determination of cd40 protein-umbelliferone compound interaction using differential fluorescence scanning

Détection haute sensibilité de la liaison de protéines composées par fluorescence

Vicatia thibetica H. Boissieu, a plant in the umbelliferous family, is commonly used in Tibetan medicine and represents one of the essential components of the five basic ingredients (Polygonatum sibiricum Delar. ex Redoute, Asparagus cochinchinensis (Lour.) Merr, Vicatia thibetica H. Boissieu, Oxybaphus himalaicus Edgew., and Gymnadenia conopsea (L.) R. Br.)1. Mainly distributed in southwest China, such as northwest Yunnan, western Sichuan, Tibet, and other areas, its dried root serves as a local substitute for Angelica1. The root, known for its fragrance, is often utilized as a stew seasoning, and the leaves, referred to as Tibetan celery, contribute to delectable dishes. Thus, Vicatia thibetica holds significance not only as a unique medicinal plant but also as a food source in Tibet.
Both domestic and international research indicates that Vicatia thibetica possesses blood-replenishing and qi (power or energy)-invigorating properties, beneficial for regulating menstruation. It is employed to address symptoms of irregular menstrual dysmenorrhea caused by palpitation and blood deficiency, exhibiting pharmacological effects such as antioxidant regulation of body immunity2. The alcohol extract of Vicatia thibetica has demonstrated the ability to restore body mass and organ index in immunocompromised mice induced by cyclophosphamide. Additionally, it increases the activity of superoxide dismutase in serum and reduces the content of malondialdehyde, suggesting an improvement in antioxidant capacity and a balancing effect on lipid peroxidation2,3. Simultaneously, it enhances the number of blood cells and hemoglobin, which not only objectively reflects the body&#39;s hematopoietic function but also plays a crucial role in the immune system2,3.
Umbelliferone, characterized by acicular crystals and a bitter taste, possesses a small molecular weight, is volatile, and can be distilled with steam. It sublimates easily, has low solubility in water, and high solubility in organic matter. As one of the main chemical components of Vicatia thibetica, umbelliferone exhibits immunomodulatory effects on cellular immunity, humoral immunity, and non-specific immunity in hydrocortisone-induced immunosuppression mouse models4,5.
The cell surface molecule CD40, a member of the tumor necrosis factor receptor superfamily, is widely expressed in immune cells6. Its homologous ligand, CD154, also known as CD40L, is a type II transmembrane protein expressed by activated T lymphocytes. CD40 activation has the capacity to up-regulate the expression of co-stimulatory molecules on the surface of dendritic cells (DC) and monocytes. This process promotes the antigen presentation function of major histocompatibility complex (MHC) molecules and further activates CD8+ T cells7.
Macrophages play a crucial role in the formation and regulation of the tumor microenvironment, and CD40 activation can enhance the remodeling of the tumor microenvironment by macrophages8. CD40 signal activation significantly influences the proliferation and activation of B cells. B cells, when activated by CD40, can function as effective antigen-presenting cells. They present antigens, generate effector T cell activity, and thereby contribute to anti-tumor effects9. Moreover, CD40 activation in tumor cells can induce apoptosis and inhibit tumor growth10. CD40 primarily transduces signals by controlling the activity of non-receptor tyrosine protein kinases, including Lyn, Fyn, Syk, and others. Additionally, it has the ability to stimulate Bcl-xL, Cdk4, and Cdk6 proteins, activate Rel/NF-kB transcription factors, and phosphorylate CG-2 and PI3K10.
The Differential Scanning Fluorescence (DSF) method is widely employed to assess the impact of various environmental conditions, such as buffer composition, temperature, and small molecule ligands, on the thermal stability of protein structures. The commonly utilized dye for DSF is an orange, environmentally sensitive, hydrophobic dye. Under normal conditions, the protein structure is folded, concealing its hydrophobic segment internally. As the temperature increases, the protein&#39;s hydrophobic region becomes more exposed, resulting in a gradual breakdown of the natural protein structure. The dye selectively binds to this exposed protein portion, amplifying its fluorescence. Tm values are then calculated by monitoring changes in the fluorescence signal detection11. To a certain extent, variations in Tm values can gauge shifts in protein stability due to mutations, changes in buffers, or ligand binding. Furthermore, it can indicate structural alterations during the protein folding process12. This approach offers precise data, a broad temperature range, high sensitivity, and minimal protein sample loss13.
In this study, fluorescence determination was conducted using a fluorescent microplate reader instead of a fluorescence quantitative polymerase chain reaction (PCR) apparatus. This modification enables DSF detection in laboratories lacking a fluorescent quantitative PCR instrument, making the method less complex and reducing the steps required for instrument setup, thereby simplifying the experimental process. However, there are certain drawbacks to this approach. While the complexity is reduced, the procedure becomes more cumbersome. Manual fluorescence detection at different temperatures is necessary, and automatic and continuous collection of fluorescence from the system cannot be achieved. Thus, this study utilized the DSF technique to explore the interaction between CD40 protein and umbelliferone, providing novel insights into the molecular mechanisms of Tibetan medicine.

Pleins feux sur l’auteur : Rationaliser les méthodes de PCR pour une accessibilité et une efficacité accrues

Détection de l’interaction protéine-ombellifère CD40 par fluorescence différentielle

Our protocol provides a significant advantage by allowing use without specialized equipment. Unlike traditional fluorescence quantitative PCR methods, it simplifies the experimental process, requiring fewer steps and reducing overall complexity, making it safer and easier to conduct. This experiment requires the operator to be very familiar with the experimental procedure and the fluorescent microplate reader, and not to waste too much time during the solution heating and the fluorescence detection.We will focus on the molecular mechanism by which umbelliferone binds to CD14 protein to produce immune effects.

Determination of CD40 Protein-Umbelliferone Compound Interaction Using Differential Fluorescence Scanning

Pour commencer, ajoutez un colorant orange 500x à une solution PBS pour obtenir différents ratios de colorant, lancez le logiciel d’acquisition de données sur le bureau. Cliquez sur Instrument, puis choisissez le modèle de microplaque correspondant, puis sélectionnez OK. Cliquez sur les paramètres d’acquisition et choisissez Fluorescence.Sous l’interface de longueur d’onde, réglez la fonction lambda sur 470 nanomètres et 570 nanomètres, puis appuyez sur OK. Pipeter 100 microlitres de chaque concentration de colorant orange dans les puits d’une plaque à 96 puits. Placez la plaque dans l’étage de détection de l’instrument de lecture de microplaques fluorescentes.Cliquez sur l’icône Lire sur le logiciel et mesurez la fluorescence de chaque concentration de colorant 13 fois à température ambiante avec un intervalle de deux minutes. Après chaque détermination, cliquez sur l’icône Exporter, puis sélectionnez Exporter au format XML XLS TXT. Choisissez Toutes les plaques.Sélectionnez ensuite Plaque dans le format de sortie, puis cliquez sur OK pour enregistrer les données au format XLS pour une analyse plus approfondie. Maintenant, allumez le chauffage numérique en secouant le bain sec. Réglez la température de chauffage à 35 degrés Celsius et le temps de chauffage à deux minutes.Préparez le taux de dilution optimal du colorant dans un microtube à centrifuger de 1,5 millilitre. Ensuite, placez-le dans le bain sec de chauffage numérique en secouant pendant deux minutes. Maintenant, ajoutez 100 microlitres de PBS et des solutions de coloration dans les puits de la plaque.Placez la plaque dans l’étage de détection de l’instrument. Cliquez sur l’icône Lire dans le logiciel d’acquisition de données. Réglez la température de chauffage à 40 degrés Celsius et le temps de chauffage à deux minutes.Maintenant, pipetez la solution de colorant dans la plaque dans le microtube à centrifuger de 1,5 millilitre. Chauffez-le dans le chauffage numérique en secouant le bain sec pendant deux minutes. Répétez l’ajout des solutions de colorant, en exportant les données résultantes pour tester l’absorbance de la solution de colorant à des incréments de température de cinq degrés de 35 à 95 degrés Celsius.Combinez la solution protéique CD40 avec le PBS pour obtenir des concentrations finales de 5, 10, 15 et 20 microgrammes par microlitre. Ensuite, combinez le colorant orange avec le PBS pour obtenir une concentration finale de un est à 500. Évaluer l’absorbance de la solution de protéine CD40 à des incréments de température de cinq degrés de 35 à 95 degrés Celsius.Enregistrez les données sous forme de fichiers XLS, puis calculez le point de fusion ou la valeur TM à l’aide du logiciel d’analyse de données. Ensuite, mélangez CD40 et Umbelliferone dans la solution PBS. Ensuite, pipetez le colorant orange dans le PBS pour obtenir une concentration de solution de un est à 500.Mesurez l’absorbance de la solution de complexes ombellifères CD40 à des incréments de température de cinq degrés de 35 à 95 degrés Celsius. Le colorant orange a toujours montré une excitation de fluorescence stable à la fois à température ambiante et à des températures élevées. Un rapport de dilution optimal de un est à 500 a été déterminé.Une valeur TM stable de 51,82 degrés Celsius était détectable à une concentration de 15 microgrammes par millilitre, qui a diminué à 45,79 degrés Celsius avec une augmentation de la concentration en protéine CD40.

Research

JoVE Journal

Medicine

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence

Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. ...

Recherche

Médecine