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（由适用提供）：表观遗传学的研究导致发现了大型多蛋白质复合物，这些复合物催化了组蛋白修饰及其去除，影响核渗透压的重塑，从而促进或干扰转录对基因的转录访问。其中之一，即反射性的COREST复合物，包括LSD1组蛋白脱甲基酶，HDAC1（组蛋白脱乙酰基酶1）和脚手架蛋白Corest1。 Corest综合体尚未以完整形式的生化特征化。该项目的目的是为Corest综合体提供更深入的生化理解。通过对corest复合物的动力学分析，并通过翻译后修饰的肽底物对蛋白质蛋白质相互作用的调节作用揭示了交叉对话。我们假设COREST复合物将更有效地处理多重修饰的组蛋白尾部，即LSD1和HDAC1酶自行起作用。 AIM 1将通过评估复合物在翻译后修饰的肽底物上的酶促活性来关注底物特异性。 AIM 2将通过分析可以停靠抑制剂部分的混合底物抑制剂辣椒来探索主动部位协调 HDAC1或LSD1的活性位点以及伴侣酶中的底物部分。此外，通过改变同一胡椒的这两组之间的距离，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促特异性中的作用。在AIM 3中，我们计划将这些研究扩展到具有特定PTM的工程设计的更加相关的核病组底物。这些实验可以揭示Corest复合物与这种更自然的底物形式的特殊特征。基因表达调节很少涉及单个酶催化修饰，并且整体上研究完整的复合物可能会更准确地了解其功能。此外，通过表观遗传机制对基因表达进行沉默是癌症和其他疾病的标志，该实验室的大量工作和其他疾病一直集中在发现针对LSD1和HDACS的新型抑制剂。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节的基本理解，而且可以为改善治疗铺平道路。