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

蛍光を用いた化合物-タンパク質結合の高感度検出

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.

著者スポットライト:アクセシビリティと効率性を向上させるためのPCR法の合理化(英語)

示差走査蛍光法によるCD40タンパク質-ウンベリフェロン相互作用の検出

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

まず、PBS溶液に500倍のオレンジ色素を添加してさまざまな色素比を実現し、デスクトップでデータ収集ソフトウェアを起動します。「装置」をクリックし、対応するマイクロプレートモデルを選択して、「OK」を選択します。Acquisition settingsをクリックし、Fluorescenceを選択します。波長インターフェースの下で、ラムダ 1 を 470 ナノメートルと 570 ナノメートルに設定し、[OK] を押します。オレンジ色の染料の各濃度の100マイクロリットルを96ウェルプレートのウェルにピペットで移します。プレートを蛍光マイクロプレートリーダー装置の検出ステージに置きます。ソフトウェアのReadアイコンをクリックし、各色素濃度の蛍光を室温で2分間隔で13回測定します。各決定後、「エクスポート」アイコンをクリックし、「XML XLS TXTにエクスポート」を選択します。[すべてのプレート] を選択します。次に、出力形式で[プレート]を選択し、[OK]をクリックしてデータをXLS形式で保存し、さらに分析します。次に、デジタル加熱シェーキングドライバスをオンにします。加熱温度を摂氏35度に、加熱時間を2分に設定します。1.5ミリリットルのマイクロ遠心チューブで染料の最適な希釈比を調製します。次に、デジタル加熱振とうドライバスに2分間入れます。次に、PBSと染色液の両方を100マイクロリットルプレートのウェルに加えます。プレートを検出ステージにセットします。Data AcquisitionソフトウェアのReadアイコンをクリックします。加熱温度を摂氏40度に、加熱時間を2分に設定します。次に、プレート内の染料溶液をピペットで1.5ミリリットルのマイクロ遠心チューブに戻します。デジタル加熱振とうドライバスで2分間加熱します。色素溶液の添加を繰り返し、結果のデータをエクスポートして、摂氏35度から95度までの5度の温度刻みで色素溶液の吸光度をテストします。CD40タンパク質溶液をPBSと組み合わせ、マイクロリットルあたり5、10、15、および20マイクログラムの最終濃度を取得します。次に、PBSとオレンジ染料を組み合わせ、1の最終濃度が500になるようにします。CD40タンパク質溶液の吸光度を、摂氏35度から95度まで5度刻みで評価します。データをXLSファイルとして保存し、Data Analysisソフトウェアを使用して融点またはTM値を計算します。次に、CD40とウンベリフェロンをPBS溶液に混合します。次に、オレンジ色の染料をPBSにピペットで移し、溶液濃度が1になるようにします。CD40 Umbelliferone複合体溶液の吸光度を、摂氏35度から95度までの5度の温度刻みで測定します。オレンジ色の色素は、室温と高温の両方で一貫して安定した蛍光励起を示しました。1つの最適な希釈倍率は500に対して、決定した。1ミリリットルあたり15マイクログラムの濃度で摂氏51.82度の安定したTM値が検出されましたが、CD40タンパク質濃度の増加に伴って摂氏45.79度に減少しました。

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. ...

医学