Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs
Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs
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DOI：
10.1021/jacs.7b11639
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发表时间：
2018-03-28
影响因子：
15
通讯作者：
Woo, Christina M.
中科院分区：
文献类型：
--
作者：
Gao, Jinxu;Mfuh, Adelphe;Woo, Christina M.
许多治疗方法会引发细胞类型特异性的多药理学，该药理学是通过小分子和整个蛋白质组之间的分子识别事件网络来执行的。然而，测量的结构，支持蛋白质组之间的分子关联，甚至常见的治疗，如非甾体抗炎药（NSAID），是有限的，不能映射的小分子相互作用组。为了解决这一差距，我们开发了一个平台，称为小分子相互作用组映射通过光亲和标记（SIM-PAL），并将其应用于特定的NSAID结合位点的细胞内直接表征。SIM-PAL使用（1）NSAID衍生物在整个蛋白质组中的光化学缀合和（2）缀合肽的富集和同位素重新编码，用于（3）基于靶向质谱的分配。使用SIM-PAL，我们鉴定了由超过1000种显著富集的蛋白质组成的NSAID相互作用组，并直接表征了近200种缀合肽，其代表光NSAID与来自Jurkat和K562细胞的蛋白质的直接结合位点。富集的蛋白质通常被鉴定为复合物的一部分，包括NSAID活性的已知靶标（例如，NF-κ B）和新的相互作用（例如，AP-2，蛋白酶体）。共轭肽揭示了直接的NSAID结合位点从细胞表面到细胞核和一个特定的结合位点热点的组蛋白H2 A和H2 B上的三个光-NSAID。通过细胞热位移测定，NSAID结合稳定考克斯-2和组蛋白H2 A。由于蛋白质复合物的小分子稳定是功能调节机制的增益，因此可以想象NSAID通过这些更广泛的蛋白质组相互作用影响生物过程。SIM-PAL能够表征NSAID结合位点热点，并且适于绘制几乎任何感兴趣分子的全局结合位点。
Many therapeutics elicit cell-type specific poly-pharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-kappa B) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.