Abstract：
It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. 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 in vivo. 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.
摘要：
传统的亲和方法在体内捕获酶-翻译后修饰（PTM）底物的瞬时相互作用是一个挑战。在这里，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直接研究活细胞中酶-PTM底物的瞬时相互作用。作为概念的证明，ProLORT允许对已知的HDAC8基板进行稳健评估，组蛋白H3K9ac.通过利用这种方法，我们鉴定出大量HDAC8靶向的推定乙酰化蛋白,并进一步证实CTTN是体内真正的底物.接下来,我们证明HDAC8通过CTTN在赖氨酸144处的脱乙酰作用促进细胞运动，从而减弱其与F-肌动蛋白的相互作用，扩大HDAC8的基本监管机制。我们开发了一种通用策略来描述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。
