天津医药

天津医药 ›› 2022, Vol. 50 ›› Issue (7): 673-677.doi: 10.11958/20212782

• 细胞与分子生物学 •    下一篇

铜绿假单胞菌信号分子3-O-C12-HSL与PPARγ-LBD的结合特性研究

张云燕，马刘合一，陈敏怡，李有强，刘润梅，罗冬元   

  1. 1广州医科大学，广州市妇女儿童医疗中心口腔科（邮编510120）；2华南理工大学医学院；3南方医科大学附属何贤纪念医院检验科；4广州医科大学，广州市妇女儿童医疗中心医务部
  • 收稿日期:2021-12-17 修回日期:2022-02-28 出版日期:2022-07-15 发布日期:2022-07-15
  • 基金资助:
    国家自然科学基金青年项目（81801973）；广州市基础研究计划基础与应用基础研究项目（202102080539）

Ligand binding characteristics of signal molecule 3-O-C12-HSL and PPAR γ-LBD in pseudomonas aeruginosa

ZHANG Yunyan, MA Liuheyi, CHEN Minyi, LI Youqiang, Liu Runmei, LUO Dongyuan   

  1. 1 Department of Stomatology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510120, China; 2 School of Medicine, South China University of Technology; 3 Department of Laboratory Medicine, He Xian Memorial Hospital, Southern Medical University; 4 Medical Section, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University
  • Received:2021-12-17 Revised:2022-02-28 Published:2022-07-15 Online:2022-07-15

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摘要： 目的　制备纯化的人过氧化物酶体增殖物激活受体-配体结合域（PPARγ-LBD）肽段并观察铜绿假单胞菌信号分子N-3-氧代十二烷酰-L-同型丝氨酸内酯（3-O-C12-HSL）与PPARγ-LBD的结合强度。方法　采用分子对接的方式预测3-O-C12-HSL与PPARγ的结合位点。合成PPARγ-LBD基因序列并插入pET28b质粒，鉴定后通过异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达重组肽段，进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）及Western blot分析。表面等离子体共振技术检测3-O-C12-HSL与PPARγ-LBD的结合动力学参数。结果　分子对接结果显示3-O-C12-HSL与PPARγ-LBD区的Tyr473和Tyr327形成氢键，3-O-C12-HSL末端柔性碳链与PPARγ-LBD区的Met329、Leu330等残基形成疏水结合。制备获得人PPARγ-LBD肽段，经SDS-PAGE及Western blot检测在25~35 ku有一条蛋白的印迹，与PPARγ-LBD分子质量一致。3-O-C12-HSL与PPARγ结合的亲和力常数为1.65×10-4 mol/L，结合速率常数为1.06×102/ms，解离速率常数为1.75×10-2/s。结论　3-O-C12-HSL与PPARγ-LBD功能区结合能力强于PPARγ特异性拮抗剂GW9662，为制备针对3-O-C12-HSL的特异性靶向药物提供了实验依据。

关键词: 铜绿假单胞菌, PPARγ, 分子对接模拟, 表面等离子体共振技术, N-3-氧代十二烷酰-L-同型丝氨酸内酯, 配体结合区域

Abstract: Objective　To prepare purified human peroxisome proliferator-activated receptor-ligand binding domain (PPARγ-LBD) peptide segment and study the binding strength of N-3-oxododecanoyl-l-homoserine lactone (3-O-C12-HSL) to PPARγ-LBD. Methods　The binding site of 3-O-C12-HSL to PPARγ was predicted by molecular docking. PPARγ-LBD gene was synthesized and inserted into pET28b plasmid. The recombinant peptide fragment was induced by IPTG after identification and analyzed by SDS-PAGE and Western blot assay. The binding kinetic parameters of 3-O-C12-HSL and PPARγ-LBD were measured by surface plasmon resonance technique. Results　Results of molecular docking showed that 3-O-C12-HSL formed hydrogen bond with Tyr473 and Tyr327 of PPARγ-LBD, and the terminal flexible carbon chain of 3-O-C12-HSL formed hydrophobic interaction with the residues of Met329 and Leu330 of PPARγ-LBD. The human PPARγ-LBD peptide fragment was prepared, and the molecular weight of PPARγ-LBD was 25~35 ku consistent with that of Western blot assay and SDS-PAGE. The binding affinity constant of 3-O-C12-HSL with PPARγ was 1.65×10-4 mol/L, the binding rate constant was 1.06×102/ms, and the dissociation rate constant was 1.75×10-2/s. Conclusion　The binding ability of 3-O-C12-HSL to PPARγ-LBD functional region is stronger than that of PPARγ-specific antagonist GW9662, which provides experimental basis for preparing specific targeted drugs against 3-O-C12-HSL.

Key words: Pseudomonas aeruginosa, PPAR gamma, molecular docking simulation, surface plasmon resonance technique, N-3-oxododecanoyl-L-homoserine lactone, ligand binding domain