Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases.
Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases.
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DOI：
10.1021/acschembio.8b01038
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发表时间：
2019-02
影响因子：
4
通讯作者：
Yen-Chih Wang;Nathan P. Westcott;Matthew E. Griffin;H. Hang
中科院分区：
文献类型：
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作者：
Yen-Chih Wang;Nathan P. Westcott;Matthew E. Griffin;H. Hang
肽聚糖片段γ-d-谷氨酰基-中位二氨基戊二酸(IE-DAP)和胞壁二肽(MDP)是微生物特有的代谢产物，可以激活细胞内的模式识别受体，刺激免疫信号通路。虽然广泛的结构活性研究表明，这些细菌细胞壁代谢物可以触发NOD1和NOD2依赖的信号转导，但它们与哺乳动物细胞中的这些天然免疫受体或其他蛋白质的直接结合尚未建立。为了表征这些基本的微生物代谢物-宿主相互作用，我们合成了一系列肽聚糖代谢物光亲和力报告，并在哺乳动物细胞中评估了它们与NOD1和NOD2的交联性。我们发现，活性的ie-DAP和MDP光亲和报告分别选择性地交联NOD1和NOD2，而不是它们的非活性突变体。我们还发现了MDP报告分子与Arf GTP酶的交联，它与GTP结合的Arf6结合最为显著，并在MDP刺激下与NOD2共沉淀。值得注意的是，与克罗恩病相关的功能丧失的NOD2突变体取消了MDP与NOD2和Arf6的结合。我们的研究表明，肽聚糖代谢产物光亲和记者可以捕获细胞中它们的同源免疫受体，并揭示意外的配体诱导的与其他细胞辅因子的相互作用。这些光亲和记者应该为发现和表征其他肽聚糖代谢产物相互作用的蛋白质提供有用的工具。
The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells. We show that active iE-DAP and MDP photoaffinity reporters selectively cross-linked NOD1 and NOD2, respectively, and not their inactive mutants. We also discovered MDP reporter cross-linking to Arf GTPases, which interacted most prominently with GTP-bound Arf6 and coimmunoprecipitated with NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was abrogated with loss-of-function NOD2 mutants associated with Crohn's disease. Our studies demonstrate peptidoglycan metabolite photoaffinity reporters can capture their cognate immune receptors in cells and reveal unpredicted ligand-induced interactions with other cellular cofactors. These photoaffinity reporters should afford useful tools to discover and characterize other peptidoglycan metabolite-interacting proteins.