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

형광을 이용한 화합물 단백질 결합의 고감도 검출

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.

저자 스포트라이트: 접근성 및 효율성 향상을 위한 PCR 방법 간소화

Differential Scanning Fluorescence를 통한 CD40 단백질-Umbelliferone 상호작용 검출

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

시작하려면 PBS 솔루션에 500x 오렌지 염료를 추가하여 다양한 염료 비율을 달성하고 데스크탑에서 데이터 수집 소프트웨어를 실행합니다. Instrument(기기)를 클릭한 다음 해당 마이크로플레이트 모델을 선택하고 Okay(확인)를 선택합니다. Acquisition settings(획득 설정)를 클릭하고 Fluorescence(형광)를 선택합니다.wavelength interface(파장 인터페이스)에서 lambda one을 470 nanometers 및 570 nanometers로 설정한 다음 Okay를 누릅니다. 오렌지 염료의 각 농도 100 마이크로 리터를 96 웰 플레이트의 웰에 피펫팅합니다. 플레이트를 형광 마이크로플레이트 리더 기기의 검출 단계에 놓습니다.소프트웨어에서 읽기 아이콘을 클릭하고 2분 간격으로 실온에서 각 염료 농도의 형광을 13회 측정합니다. 각 결정 후 내보내기 아이콘을 클릭한 다음 XML XLS TXT로 내보내기를 선택합니다. 모든 플레이트를 선택합니다.그런 다음 출력 형식에서 플레이트를 선택하고 확인을 클릭하여 추가 분석을 위해 데이터를 XLS 형식으로 저장합니다. 이제 디지털 가열 쉐이킹 건식 욕조를 켭니다. 가열 온도는 섭씨 35도, 가열 시간은 2분으로 설정합니다.1.5 밀리리터 마이크로 원심분리기 튜브에서 염료의 최적 희석 비율을 준비합니다. 그런 다음 디지털 가열 쉐이킹 건욕에 2분 동안 넣습니다. 이제 PBS와 염색 용액 100마이크로리터를 플레이트의 웰에 추가합니다.플레이트를 감지 단계에 놓습니다.tag기기의 s. 데이터 수집 소프트웨어에서 읽기 아이콘을 클릭합니다. 가열 온도는 섭씨 40도, 가열 시간은 2분으로 설정합니다.이제 플레이트의 염료 용액을 1.5밀리리터 마이크로 원심분리기 튜브에 다시 피펫팅합니다. 디지털 가열 쉐이킹 건욕에서 2분 동안 가열합니다. 염료 용액의 추가를 반복하여 결과 데이터를 내보내 섭씨 35도에서 95도까지 5도의 온도 증가에서 염료 용액의 흡광도를 테스트합니다.CD40 단백질 용액과 PBS를 결합하여 마이크로리터당 5, 10, 15 및 20마이크로그램의 최종 농도를 얻습니다. 다음으로, 오렌지 염료와 PBS를 결합하여 최종 농도가 500이 되는 것을 얻습니다. 섭씨 35도에서 95도까지 5도의 온도 증가에서 CD40 단백질 용액의 흡광도를 평가합니다.데이터를 XLS 파일로 저장한 다음 데이터 분석 소프트웨어를 사용하여 융점 또는 TM 값을 계산합니다. 다음으로, CD40와 Umbelliferone를 PBS 용액에 혼합합니다. 그런 다음 오렌지 염료를 PBS에 피펫팅하여 용액 농도가 500이 되도록 합니다.CD40 Umbelliferone 복합체 용액의 흡광도를 섭씨 35도에서 95도까지 5도 온도 증분으로 측정합니다. 주황색 염료는 실온과 고온 모두에서 안정적인 형광 여기(excitation)를 일관되게 나타냈습니다. 최적 희석 비율은 500으로 결정되었다.섭씨 51.82도의 안정적인 TM 값은 밀리리터당 15마이크로그램의 농도에서 검출할 수 있었으며, CD40 단백질 농도가 증가함에 따라 섭씨 45.79도로 감소했습니다.

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. ...

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