Chemoproteomics of microbiota-derived indole metabolites in mammalian cells

The microbiota generates diverse metabolites to modulate host physiology and disease, but their protein targets and mechanisms of action have not been fully elucidated. To address this challenge, we focused on microbiota-derived indole metabolites and develop photoaffinity chemical probes for proteomic studies. We identified many potential indoleinteracting proteins, including metabolic enzymes, transporters, immune sensors, and Gprotein coupled receptors. Notably, we discovered aromatic monoamines can activate - arrestin recruitment by the orphan receptor GPRC5A. Metabolomic and functional profiling of microbiota species revealed specific aromatic amino acid decarboxylaseexpressing bacteria producing monoamine agonists for GPRC5A. Structure-activity relationship studies of synthetic aromatic monoamines identified 7-fluorotryptamine as a more potent activator of -arrestin recruitment by GPRC5A. Our results highlight the utility of chemoproteomics to identify novel microbiota metabolite-interacting proteins and discover potential microbiota-derived small molecule agonists for orphan receptors.微生物群可产生多种代谢物以调控宿主生理状态与疾病进程，但其蛋白靶点及作用机制尚未完全阐明。为应对这一研究挑战，我们聚焦于微生物群衍生的吲哚类代谢物，并开发了用于蛋白质组学研究（proteomic studies）的光亲和化学探针（photoaffinity chemical probes）。我们鉴定出多种潜在的吲哚互作蛋白，涵盖代谢酶、转运蛋白、免疫感受器以及G蛋白偶联受体（G protein-coupled receptors）。值得注意的是，我们发现芳香族单胺可激活孤儿受体GPRC5A对β抑制蛋白（β-arrestin）的招募过程。通过对微生物群物种开展代谢组学与功能谱分析，我们发现了一类特异性表达芳香族氨基酸脱羧酶（aromatic amino acid decarboxylase）的细菌，其可产生靶向GPRC5A的单胺类激动剂。针对合成芳香族单胺开展的构效关系（structure-activity relationship）研究显示，7-氟色胺（7-fluorotryptamine）是一种更为高效的GPRC5A介导的β抑制蛋白招募激活剂。本研究结果证实了化学蛋白质组学（chemoproteomics）在鉴定新型微生物群代谢物互作蛋白，以及挖掘潜在微生物群衍生的孤儿受体小分子激动剂方面的应用价值。