It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates . Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate . Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.

中文翻译：

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基于邻近标记的正交捕获策略识别 HDAC8 通过调节 Cortactin 乙酰化来促进细胞运动

捕获酶翻译后修饰（PTM）底物的瞬时相互作用对传统亲和方法来说是一个挑战。在此，我们提出了一种称为基于邻近标记的正交陷阱方法（ProLORT）的策略，依靠 APEX2 催化的邻近标记和正交陷阱管道以及定量蛋白质组学来直接研究活细胞中酶-PTM 底物的瞬时相互作用组。作为概念证明，ProLORT 可以对已知的 HDAC8 底物组蛋白 H3K9ac 进行稳健评估。通过利用这种方法，我们鉴定了许多假定的 HDAC8 靶向的乙酰化蛋白，并进一步证实 CTTN 是真正的底物。接下来，我们证明 HDAC8 通过 CTTN 在赖氨酸 144 处的去乙酰化来促进细胞运动，从而减弱其与 F-肌动蛋白的相互作用，从而扩展 HDAC8 的潜在调节机制。我们开发了一种通用策略来分析 PTM 介导的瞬时酶-底物相互作用，为识别活细胞中酶调节的时空 PTM 网络提供了强大的工具。