RESUMEN
Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M-1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M-1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M-1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.
Asunto(s)
Aptámeros de Nucleótidos , PPAR gamma , PPAR gamma/química , PPAR gamma/metabolismo , Ligandos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Unión Proteica , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/análogos & derivados , Humanos , CalorimetríaRESUMEN
Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize ß2-adrenergic receptor (ß2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged ß2-adrenoceptor (ß2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, ß2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on ß2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M-1 respectively, which were two orders of magnitude higher than those on the Halo-ß2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.
Asunto(s)
Escherichia coli , Receptores Adrenérgicos beta 2 , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/química , Escherichia coli/química , Péptidos/química , Péptidos/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Adsorción , Unión ProteicaRESUMEN
The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Maleatos , Receptores Histamínicos H1 , Ligandos , Maleatos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Humanos , Histidina/química , Animales , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Células CHO , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Antagonistas de los Receptores Histamínicos H1/química , Poliestirenos/química , Cricetulus , Oligopéptidos/químicaRESUMEN
Immobilization of functional protein, especially G protein-coupled receptors (GPCRs), is particularly significant in various fields such as the development of assays for diagnosis, lead compound screening, as well as drug-protein interaction analysis. However, there are still some challenges with the immobilized proteins such as undefined loads, orientations, and the loss of activity. Herein, we introduced a DNA conjugation strategy into the immobilization of Cysteinyl leukotriene receptor 1(CysLTR1) which enables exquisite molecular control and higher activity of the receptor. We used the bacterial relaxases VirD2 as an immobilized tag fused at the C terminus of CysLTR1. Tyrosine residue(Y29) at the core binding site of the VirD2 tag can react with the single-strand piece of DNA(T-DNA) in the form of a covalent bond. Inspired by this strategy, we developed a new immobilization method by mixing the T-DNA-modified silica gel with the cell lysate containing the expressed VirD2-tagged CysLTR1 for 1 hour. We found that the successful formation of DNA-protein conjugate enables the immobilization of CysLTR1 fast, site-specific, and with minimal loss of activity. The feasibility of the immobilized CysLTR1 was evaluated in drug-protein binding interaction by frontal analysis and adsorption energy distribution analysis. The binding of pranlukast, zafirlukast, and MK571 to the immobilized CysLTR1 was realized, and the association constants presented good agreement between the two methods. Rosmarinic acid was retained in the immobilized CysLTR1 column, and the in-vitro test revealed that the compound binds to the receptor in one type of binding site mode. Despite these results, we concluded that the DNA-protein conjugate strategy will probably open up the possibilities for capturing other functional proteins in covalent and site-specific modes from the complex matrices and the immobilized receptor preserves the potential in fishing out lead compounds from natural products.
Asunto(s)
Proteínas Inmovilizadas , Receptores de Leucotrienos , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrienos/química , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Humanos , ADN/química , ADN/metabolismo , Sitios de Unión , Unión ProteicaRESUMEN
Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.
Asunto(s)
Criogeles , Oxidación-Reducción , Polietilenglicoles , Criogeles/química , Polietilenglicoles/química , Animales , Concanavalina A/química , Concanavalina A/metabolismo , Metacrilatos/química , Ratones , Manosa/química , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Compuestos de Sulfhidrilo/química , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas/química , Proteínas/metabolismo , Biotina/química , Biotina/metabolismo , Biotina/análogos & derivados , PorosidadRESUMEN
In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.
Asunto(s)
Proteína Transportadora de Acilo , Panteteína , Cinética , Panteteína/química , Panteteína/metabolismo , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Escherichia coli/metabolismo , alfa-Amilasas/metabolismo , Proteínas Inmovilizadas/metabolismoRESUMEN
Robust regulatory signals in the cell often depend on interactions between short linear motifs (SLiMs) and globular proteins. Many of these interactions are poorly characterized because the binding proteins cannot be produced in the amounts needed for traditional methods. To address this problem, we developed a single-molecule off-rate (SMOR) assay based on microscopy of fluorescent ligand binding to immobilized protein partners. We used it to characterize substrate binding to the Anaphase-Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that triggers chromosome segregation. We find that SLiMs in APC/C substrates (the D box and KEN box) display distinct affinities and specificities for the substrate-binding subunits of the APC/C, and we show that multiple SLiMs in a substrate generate a high-affinity multivalent interaction. The remarkably adaptable substrate-binding mechanisms of the APC/C have the potential to govern the order of substrate destruction in mitosis.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Humanos , Proteínas Inmovilizadas/metabolismo , Ligandos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Proteolisis , Especificidad por SustratoRESUMEN
The hedgehog (Hh) and Wnt pathways, crucial for the embryonic development and stem cell proliferation of Metazoa, have long been known to have similarities that argue for their common evolutionary origin. A surprising additional similarity of the two pathways came with the discovery that WIF1 proteins are involved in the regulation of both the Wnt and Hh pathways. Originally, WIF1 (Wnt Inhibitory Factor 1) was identified as a Wnt antagonist of vertebrates, but subsequent studies have shown that in Drosophila, the WIF1 ortholog serves primarily to control the distribution of Hh. In the present, work we have characterized the interaction of the human WIF1 protein with human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein binds human Shh with high affinity and inhibits its signaling activity efficiently. Our observation that the human WIF1 protein is a potent antagonist of human Shh suggests that the known tumor suppressor activity of WIF1 may not be ascribed only to its role as a Wnt inhibitor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Animales , Línea Celular , Proteínas Hedgehog/metabolismo , Humanos , Proteínas Inmovilizadas/metabolismo , Cinética , Ratones , Células 3T3 NIH , Unión Proteica , Transducción de SeñalRESUMEN
Binding and unbinding of transcription factors to DNA are kinetically controlled to regulate the transcriptional outcome. Control of the release of the transcription factor NF-κB from DNA is achieved through accelerated dissociation by the inhibitor protein IκBα. Using single-molecule FRET, we observed a continuum of conformations of NF-κB in free and DNA-bound states interconverting on the subseconds to minutes timescale, comparable to in vivo binding on the seconds timescale, suggesting that structural dynamics directly control binding kinetics. Much of the DNA-bound NF-κB is partially bound, allowing IκBα invasion to facilitate DNA dissociation. IκBα induces a locked conformation where the DNA-binding domains of NF-κB are too far apart to bind DNA, whereas a loss-of-function IκBα mutant retains the NF-κB conformational ensemble. Overall, our results suggest a novel mechanism with a continuum of binding modes for controlling association and dissociation of transcription factors.
Asunto(s)
ADN/genética , Interferones/genética , Inhibidor NF-kappaB alfa/genética , Factor de Transcripción ReIA/genética , Transcripción Genética , Animales , Avidina/química , Sitios de Unión , Biotina/química , ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Proteínas Inmovilizadas/metabolismo , Interferones/química , Interferones/metabolismo , Secuencias Invertidas Repetidas , Ratones , Simulación de Dinámica Molecular , Inhibidor NF-kappaB alfa/química , Inhibidor NF-kappaB alfa/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Imagen Individual de Molécula/métodos , Factor de Transcripción ReIA/química , Factor de Transcripción ReIA/metabolismoRESUMEN
Soy diet is thought to help prevent cardiovascular diseases in humans. Isoflavone, which is abundant in soybean and other legumes, has been reported to possess antiplatelet activity and potential antithrombotic effect. Our study aims to elucidate the potential target of soy isoflavone in platelet. The anti-thrombosis formation effect of genistein and daidzein was evaluated in ex vivo perfusion chamber model under low (300 s-1) and high (1800 s-1) shear forces. The effect of genistein and daidzein on platelet aggregation and spreading was evaluated with platelets from both wildtype and GPIbα deficient mice. The interaction of these soy isoflavone with 14-3-3ζ was detected by surface plasmon resonance (SPR) and co-immunoprecipitation, and the effect of αIIbß3-mediated outside-in signaling transduction was evaluated by western blot. We found both genistein and daidzein showed inhibitory effect on thrombosis formation in perfusion chamber, especially under high shear force (1800 s-1). These soy isoflavone interact with 14-3-3ζ and inhibited both GPIb-IX and αIIbß3-mediated platelet aggregation, integrin-mediated platelet spreading and outside-in signaling transduction. Our findings indicate that 14-3-3ζ is a novel target of genistein and daidzein. 14-3-3ζ, an adaptor protein that regulates both GPIb-IX and αIIbß3-mediated platelet activation is involved in soy isoflavone mediated platelet inhibition.
Asunto(s)
Proteínas 14-3-3/metabolismo , Plaquetas/metabolismo , Glycine max/química , Isoflavonas/farmacología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Transducción de Señal , Animales , Fibrinógeno/metabolismo , Genisteína/química , Genisteína/farmacología , Proteínas Inmovilizadas/metabolismo , Isoflavonas/química , Masculino , Ratones Endogámicos C57BL , Agregación Plaquetaria/efectos de los fármacos , Trombosis/patologíaRESUMEN
Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.
Asunto(s)
Cromatografía de Afinidad/métodos , Electroforesis Capilar/métodos , Lectinas/metabolismo , Orosomucoide , Glicoproteínas/análisis , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Lectinas/química , Ácido N-Acetilneuramínico/química , Orosomucoide/análisis , Orosomucoide/química , Orosomucoide/aislamiento & purificación , Polisacáridos/químicaRESUMEN
A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.
Asunto(s)
Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Proteínas Inmovilizadas/química , Proteínas de la Membrana/química , Nanoestructuras/química , Animales , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Inmovilizadas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch's membrane, are essential for RPE cell health and function, but the signals induced by Bruch's membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch's membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and ß1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.
Asunto(s)
Integrina alfa5beta1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Comunicación Celular , Muerte Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas Inmovilizadas/metabolismo , Unión Proteica , Telomerasa/metabolismoRESUMEN
The TEAD (Sd in drosophila) transcription factors are essential for the Hippo pathway. Human VGLL4 and drosophila Tgi bind to TEAD/Sd via two distinct binding sites. These two regions are separated by few amino acids in VGLL4 but they are very distant from each other in Tgi. This difference prompted us to study whether it influences the interaction with TEAD4/Sd. We show that the full-length VGLL4/Tgi proteins behave as intrinsically disordered proteins. They have a similar affinity for TEAD4/Sd revealing that the length of the region between the two binding sites has little effect on the interaction. One of their two binding sites (high-affinity site) binds to TEAD4/Sd 100 times more tightly than to the other site, and size exclusion chromatography experiments reveal that VGLL4/Tgi only form trimeric complexes with TEAD4/Sd at high protein concentrations. In solution, therefore, VGLL4/Tgi may predominantly interact with TEAD4/Sd via their high-affinity site to create dimeric complexes. In contrast, when TEAD4/Sd molecules are immobilized on sensor chips used in Surface Plasmon Resonance experiments, one VGLL4/Tgi molecule can bind simultaneously with an enhanced affinity to two immobilized molecules. This effect, due to a local increase in protein concentration triggered by the proximity of the immobilized TEAD4/Sd molecules, suggests that in vivo VGLL4/Tgi could bind with an enhanced affinity to two nearby TEAD/Sd molecules bound to DNA. The presence of two binding sites in VGLL4/Tgi might only be required for the function of these proteins when they interact with TEAD/Sd bound to DNA.
Asunto(s)
Proteínas Portadoras/química , ADN/química , Proteínas de Drosophila/química , Proteínas Intrínsecamente Desordenadas/química , Factores de Transcripción de Dominio TEA/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN/genética , ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Vía de Señalización Hippo/genética , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Proteínas Inmovilizadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción de Dominio TEA/genética , Factores de Transcripción de Dominio TEA/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.
Asunto(s)
Dosificación de Gen , Inmunoensayo/métodos , Proteínas/metabolismo , Análisis de la Célula Individual , Anticuerpos/metabolismo , Calibración , Línea Celular Tumoral , Supervivencia Celular , Análisis de Datos , Oro/química , Humanos , Proteínas Inmovilizadas/metabolismo , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Coloración y EtiquetadoRESUMEN
Mammalian olfactory receptors (ORs) constitute the largest family of G-protein-coupled receptors, with up to about 1000 different genes per species, each having specific odorant ligands. ORs could be used as sensing elements of highly specific and sensitive bioelectronic hybrid devices such as bioelectronic noses. After optimized immobilization onto the device, natural ORs provide molecular recognition of various odors with their intrinsic sensitivity, discrimination, and detection properties. However, the main difficulties are related to the low expression level of recombinant ORs, their stability and potential loss of activity. Such drawbacks can be successfully overcome in bioelectronic noses integrating nanosomes (nanometric membrane vesicles carrying ORs) that are stably immobilized through a specific antibody. The advantages of such a platform rely on the fact that ORs stay in the natural membrane environment, and thus preserve their full activity. Thanks to their small sizes, nanosomes offer potential for micro- and nano-scale sensor development. In this paper, we summarize the key elements regarding nanosomes production and manipulation and provide an example of their immobilization onto a gold sensor surface. Rat ORI7 is used as a representative OR that can be functionally expressed in Saccharomyces cerevisiae. The receptor was not purified but only nanosomes were prepared. Nanosomes were immobilized onto functionalized gold surface using the anti-I7 antibody. Utilization of the antibody provides enrichment of ORI7 on the sensor surface but also uniform and appropriate orientation of the receptors. These features are crucial in optimization of bioelectronic nose' analytical performances.
Asunto(s)
Técnicas Biosensibles/métodos , Oro/química , Proteínas Inmovilizadas/metabolismo , Proteínas de la Membrana/metabolismo , Odorantes/análisis , Receptores Odorantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Ratas , Receptores Odorantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Toxic aggregates of amyloid-beta (Aß) have importance in the pathology of Alzheimer's disease, and inhibition of aggregate formation is considered to be a promising strategy for drug development. Here, we report a simple and rapid surface plasmon resonance (SPR) assay method that can identify potential Aß aggregation inhibitors. Our assay is based on the SPR shifting of the Aß-gold nanoparticle (Aß-GNP) aggregates by size under the influence of an Aß aggregation inhibitor. This user-friendly assay features a short assay time with a low reagent consumption that can be easily adapted as a high-throughput screen. We demonstrated that an effective Aß aggregation inhibitor induces the blue-shifted SPR peaks of the Aß-GNP aggregates by hindering the formation of long fibrillar aggregates. Moreover, the blue shifting was correlated to the efficacy and concentrations of an Aß aggregation inhibitor. Overall, our findings suggest that our simple SPR assay can be a powerful tool to screen small molecules targeting Aß aggregation.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Fragmentos de Péptidos/antagonistas & inhibidores , Fenoles/análisis , Multimerización de Proteína/efectos de los fármacos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Oro/química , Concentración de Iones de Hidrógeno , Proteínas Inmovilizadas/antagonistas & inhibidores , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Nanopartículas del Metal/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fenoles/química , Resonancia por Plasmón de SuperficieRESUMEN
Targeting cell surface receptors for specific drug delivery in cancer has garnered lot of attention. Urokinase plasminogen activator receptor (uPAR), a surface biomarker, is overexpressed on many tumours including breast, colorectal, prostate, and ovarian cancers. Binding of growth factor domain (GFD) of urokinase plasminogen activator (uPA) with uPAR lead to its close conformation, and allow somatomedin B domain (SMB) of vitronectin binding by allosteric modulation. In-silico docking of uPAR with GFD and SMB peptides was performed to identify potential binding affinity. Herein, we report fluorescently labeled peptide functionalized AuNPs with a mixed self-assembled monolayer of intercalating chitosan polymer for efficient targeting and imaging of uPAR-positive cells. The biophysical characterization of nanoconjugates and uPAR-specific targeting was assessed by FACS, cell adhesion, and fluorescence imaging. AuNPs/chitosan/GFD+SMB peptides showed higher uptake as compared to AuNPs/chitosan/GFD, and AuNPs/chitosan/SMB that can be utilized as a tool for molecular targeting and imaging in metastasis.
Asunto(s)
Quitosano/química , Nanopartículas del Metal/química , Neoplasias/diagnóstico por imagen , Péptidos/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Línea Celular Tumoral , Quitosano/toxicidad , Oro/química , Oro/toxicidad , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Proteínas Inmovilizadas/toxicidad , Nanopartículas del Metal/toxicidad , Microscopía Fluorescente , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Péptidos/metabolismo , Péptidos/toxicidad , Unión ProteicaRESUMEN
Nanoparticles (NPs) promise a huge potential for clinical diagnostic and therapeutic applications. However, nano-bio (e.g., the NP-cell membrane) interactions and underlying mechanisms are still largely elusive. In this study, two types of congeneric peptides, namely PGLa and magainin 2 (MAG2), with similar membrane activities were employed as model ligands for NP decoration, and the diffusion behaviours (including both translation and rotation) of the ligand-decorated NPs on a lipid bilayer membrane were studied via molecular dynamics simulations. It was found that, although both PGLa- and MAG2-coated NPs showed alternatively "hopping" and "jiggling" diffusions, the PGLa-coated ones had an enhanced circling at the hopping stage, while a much confined circling at the jiggling stage. In contrast, the MAG2-coated NPs demonstrated constant circling tendencies throughout the diffusion process. Such differences in the coupling between translational and rotational dynamics of these two types of NPs are ascribed to the different ligand-lipid interactions of PGLa and MAG2, in which the PGLa ligands prefer to vertically insert into the membrane, while MAG2 tends to lie flat on the membrane surface. Our results are helpful for the understanding the underlying associations between the NP motions and their interfacial membrane interactions, and shed light on the possibility of regulating NP behaviours on a cellular surface for better biomedical uses.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Inmovilizadas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Magaininas/metabolismo , Nanopartículas/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Proteínas Inmovilizadas/química , Ligandos , Membrana Dobles de Lípidos/química , Magaininas/química , Simulación de Dinámica Molecular , Nanopartículas/químicaRESUMEN
BACKGROUND: Due to educational, social and economic reasons, more and more women are delaying childbirth. However, advanced maternal age is associated with several adverse pregnancy outcomes, and in particular a high risk of Down's syndrome (DS). Hence, it is increasingly important to be able to detect fetal Down's syndrome (FDS). METHODS: We developed an effective, highly sensitive, surface plasmon resonance (SPR) biosensor with biochemically amplified responses using carboxyl-molybdenum disulfide (MoS2) film. The use of carboxylic acid as a surface modifier of MoS2 promoted dispersion and formed specific three-dimensional coordination sites. The carboxylic acid immobilized unmodified antibodies in a way that enhanced the bioaffinity of MoS2 and preserved biorecognition properties of the SPR sensor surface. Complete antigen pregnancy-associated plasma protein-A2 (PAPP-A2) conjugated with the carboxyl-MoS2-modified gold chip to amplify the signal and improve detection sensitivity. This heterostructure interface had a high work function, and thus improved the efficiency of the electric field energy of the surface plasmon. These results provide evidence that the interface electric field improved performance of the SPR biosensor. RESULTS: The carboxyl-MoS2-based SPR biosensor was used successfully to evaluate PAPP-A2 level for fetal Down's syndrome screening in maternal serum samples. The detection limit was 0.05 pg/mL, and the linear working range was 0.1 to 1100 pg/mL. The women with an SPR angle >46.57 m° were more closely associated with fetal Down's syndrome. Once optimized for serum Down's syndrome screening, an average recovery of 95.2% and relative standard deviation of 8.5% were obtained. Our findings suggest that carboxyl-MoS2-based SPR technology may have advantages over conventional ELISA in certain situations. CONCLUSION: Carboxyl-MoS2-based SPR biosensors can be used as a new diagnostic technology to respond to the increasing need for fetal Down's syndrome screening in maternal serum samples. Our results demonstrated that the carboxyl-MoS2-based SPR biosensor was capable of determining PAPP-A2 levels with acceptable accuracy and recovery. We hope that this technology will be investigated in diverse clinical trials and in real case applications for screening and early diagnosis in the future.