Understanding the coupling specificity between G protein-coupled receptors （GPCRs） and specific classes of G proteins is important for further elucidation of receptor functions within a cell. Increasing information ...Understanding the coupling specificity between G protein-coupled receptors （GPCRs） and specific classes of G proteins is important for further elucidation of receptor functions within a cell. Increasing information on GPCR sequences and the G protein family would facilitate prediction of the coupling properties of GPCRs. In this study, we describe a novel approach for predicting the coupling specificity between GPCRs and G proteins. This method uses not only GPCR sequences but also the functional knowledge generated by natural language processing, and can achieve 92.2% prediction accuracy by using the C4.5 algorithm. Furthermore, rules related to GPCR-G protein coupling are generated. The combination of sequence analysis and text mining improves the prediction accuracy for GPCR-G protein coupling specificity, and also provides clues for understanding GPCR signaling.展开更多
GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the int...GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the intrinsic activities of them are seldom simultaneously determined during the screening.Taking beta2-adrenoceptor(β2AR)as a probe,this work created a strategy for screening GPCR ligands with simultaneous characterization of their downstream G protein binding responses associated with GTP.The strategy included(i)the design and expression of a protein miniature formed byβ2AR and G proteinα-subunit(Gαs)using circularly permuted HaloTag(cpHalo)as a flexible linker;(ii)immobilization of the miniature onto silica gel by a click dehalogenation reaction;(iii)systematic characterization of the immobilized miniature by fluorescent and chromatographic studies,and(iv)simulating of ligand-inducedβ2AR-Gαs signaling cascade by chromatographic assays using GTP as an indicator.The immobilized miniature exhibited specificity toβ2AR and Gαs antibodies and ligands.The specificity is stable at least within fifteen days with the variation less than 1%.The intrinsic activities ofβ2AR ligands were distinguished by the changes of GTP chromatographic behaviors on Gαs-cpHalo-β2AR column.Agonists strengthened the binding affinity and kinetics of GTP with Gαs,while antagonist did not give any effect on them.With the intrinsic activity evaluation,we believe,it will improve the attributes of chromatographic methods for drug discovery efforts with minimizing false-positive results.展开更多
文摘Understanding the coupling specificity between G protein-coupled receptors （GPCRs） and specific classes of G proteins is important for further elucidation of receptor functions within a cell. Increasing information on GPCR sequences and the G protein family would facilitate prediction of the coupling properties of GPCRs. In this study, we describe a novel approach for predicting the coupling specificity between GPCRs and G proteins. This method uses not only GPCR sequences but also the functional knowledge generated by natural language processing, and can achieve 92.2% prediction accuracy by using the C4.5 algorithm. Furthermore, rules related to GPCR-G protein coupling are generated. The combination of sequence analysis and text mining improves the prediction accuracy for GPCR-G protein coupling specificity, and also provides clues for understanding GPCR signaling.
基金National Natural Science Foundation of China(Nos.22374116,22074118,82174088)Natural Science Basic Research Program of Shaanxi(2024JC-TBZC-21)Shaanxi Administration of Traditional Chinese Medicine(No.2022-SLRH-YQ-007)。
文摘GPCRs are dominant targets for approved drugs and the discovery of lead compound targeting them is still challengeable.Affinity-based screening technique is a promising platform to uncover GPCR ligands.However,the intrinsic activities of them are seldom simultaneously determined during the screening.Taking beta2-adrenoceptor(β2AR)as a probe,this work created a strategy for screening GPCR ligands with simultaneous characterization of their downstream G protein binding responses associated with GTP.The strategy included(i)the design and expression of a protein miniature formed byβ2AR and G proteinα-subunit(Gαs)using circularly permuted HaloTag(cpHalo)as a flexible linker;(ii)immobilization of the miniature onto silica gel by a click dehalogenation reaction;(iii)systematic characterization of the immobilized miniature by fluorescent and chromatographic studies,and(iv)simulating of ligand-inducedβ2AR-Gαs signaling cascade by chromatographic assays using GTP as an indicator.The immobilized miniature exhibited specificity toβ2AR and Gαs antibodies and ligands.The specificity is stable at least within fifteen days with the variation less than 1%.The intrinsic activities ofβ2AR ligands were distinguished by the changes of GTP chromatographic behaviors on Gαs-cpHalo-β2AR column.Agonists strengthened the binding affinity and kinetics of GTP with Gαs,while antagonist did not give any effect on them.With the intrinsic activity evaluation,we believe,it will improve the attributes of chromatographic methods for drug discovery efforts with minimizing false-positive results.
文摘目的研究使用En Vision多标记读板仪检测细胞内钙信号的可行性。方法分别用卡巴胆碱和凝血酶刺激LN229、SKN-MC细胞或者磷脂酶C抑制剂U73122预处理的LN229细胞,使用En Vision多标记读板仪检测细胞内钙信号强度的变化。结果卡巴胆碱诱导的LN229细胞内钙信号变化呈现浓度依赖性;卡巴胆碱和凝血酶诱导的细胞内钙信号变化趋势不同;凝血酶诱导的钙信号变化具有细胞特异性;U73122对凝血酶诱导的钙信号的抑制作用呈现剂量依赖性。结论 En Vision多标记读板仪作为细胞内钙信号检测仪器,具有快速、可重复性好和灵敏度高等优势,适用于钙信号相关的研究和小分子抑制剂的筛选等领域的工作。
