Identification of Histone Deacetylase Substrates using Trapping Mutants

总结 组蛋白脱乙酰酶（HDAC）蛋白是表观遗传调节剂，其具有明确且充分研究的作用 在基因表达调控中。HDAC蛋白通过脱乙酰化乙酰赖氨酸影响基因表达 核小体组蛋白底物上的残基。近年来，各种各样的乙酰化蛋白质已经被发现， 这表明HDAC蛋白除了组蛋白之外还可能使底物脱乙酰化。 通过非组蛋白底物，HDAC蛋白可能在细胞外的细胞过程中发挥扩大的作用。 包括细胞信号和通讯。不幸的是，识别非- 组蛋白HDAC底物在很大程度上是偶然的，因为系统地 在细胞中发现HDAC脱乙酰化的靶点是缺乏的。 本申请概述了HDAC蛋白质的“底物捕获”突变体的开发， 的发现在之前的工作中，我们创造了大约70个无活性的HDAC 1突变体，这些突变体具有稳定表达HDAC 1的潜力。 结合底物并促进其纯化和鉴定。我们在这里提出令人兴奋的初步 结果使用捕获策略鉴定和验证了几种新型HDAC 1底物。我们进一步 本申请的目的是筛选另外的突变体以获得最佳的底物结合特性， 随后在哺乳动物细胞中鉴定HDAC 1的底物（具体目标1）。考虑到高 由于HDAC家族中的序列相似性，我们将这些研究扩展到HDAC 6，因为其 细胞质定位提示非基因调节功能（特异性目的2）。最后，为了更清楚地 在捕获实验中区分活性位点结合底物与相关蛋白质，我们将 产生HDAC 1选择性抑制剂，用作竞争性对照化合物（具体目标3）。总的来说， 这一应用的重要成果是创造了一种诱捕突变体的策略， HDAC底物的鉴定。随着第一个系统工具的可用性，以确定基板， 我们的长期目标是应用该技术来破译细胞中HDAC蛋白的底物特异性， 生物学鉴于HDAC蛋白在表观遗传学中的作用及其在细胞信号传导中可能的作用，底物 捕获突变体有可能增加我们对细胞生物学的理解， 针对HDAC蛋白的设计工作。

Summary Histone Deacetylase (HDAC) proteins are epigenetic modulators that have a clear and well studied role in gene expression regulation. HDAC proteins influence gene expression by deacetylating acetyl-lysine residues on nucleosomal histone protein substrates. Recently a wide variety of acetylated proteins have been discovered, which suggests that HDAC proteins likely deacetylate substrates in addition to histones. Through non-histone substrates, HDAC proteins may play an expanded role in cellular processes outside of gene expression, including cell signaling and communication. Unfortunately, identification of non- histone HDAC substrates has been largely serendipitous because facile methods to systematically discover in cellulo targets of HDAC deacetylation are lacking. This application outlines development of “substrate trapping” mutants of HDAC proteins for substrate discovery. In prior work, we created roughly 70 inactive HDAC1 mutants that have the potential to stably bind substrates and facilitate their purification and identification. We present here exciting preliminary results identifying and validating several novel HDAC1 substrates using the trapping strategy. Our further goals in this application are to screen additional mutants for optimal substrate binding properties, with subsequent identification of substrates of HDAC1 in mammalian cells (Specific Aim 1). Given the high sequence similarity within the HDAC family, we will extend these studies to HDAC6 because its cytoplasmic localization suggests a non-gene regulatory function (Specific Aim 2). Finally, to more clearly distinguish active site binding substrates from associated proteins in the trapping experiments, we will generate HDAC1-selective inhibitors for use as competitive control compounds (Specific Aim 3). In total, the significant outcome of this application is the creation of a trapping mutant strategy for the unbiased identification of HDAC substrates. With the availability of the first systematic tool to identify substrates, our long-term goal is to apply the technology to decipher the substrate specificity of HDAC proteins in cell biology. Given the role of HDAC proteins in epigenetics, and their possible role in cell signaling, substrate trapping mutants have the potential to augment our understanding of cell biology and embolden drug design efforts targeting HDAC proteins.