Chemical Approaches to Understanding Reversible Lysine Modifications

理解可逆赖氨酸修饰的化学方法

• 批准号: 10621611
• 负责人: PHILIP A COLE
• 金额: $ 44.75万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10621611
• 关键词: Acetylation; Acetyltransferase; Biochemical; Biology; Cells; Chemicals; Chromatin; Complex; Deacetylase; Development; Diagnosis; Disease; EP300 gene; Engineering; Enzymes; Epigenetic Process; Gatekeeping; Gene Expression; Gene Expression Regulation; Gene Silencing; Generations; Genes; HDAC1 gene; Histone H3; Histones; Human; KDM1A gene; Knowledge; Ligation; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modification; Molecular; National Institute of General Medical Sciences; Nucleosomes; PRC1 Protein; Paste substance; Pattern; Phase; Post-Translational Protein Processing; Predisposition; Production; Protein Isoforms; Proteins; Proteomics; Research; Research Personnel; SAGA; Science; Site; Tail; Therapeutic; Training; Writing; analog; cell type; combat; demethylation; design; diversity and inclusion; experimental study; histone modification; inhibitor; innovation; interest; next generation; novel therapeutic intervention; pharmacologic; pre-clinical; programs; response; sortase; ubiquitin isopeptidase

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.

摘要 这是一个R35 NIGMS应用程序，旨在接替R37 GM 62437，涉及开发和 应用化学方法提高我们对组蛋白翻译后修饰的理解 （PTM）作用于赖氨酸残基以及附着和去除它们的酶（“写入器”和“橡皮擦”）。我们的实验室有一个 在组蛋白修饰的化学生物学方面的技术创新记录，包括二价类似物设计 蛋白质半合成我们的二价化合物方法已经导致产生了有效的， p300和CBP（p300/CBP）乙酰转移酶和LSD 1-HDAC 1-CoREST（LHC）的选择性抑制剂 脱甲基酶/脱乙酰酶基因沉默复合物。我们最新一代的化合物，用于p300/CBP的A485和 corin的LHC已经成为非常有用和流行的药理学探针，用于分析这些组蛋白， 修饰酶在机械和临床前治疗实验中的应用。我们的蛋白质半合成方法 包括表达的蛋白质连接、工程化分选酶催化的组蛋白产生和Cys修饰， 已证明引入酰基-赖氨酸模拟物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， 对擦除酶包括脱乙酰酶、脱甲基酶和脱泛素酶同种型的易感性。值得注意的是， 我发现了一个新的组蛋白标记（PTM）串扰的案例--组蛋白H3的一个明显的看门人功能 在Lys 14处的乙酰化阻断H3甲基-Lys 4的LSD 1去甲基化。在我们研究的下一阶段 计划，我们将开发和应用新的化学方法，以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“剪切和粘贴”质量的标记串扰 spec蛋白质组学我们将探讨酶核小体相互作用的结构和功能特征， LSD 1，PRC 1，佐贺，HDAC 1和Sirt 6复合物的背景部分通过将化学弹头并入 设计核小体。此外，我们将应用一种新设计的分选酶来分离组蛋白H3。 从细胞染色质的尾部定量读出不同细胞类型中PTM的模式， 通过使用串联质谱标签质谱法测定药理学药剂。在完成这项研究后， 通过努力，我们的发现将拓宽对组蛋白赖氨酸修饰如何被“写入”和“擦除”的认识， 特定PTM模式如何调节基因表达和细胞命运。此外，这些研究应该为 新的治疗策略，以打击各种疾病的表观遗传失调的方式。本研究 该计划还将培训对蛋白质感兴趣的下一代生化研究人员 科学，并以多样性和包容性的承诺为指导。