Identification of Histone Deacetylase Substrates using Trapping Mutants

使用捕获突变体鉴定组蛋白脱乙酰酶底物

• 批准号: 9355222
• 负责人: Mary Kay H Pflum
• 金额: $ 26.22万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-20 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9355222
• 关键词: Acetylation; Active Sites; Address; Area; Binding; Binding Sites; Biological; Biology; Catalytic Domain; Cell Communication; Cell Line; Cell physiology; Cells; Cellular biology; Clinical; Cytoplasm; Data; Deacetylation; Development; Drug Design; Enzymes; Epigenetic Process; Event; Excision; Family; Family member; Future; Gene Expression; Gene Expression Regulation; Goals; HDAC1 gene; HDAC6 gene; Histone Deacetylase; Histone Deacetylase Inhibitor; Histones; In Vitro; Lysine; Mammalian Cell; Mediating; Methods; Monitor; Outcome; Pharmaceutical Preparations; Play; Property; Protein Isoforms; Proteins; Reaction; Research; Role; Signal Transduction; Substrate Specificity; Technology; Translational Research; Tubulin; Work; experimental study; inhibitor/antagonist; innovation; insight; mutant; new technology; novel; tool; validation studies

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.

摘要 组蛋白脱乙酰酶(HDAC)蛋白是表观遗传调节剂，具有明确的和被广泛研究的作用 在基因表达调控中。HDAC蛋白通过去乙酰化赖氨酸影响基因表达 核小体组蛋白底物上的残留物。最近，各种各样的乙酰化蛋白质已经 这表明，除了组蛋白外，HDAC蛋白还可能对底物进行脱乙酰化。 通过非组蛋白底物，HDAC蛋白可能在细胞外的过程中发挥更大的作用 基因表达，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，因为简单的方法系统地 在细胞内发现缺乏HDAC脱乙酰靶。 本应用概述了HDAC蛋白底物“底物捕捉”突变体的研究进展 发现号。在之前的工作中，我们创造了大约70个不活跃的HDAC1突变体，它们有可能稳定地 结合底物并促进其提纯和鉴定。我们在这里呈现激动人心的预赛 结果利用捕获策略鉴定和验证了几种新的HDAC1底物。我们的进一步 本申请的目标是筛选更多的突变体，以获得最佳的底物结合性能， 哺乳动物细胞中HDAC1底物的后续鉴定(特定目标1)。考虑到高价 在HDAC家族内的序列相似性，我们将把这些研究扩展到HDAC6，因为它 细胞质定位提示非基因调控功能(特异性目标2)。最后，为了更清楚地 在捕获实验中区分活性结合底物和相关蛋白质，我们将 产生HDAC1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， 这个应用的重要结果是为不偏不倚的人创建了诱捕突变策略 HDAC底物的识别。随着识别底物的第一个系统工具的可用， 我们的长期目标是应用这项技术来破译细胞中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，以及它们在细胞信号、底物中的可能作用 诱捕突变体有可能增加我们对细胞生物学的理解，并鼓励药物 针对HDAC蛋白的设计努力。