Program in Chemical Biology, ‡Department of Chemistry, and §Department of Biological Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
J Am Chem Soc. 2017 Nov 15;139(45):16222-16227. doi: 10.1021/jacs.7b07603. Epub 2017 Nov 1.
The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme-substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, p-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical crosslinking approach identified novel HDAC8-specific substrates with high catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery.
组蛋白去乙酰化酶家族由 18 种酶组成，它们催化乙酰化赖氨酸残基的去乙酰化；然而，每种同工酶的特异性和底物谱仍然很大程度上未知。由于酶-底物的瞬时相互作用，传统的共免疫沉淀方法经常无法识别酶特异性底物。此外，补偿机制通常限制了敲低或化学抑制研究的能力，使其无法实现乙酰化蛋白质组学方法观察到的显著倍数变化。此外，测量的变化并不能保证酶和底物之间存在直接联系。在这里，我们提出了一种化学交联策略，该策略将一种光反应性、非天然氨基酸对苯甲酰基-l-苯丙氨酸引入结构特征明确的同工酶组蛋白去乙酰化酶 8（HDAC8）的各种位置。在共价捕获、共免疫沉淀和质谱分析后，我们从人细胞裂解物中鉴定出了一组 HDAC8 的底物，并用催化周转进一步验证。总的来说，这种化学交联方法鉴定了具有高催化效率的新型 HDAC8 特异性底物，从而为无偏的去乙酰化酶底物发现提供了一种通用策略。
