A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome.
A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome.
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DOI：
10.1016/j.chembiol.2020.05.010
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发表时间：
2020-08-20
影响因子：
8.6
通讯作者：
Niethammer P
中科院分区：
文献类型：
--
作者：
Jelcic M;Wang K;Hui KL;Cai XC;Enyedi B;Luo M;Niethammer P
ATP是一种重要的能量代谢产物和变构信号，在健康和疾病中起重要作用。ATP相互作用蛋白，如P2受体，控制炎症，细胞死亡，迁移和伤口愈合。然而，识别变构ATP位点仍然具有挑战性，我们目前的ATP控制途径的库存可能是不完整的。在这里，我们开发和验证mipATP作为一种微创的光亲和探针的ATP相互作用的蛋白质。其N6官能化允许通过UV交联和通过“点击”化学缀合至报告标签的靶富集。添加物是紧凑的，允许mipATP在体外和体内完全保留天然ATP的钙信号传导反应。在A549细胞裂解物和膜级分中特异性富集已知核苷酸结合剂的mipATP。此外，它检索了未注释的ATP相互作用物，如FAS受体，CD 44和各种SLC转运蛋白。因此，mipATP是一个有前途的工具，以确定变构ATP位点的蛋白质组。Jelcic等人开发并验证了一种微创光亲和ATP（mipATP）探针，该探针保留了体内和体外天然ATP的信号传导功能，并提供了mipATP可用于使用蛋白质组学筛选绘制ATP-蛋白质相互作用空间的原理证明。
ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV-crosslinking and conjugation to reporter tags by “click” chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome. Jelcic et al. developed and verified a minimally invasive photoaffinity ATP (mipATP) probe that retains the signaling functions of native ATP in vivo and in vitro and provide proof-of-principle that mipATP can be used to map ATP-protein interaction space using proteomic screens.
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作者：
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通讯作者：
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通讯作者：
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