only promoted the proper folding of LBD-hERα-mTq2, but also prevented the proteolysis of poorly folded recombinant proteins. We established a FRET-based binding assay between a fluorescent estrogen, coumestrol, and the LBD-hERα-mTq2, in which the formation of the complex exhibits a significant degree of FRET. A subsequent competitive binding assay with diethylstilbestrol demonstrates that our system successfully functions as a simple and reliable bioassay to detect estrogenic EDCs.
Asunto(s)
Cumestrol , Escherichia coli , Receptor alfa de Estrógeno , Etanol , Proteínas Fluorescentes Verdes , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/biosíntesis , Etanol/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Cumestrol/metabolismo , Cumestrol/química , Unión Competitiva , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Ligandos , Expresión Génica , Dominios ProteicosRESUMEN
Developing new anti-atherosclerotic agents and exploring their mechanistic actions is required. This study defined the molecular mechanism of icariin (ICA) in human aortic smooth muscle cells (HA-VSMCs) proliferation and migration by focusing on lncRNA H19 mediated the ability of the HuR protein to bind target mRNAs. The levels of lncRNA H19, cyclin D1 and matrix metalloproteinase-9 (MMP-9) were measured via qPCR or western blot. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS), flow cytometry and transwell assays were performed to determine the functions of lncRNA H19 in cell proliferation and migration. RNA pull down and RIP were performed to verify the interaction between lncRNA H19 and HuR, or interaction between HuR and target mRNAs. LncRNA H19 expression was modulated in response to treatment with ICA. LncRNA H19 overexpression inhibited the proliferation and migration of ox-LDL-induced HA-VSMCs. ICA attenuated cell proliferation and migration, which was reversed by lncRNA H19 knockdown. LncRNA H19 overexpression reduced the expression of cyclin D1/MMP-9, and restrained the stability of cyclin D1/MMP-9 mRNAs. Moreover, lncRNA H19 was found to bind with HuR to decrease the mRNA stability of cyclin D1/MMP-9 mRNAs. In addition, ICA suppressed the expression of cyclin D1/MMP-9, this effect was partly reversed by lncRNA H19 knockdown. We propose that lncRNA H19 serves as an endogenous competing RNA to disable HuR, restricting its availability to target cyclin D1/MMP-9 mRNAs, generally repressing HA-VSMCs proliferation and migration. ICA inhibits proliferation and migration of HA-VSMCs, altering the expression of cyclin D1 and MMP-9, in a lncRNA H19/HuR dependent manner.
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Movimiento Celular , Proliferación Celular , Proteína 1 Similar a ELAV , Flavonoides , Músculo Liso Vascular , Miocitos del Músculo Liso , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Movimiento Celular/efectos de los fármacos , Flavonoides/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/citología , Ciclina D1/metabolismo , Ciclina D1/genética , Unión Competitiva , Células CultivadasRESUMEN
Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and deregulated in several pathologies, including cancer. Many studies point to their activity as microRNAs (miRNAs) and protein sponges; however, we propose a function based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely tumor-associated circHIPK3 directly interacts in vivo with the BRCA1 mRNA through the back-splicing region in human cancer cells. This interaction increases BRCA1 translation by competing for the binding of the fragile-X mental retardation 1 protein (FMRP) protein, which we identified as a BRCA1 translational repressor. CircHIPK3 depletion or disruption of the circRNA-mRNA interaction decreases BRCA1 protein levels and increases DNA damage, sensitizing several cancer cells to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, blocking FMRP interaction with BRCA1 mRNA with locked nucleic acid (LNA) restores physiological protein levels in BRCA1 hemizygous breast cancer cells, underscoring the importance of this circRNA-mRNA interaction in regulating DNA-damage response.
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Proteína BRCA1 , Daño del ADN , Ribonucleoproteína 1 Mensajera del X Frágil , Proteínas Serina-Treonina Quinasas , ARN Circular , ARN Mensajero , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , ARN Circular/metabolismo , ARN Circular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Ribonucleoproteína 1 Mensajera del X Frágil/metabolismo , Ribonucleoproteína 1 Mensajera del X Frágil/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Unión Competitiva , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , ARN/metabolismo , ARN/genética , Células HEK293 , Unión Proteica , Línea Celular Tumoral , Biosíntesis de Proteínas , Células MCF-7 , FemeninoRESUMEN
Understanding the interaction between pharmaceuticals and serum proteins is crucial for optimizing therapeutic strategies, especially in patients with coexisting chronic diseases. The primary goal of this study was to assess the potential changes in binding affinity and competition between glipizide (GLP, a second-generation sulfonylurea hypoglycemic drug) and losartan (LOS, a medication commonly prescribed for hypertension, particularly for patients with concurrent diabetes) with non-glycated (HSA) and glycated (gHSAGLC, gHSAFRC) human serum albumin using multiple spectroscopic techniques (fluorescence, UV-visible absorption, and circular dichroism spectroscopy). The results indicated that FRC is a more effective glycation agent for HSA than GLC, significantly altering the albumin structure and affecting the microenvironment around critical amino acid residues, Trp-214 and Tyr. These modifications reduce the binding affinity of LOS and GLP to gHSAGLC and gHSAFRC, compared to HSA, resulting in less stable drug-protein complexes. The study revealed that LOS and GLP interact nonspecifically with the hydrophobic regions of the albumin surface in both binary (ligand-albumin) and ternary systems (ligand-albumin-ligandconst) and specifically saturate the binding sites within the protein molecule. Furthermore, the presence of an additional drug (GLP in the LOS-albumin complex or LOS in the GLP-albumin complex) complicates the interactions, likely leading to competitive binding or displacement of the initially bound drug in both non-glycated and glycated albumins. Analysis of the CD spectra suggests mutual interactions between GLP and LOS, underscoring the importance of closely monitoring patients co-administered these drugs, to ensure optimal therapeutic efficacy and safety.
Asunto(s)
Unión Competitiva , Glipizida , Albúmina Sérica Glicada , Losartán , Unión Proteica , Albúmina Sérica , Losartán/química , Losartán/metabolismo , Humanos , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Glipizida/química , Glipizida/metabolismo , Sitios de Unión , Productos Finales de Glicación Avanzada/metabolismo , Productos Finales de Glicación Avanzada/química , Dicroismo Circular , Albúmina Sérica Humana/química , Albúmina Sérica Humana/metabolismo , Espectrometría de Fluorescencia , Hipoglucemiantes/química , Hipoglucemiantes/metabolismoRESUMEN
The ability to accurately measure drug-target interaction is critical for the discovery of new therapeutics. Classical pharmacological bioassays such as radioligand or fluorescent ligand binding assays can define the affinity or Kd of a ligand for a receptor with the lower the Kd, the stronger the binding and the higher the affinity. However, in many drug discovery laboratories today, the target of interest if often artificially upregulated by means of transfection to modify the host cell's genetic makeup. This then potentially invalidates the assumptions of classical pharmacology affinity calculations as the receptor of interest is no longer at normal physiological densities. The CXCR4 receptor is expressed on many different cancer cell types and is associated with metastasis and poor prognosis. Therefore, the CXCR4 receptor is a desirable target for novel therapeutics. In this study, we explore the applicability of the newly developed fluorescently tagged CXCR4 antagonists, IS4-FAM as an investigative tool to study CXCR4 affinity and competitive antagonism in native, non-transfected cancer cells using confocal microscopy and flow cytometry. IS4-FAM directly labels CXCR4 in several cell lines including high CXCR4 expressing SK-MEL-28 (malignant melanoma) and PC3 (metastatic prostate cancer) and lower CXCR4 expressing THP-1 (acute monocytic leukemia) and was competitive with the established CXCR4 antagonist, AMD3100. This highlights the potential of IS4-FAM as a pharmacological tool for drug discovery in native cells lines and tissues.
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Colorantes Fluorescentes , Receptores CXCR4 , Receptores CXCR4/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Humanos , Línea Celular Tumoral , Colorantes Fluorescentes/química , Citometría de Flujo , Unión Competitiva , Microscopía Confocal , Células PC-3 , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/metabolismoRESUMEN
The pH varies in different tissues and organelles and also changes during some diseases. In this regard, the application of molecular switches that use a competition-based aptamer switch design in biological systems requires studying the thermodynamics of such systems at different pH values. In this work, we studied the binding of the classical ATP aptamer to ATP and competition strands under different pH and ionic conditions using fluorescent melting curve analysis. We have developed an original approach to processing source data from a PCR thermal cycler. It is based on constructing a thermodynamic model of the melting profile and the subsequent fit of experimental curves within this model. We have shown that this approach enables us to narrow the temperature region under study to the width of the melting region without a significant loss in the quality of the result. This impressively expands the application area of this approach compared to frequently used techniques that require mandatory measurement of the signal outside the melting region. The results obtained by the method showed that the thermodynamic parameters of the ATP aptamer and its duplexes with competition strands change depending on pH. Therefore, molecular switches that use a competition strand to the ATP aptamer may have a pH-dependent sensitivity that has not been previously considered. This should be taken into account for future rational design of similar systems.
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Adenosina Trifosfato , Aptámeros de Nucleótidos , Termodinámica , Concentración de Iones de Hidrógeno , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Unión Competitiva , Desnaturalización de Ácido NucleicoRESUMEN
Circular RNAs (circRNAs) are involved in various physiological and pathological processes in both vertebrates and invertebrates. However, most studies on circRNAs have focused on their roles as endogenous competitive RNAs. Here, we report a novel function of circRNA derived from the Fibrinogen-like protein 1 gene (circ-FGL1) that inhibits coelomocyte apoptosis via competing with the deubiquitinase AjOTUB1 to bind AjMyc in Apostichopus japonicus during Vibrio splendidus infection. The results showed that circ-FGL1 is significantly downregulated in coelomocytes of V. splendidus-induced A. japonicus and negatively regulates coelomocyte apoptosis through the AjBax-AjCyt c pathway. Mechanistically, the deubiquitinase AjOTUB1 and circ-FGL1 could interact with the transcription factor protein AjMyc in the same region with circ-FGL1/AjMyc having greater affinity. Under normal conditions, high levels of circ-FGL1 bind directly to AjMyc, inhibiting the deubiquitylation of AjMyc by AjOTUB1 and leading to the degradation of AjMyc. After V. splendidus infection, AjMyc disassociates from the depressed expression of circ-FGL1, promoting its deubiquitylation by binding to the induced deubiquitinase AjOTUB1 to inhibit its degradation. AjMyc is then transferred to the nucleus and promotes the transcription of AjCyt c and AjBax to induce coelomocyte apoptosis. The new finding will expand our present outstanding on the functional role of circRNAs and suggest new therapeutic targets for the treatment of echinoderms during bacterial invasion.
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Apoptosis , ARN Circular , Stichopus , Vibriosis , Vibrio , Animales , ARN Circular/metabolismo , ARN Circular/genética , Stichopus/microbiología , Stichopus/metabolismo , Stichopus/genética , Vibriosis/metabolismo , Unión Competitiva , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.
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Colorantes Fluorescentes , Macrófagos , Receptor de Adenosina A2B , Humanos , Células HEK293 , Receptor de Adenosina A2B/metabolismo , Ligandos , Colorantes Fluorescentes/química , Macrófagos/metabolismo , Macrófagos/inmunología , Unión Competitiva , Antagonistas del Receptor de Adenosina A2/farmacología , Agonistas del Receptor de Adenosina A2/farmacologíaRESUMEN
Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV's NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV's capsid protein.
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Unión Competitiva , Simulación de Dinámica Molecular , Señales de Localización Nuclear , alfa Carioferinas , alfa Carioferinas/metabolismo , alfa Carioferinas/química , alfa Carioferinas/antagonistas & inhibidores , Ligandos , Señales de Localización Nuclear/química , Virus de la Encefalitis Equina Venezolana/metabolismo , Virus de la Encefalitis Equina Venezolana/química , Unión Proteica , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Secuencia de AminoácidosRESUMEN
BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin. OBJECTIVES: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. METHODS: The ability of F(ab')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4. RESULTS: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition. CONCLUSION: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.
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Anticuerpos Monoclonales , Vacunas contra la COVID-19 , Heparina , Factor Plaquetario 4 , Humanos , Factor Plaquetario 4/inmunología , Heparina/efectos adversos , Heparina/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/efectos adversos , Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Valor Predictivo de las Pruebas , Anticoagulantes/efectos adversos , Anticoagulantes/inmunología , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/diagnóstico , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/efectos adversos , Unión Proteica , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/diagnóstico , Púrpura Trombocitopénica Idiopática/inducido químicamente , SARS-CoV-2/inmunología , Unión Competitiva , Púrpura Trombocitopénica Trombótica/inmunología , Púrpura Trombocitopénica Trombótica/diagnóstico , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/inducido químicamenteRESUMEN
ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.
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Sistema de Transporte de Aminoácidos ASC , Antígenos de Histocompatibilidad Menor , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/química , Cinética , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/química , Humanos , Sodio/metabolismo , Sodio/química , Animales , Unión CompetitivaRESUMEN
Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
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Quimiocina CXCL12 , Receptores CXCR4 , Humanos , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL11/metabolismo , Transducción de Señal , Ligandos , Unión CompetitivaRESUMEN
BACKGROUND: In a previous study, diethylstilbestrol (DES) was shown to induce oocyte maturation in fish. In the present study, the interaction of DES on goldfish membrane progesterone receptor α (GmPRα) was investigated using a competitive binding assay with radiolabeled steroids. The results indicate that DES exerts its effects on membrane progesterone receptor alpha (mPRα) and induces oocyte maturation through nongenomic steroid mechanisms. This study provides empirical data that demonstrate the binding between DES and GmPRα. METHODS: Binding of DES to GmPRα was achieved by using radiolabeled DES and recombinant GmPRα expressed in culture cells or purified GmPRα proteins that coupled to graphene quantum dots (GQDs). Additionally, the competitive binding of fluorescently labeled progesterone to GmPRα-expressing cells was evaluated. RESULTS: Although significant nonspecific binding of radiolabeled DES to the cell membrane that expresses GmPRα has been observed, specific binding of DES to GmPRα has been successfully identified in the presence of digitonin. Furthermore, the specific binding of DES to GmPRα was confirmed by a binding assay using GQD-GmPRα. The radiolabeled DES was shown to bind to GQD-GmPRα. Additionally, the competition for the binding of fluorescently labeled progesterone to GmPRα-expressing cells was achieved with the DES. CONCLUSIONS: The results of the experiments revealed that DES binds to GmPRα. Thus, it can be concluded that DES induces goldfish oocyte maturation by binding to GmPRα.