Investigating substrate specificity and crosstalk amongst the histone modifying complex CoREST components through peptide and inhibitor analysis

通过肽和抑制剂分析研究组蛋白修饰复杂 CoREST 成分之间的底物特异性和串扰

• 批准号: 9126833
• 负责人: Dawn Hayward
• 金额: $ 4.36万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-16 至 2019-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9126833
• 关键词: Acetylation; Active Sites; Affect; Biochemical; Biological; Cells; Collaborations; Complex; Data; Deacetylase; Deacetylation; Disease; Docking; Engineering; Enzymes; Epigenetic Process; Excision; Gene Expression; Gene Expression Regulation; Genes; Geometry; Goals; HDAC1 gene; Histone H3; Histones; Hybrids; K-18 conjugate; Kinetics; Laboratories; Length; Malignant Neoplasms; Mammalian Cell; Measures; Methylation; Modification; N-terminal; Nucleosomes; Peptides; Peptidyltransferase; Plasmids; Positioning Attribute; Process; Recombinants; Reporting; Research; Role; Scaffolding Protein; Series; Site; Specificity; Substrate Specificity; System; Tail; Therapeutic; Time; Work; analog; demethylation; histone demethylase; histone modification; hydroxamate; improved; inhibitor/antagonist; insight; novel; novel therapeutic intervention; protein complex; protein protein interaction; public health relevance; reconstitution; research study; sortase; synthetic peptide

 DESCRIPTION (provided by applicant): Research in epigenetics has led to the discovery of large multi-protein complexes that catalyze histone modifications and their removal, affecting nucleosomal remodeling that either promotes or interferes with transcriptional access to genes. One of these, the repressive CoREST complex, includes the enzymes LSD1 histone demethylase, HDAC1 (histone deacetylase 1) and the scaffolding protein CoREST1. The CoREST complex has not yet been well-characterized biochemically in its intact form. The goal of this project is to provide a deeper biochemical understanding of the CoREST complex. Through kinetic analysis of the CoREST complex with post-translationally modified peptide substrates, we expect to reveal cross-talk among histone modifications and the regulatory effects of protein-protein interactions. We hypothesize that the CoREST complex will process multiply-modified histone tails more efficiently that the LSD1 and HDAC1 enzymes acting on their own. Aim 1 will focus on substrate specificity by evaluating the enzymatic activities of the complex on post-translationally modified peptide substrates. Aim 2 will explore active site coordination by analyzing hybrid substrate-inhibitor peptides that can dock the inhibitor moiety in the active site of HDAC1 or LSD1 and the substrate moiety in the partner enzyme. Furthermore, by varying the distance between these two groups on the same peptide, we hope to determine complex orientation, geometry and the role of protein-protein interactions in enzymatic specificity. In Aim 3, we plan to extend these studies to the more physiologically relevant nucleosome substrates engineered to have specific PTMs. These experiments can reveal whether there are special features of the CoREST complex interactions with this more natural substrate form. Gene expression regulation rarely involves single enzymes catalyzing modifications and investigating intact complexes as a whole is likely to give more accurate insight into their functions. In addition, silencing of gene expression through epigenetic mechanisms is a hallmark of cancer and other diseases, and much work from this laboratory and others has been focused on discovering novel inhibitors for LSD1 and HDACs. Therefore, studies on the biological mechanisms of these complexes can not only enhance our fundamental understanding of gene regulation but it can pave the way toward improved therapeutics.

 描述（由申请人提供）：表观遗传学研究发现了催化组蛋白修饰及其去除的大型多蛋白复合物，影响核小体重塑，从而促进或干扰基因的转录访问。其中之一，抑制性CoREST复合物，包括酶LSD 1组蛋白脱甲基酶，HDAC 1（组蛋白脱乙酰酶1）和支架蛋白CoREST 1。CoREST复合物尚未以其完整形式进行良好的生物化学表征。该项目的目标是提供对CorREST复合体的更深入的生物化学理解。通过动力学分析与后修饰的肽底物的CoREST复合物，我们希望揭示组蛋白修饰和蛋白质-蛋白质相互作用的调节作用之间的串扰。我们假设CoREST复合物将比LSD 1和HDAC 1酶更有效地处理多重修饰的组蛋白尾部。目的1将通过评价复合物对后修饰肽底物的酶活性来关注底物特异性。目的2将通过分析杂合底物-抑制剂肽来探索活性位点的配位， HDAC 1或LSD 1的活性位点和伴侣酶中的底物部分。此外，通过改变同一肽上这两个基团之间的距离，我们希望确定复杂的方向，几何形状和蛋白质-蛋白质相互作用在酶特异性中的作用。在目标3中，我们计划将这些研究扩展到生理学上更相关的核小体底物，这些核小体底物被工程化以具有特定的PTM。这些实验可以揭示CoREST复合物与这种更天然的底物形式相互作用的特殊特征。基因表达调控很少涉及催化修饰的单一酶，将完整的复合物作为一个整体进行研究可能会更准确地了解它们的功能。此外，通过表观遗传机制沉默基因表达是癌症和其他疾病的标志，该实验室和其他实验室的许多工作都集中在发现LSD 1和HDAC的新型抑制剂上。因此，对这些复合物的生物学机制的研究不仅可以增强我们对基因调控的基本理解，而且可以为改进治疗方法铺平道路。