Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, United States of America.
Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, United States of America.
J Proteomics. 2023 Mar 15;274:104807. doi: 10.1016/j.jprot.2022.104807. Epub 2022 Dec 29.
Histone deacetylase 1 (HDAC1) plays a key role in diverse cellular processes. With the aberrant expression of HDAC1 linked to many diseases, including cancers, HDAC inhibitors have been used successfully as therapeutics. HDAC1 has been predominantly associated with histone deacetylation and gene expression. Recently, non-histone substrates have revealed diverse roles of HDAC1 beyond epigenetics. To augment discovery of non-histone substrates, we introduced "substrate trapping" to enrich HDAC1 substrates using an inactive mutant. Herein, we performed a series of proteomics studies to test the robustness of HDAC1 substrate trapping. Based on our recent results documenting that different HDAC1 mutants preferentially bound different substrates, which suggested that multiple mutants could be used for efficient trapping, trapping with three single point mutants simultaneously identified several potential substrates uniquely compared to a single mutant alone. However, a greater number of biologically interesting hits were observed using only a single mutant, which suggests that the C151A HDAC1 mutant is the optimal trap. Importantly, comparing independent trials with a single mutant performed by different experimentalists and HEK293 cell populations, trapping was robust and reproducible. Based on the reproducible trapping data, carnosine N-methyltransferase 1 (CARNMT1) was validated as an HDAC1 substrate. The data document that mutant trapping is an effective method for discovery of unanticipated HDAC substrates. SIGNIFICANCE: Histone deacetylase (HDAC) proteins are well established epigenetic transcriptional regulators that deacetylate histone substrates to control gene expression. More recently, deacetylation of non-histone substrates has linked HDAC activity to functions outside of epigenetics. Given the use of HDAC inhibitor drugs as anti-cancer therapeutics, understanding the full functions of HDAC proteins in cell biology is essential to future drug design. To discover unanticipated non-histone substrates and further characterize HDAC functions, inactive mutants have been used to "trap" putative substrates, which were identified with mass spectrometry-based proteomics analysis. Here multiple trapping studies were performed to test the robustness of using inactive mutants and proteomics for HDAC substrate discovery. The data confirm the value of trapping mutants as effective tools to discover HDAC substrates and link HDAC activity to unexpected biological functions.
组蛋白去乙酰化酶 1（HDAC1）在多种细胞过程中发挥着关键作用。由于 HDAC1 的异常表达与包括癌症在内的许多疾病有关，因此 HDAC 抑制剂已成功用作治疗药物。HDAC1 主要与组蛋白去乙酰化和基因表达有关。最近，非组蛋白底物揭示了 HDAC1 在表观遗传学之外的多种作用。为了增加非组蛋白底物的发现，我们引入了“底物捕获”，使用无活性突变体来富集 HDAC1 底物。在此，我们进行了一系列蛋白质组学研究，以测试 HDAC1 底物捕获的稳健性。根据我们最近的研究结果，记录了不同的 HDAC1 突变体优先结合不同的底物，这表明可以使用多个突变体进行有效的捕获，同时使用三个单点突变体进行捕获比单独使用单个突变体鉴定出几种潜在的底物独特，但是，仅使用单个突变体观察到更多有生物学意义的命中，这表明 C151A HDAC1 突变体是最佳的陷阱。重要的是，将不同实验员和 HEK293 细胞群进行的单个突变体的独立试验进行比较，捕获是稳健且可重复的。基于可重复的捕获数据，肉毒碱 N-甲基转移酶 1（CARNMT1）被验证为 HDAC1 底物。该数据表明，突变体捕获是发现意外 HDAC 底物的有效方法。意义：组蛋白去乙酰化酶（HDAC）蛋白是公认的表观遗传转录调节剂，可使组蛋白底物去乙酰化以控制基因表达。最近，非组蛋白底物的去乙酰化作用将 HDAC 活性与表观遗传学以外的功能联系起来。鉴于将 HDAC 抑制剂药物用作抗癌治疗药物，了解 HDAC 蛋白在细胞生物学中的全部功能对于未来的药物设计至关重要。为了发现意外的非组蛋白底物并进一步表征 HDAC 功能，已使用无活性突变体来“捕获”假定的底物，然后使用基于质谱的蛋白质组学分析来鉴定这些底物。在此进行了多项捕获研究，以测试使用无活性突变体和蛋白质组学进行 HDAC 底物发现的稳健性。该数据证实了捕获突变体作为发现 HDAC 底物的有效工具的价值，并将 HDAC 活性与意外的生物学功能联系起来。
