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