Chemical Approaches to Understanding Reversible Lysine Modifications

摘要 这是一个R35 NIGMS应用程序，旨在接替R37GM62437，并涉及到开发和 应用化学方法加强我们对组蛋白翻译后修饰的理解 (PTM)赖氨酸残基和附着和移除它们的酶(“写入器”和“擦除器”)。我们的实验室有一个 组蛋白修饰的化学生物学技术创新记录，包括二价类似物设计 和蛋白质半合成。我们的二价化合物方法已经导致了有效和 P300和CBP(p300/CBP)乙酰转移酶选择性抑制剂与LSD1-HDAC1-COREST(LHC) 去甲基酶/脱乙酰酶基因沉默复合体。我们最新一代的化合物，用于p300/CBP的A485和 用于LHC的Corin已经成为分析这些组蛋白的非常有用和受欢迎的药理学探针- 机械性和临床前治疗实验中的修饰酶。我们的蛋白质半合成方法 包括表达的蛋白质连接，工程索尔特酶催化的组蛋白生产和半胱氨酸修饰以 引入酰基-赖氨酸模拟物已被证明是提供位点特异性修饰蛋白质的有效方法。 近年来，我们已经展示了特定的组蛋白修饰如何影响核小体的稳定性和 对包括脱乙酰酶、脱甲基酶和脱泛素酶亚型在内的擦除酶的敏感性。值得注意的是，我们 我发现了一种新的组蛋白标记(PTM)串扰-组蛋白H3的明显把关功能 Lys14位乙酰化在阻止H3甲基Lys4的LSD1去甲基化中的作用在我们下一阶段的研究中 计划中，我们将开发和应用新的化学方法来更广泛地了解组蛋白的生物学 使用蛋白质半合成、基因编辑、结构方法和“剪切粘贴”质量来标记相声 SPEC蛋白质组学。我们将探索酶促核小体相互作用的结构和功能特征 LSD1、PRC1、SAGA、HDAC1和SIRT6复合体的背景部分是通过将化学弹头结合到 设计好的核小体。此外，我们将应用一种新设计的索尔特酶版本来分离组蛋白H3 从细胞染色质的尾巴到不同细胞类型的PTM的定量读出模式以及对 药理药剂采用串联质量标签质谱仪。在完成这项研究后 努力，我们的发现将扩大关于组蛋白Lys修饰是如何“写入”和“擦除”以及 特定的PTM模式如何调控基因表达和细胞命运。此外，这些研究应该为 为对抗各种疾病的表观遗传失调的新治疗策略开辟了道路。这项研究 该计划还将使培训对蛋白质感兴趣的下一代生化研究人员成为可能 科学，并以对多样性和包容性的承诺为指导。

Abstract This is an R35 NIGMS application that is meant to succeed R37GM62437 and concerns the development and application of chemical approaches to enhance our understanding of histone post-translational modifications (PTMs) on Lys residues and the enzymes that attach and remove them (“writers” and “erasers”). Our lab has a record of technical innovation in the chemical biology of histone modifications including bivalent analog design and protein semisynthesis. Our bivalent compound approaches have led to the generation of potent and selective inhibitors of p300 and CBP (p300/CBP) acetyltransferases and the LSD1-HDAC1-CoREST (LHC) demethylase/ deacetylase gene silencing complex. Our latest-generation compounds, A485 for p300/CBP and corin for LHC have become very useful and popular pharmacological probes for analyzing these histone- modifying enzymes in mechanistic and preclinical therapeutic experiments. Our protein semisynthetic methods including expressed protein ligation, engineered sortase-catalyzed histone production, and Cys modification to introduce acyl-Lys mimics have proven to be efficient approaches to furnish site-specifically modified proteins. In recent years we have shown how particular histone modifications influence nucleosome stability and susceptibility to eraser enzymes including deacetylase, demethylase, and deubiquitinase isoforms. Notably, we have discovered a new case of histone mark (PTM) crosstalk—an apparent gatekeeper function for histone H3 acetylation at Lys14 in blocking LSD1 demethylation of H3 methyl-Lys4. In the next phase of our research program, we will develop and apply new chemical methods to more broadly understand the biology of histone mark crosstalk using protein semisynthesis, gene editing, structural approaches, and “cut and paste” mass spec proteomics. We will explore structural and functional features of enzymatic nucleosome interactions in the context of LSD1, PRC1, SAGA, HDAC1, and Sirt6 complexes in part by incorporating chemical warheads into designer nucleosomes. In addition, we will apply a newly engineered version of sortase to isolate histone H3 tails from cellular chromatin to quantitatively readout patterns of PTMs in different cell types and in response to pharmacological agents by employing tandem mass tag mass spectrometry. Upon completion of this research effort, our findings will broaden the knowledge of how histone Lys modifications are “written” and “erased” and how specific PTM patterns regulate gene expression and cell fate. Moreover, these studies should pave the way for new therapeutic strategies to combat epigenetic dysregulation in various diseases. This research program will also enable the training of the next generation of biochemical investigators interested in protein science and guided by a commitment to diversity and inclusion.