Determining histone deacetylase 8 substrates using non-natural amino acids

使用非天然氨基酸测定组蛋白脱乙酰酶 8 底物

• 批准号: 9128444
• 负责人: Jeffrey Lopez
• 金额: $ 3.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9128444
• 关键词: Acetylation; Active Sites; Affect; Amino Acids; Binding; Biological Assay; Biological Models; Biology; Breast; Catabolism; Cell physiology; Cells; Collaborations; Colon; Colon Carcinoma; Coupled; DNA; Deacetylation; Development; Diabetes Mellitus; Disease; Enzymes; Excision; FDA approved; Family; Gene Expression; HDAC8 gene; Hereditary Disease; Histones; Homeostasis; In Vitro; Individual; Isoenzymes; Kinetics; Knock-out; Lead; Life; Link; Liquid Chromatography; Location; Lysine; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Mass Spectrum Analysis; Measures; Mental Retardation; Metals; Methods; Modeling; Neurodegenerative Disorders; Nucleosomes; Pathway interactions; Peptides; Play; Positioning Attribute; Post-Translational Protein Processing; Prostate; Protein-Protein Interaction Map; Proteins; Proteome; Research; Role; Side; Site; Stomach; Substrate Specificity; Surface; Syndrome; T-Cell Lymphoma; Techniques; Zolinza; abstracting; cancer therapy; crosslink; drug development; histone acetyltransferase; in vivo; inhibitor/antagonist; insight; malformation; malignant breast neoplasm; malignant stomach neoplasm; mutant; peptidomimetics; protein function; protein protein interaction; public health relevance; research study; tandem mass spectrometry; tool

 DESCRIPTION (provided by applicant): Lysine acetylation is a reversible post-translational modification (PTM) that affects protein function including stability, localization, and interaction with other proteins and DNA. Acetylation levels are regulated by the relative activity of histone N-acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylated lysine side chains were first identified in histones, where they are proposed to regulate gene expression altering the accessibility to the DNA in nucleosomes. However, over 9000 acetylation sites have been identified in the mammalian proteome and these proteins have been implicated in a variety of cellular processes ranging from catabolism to cell homeostasis. Aberrant acetylation has been implicated in the development of T-cell lymphoma and several cancers, including gastric, prostate, colon, and breast cancers. Defining the substrate pool of each HDAC isozyme is critical for targeted drug development and understanding which pathways are affected by inhibition of each enzyme. HDAC8, the focus of this proposal, has been characterized and studied extensively in vitro, making HDAC8 an ideal model system for developing tools to identify HDAC interaction partners. HDAC8 activity toward acetylated peptides mimicking potential substrates has been measured; however, there are very few validated in vivo substrates. Recent mass spectrometry analyses have yielded a putative in vivo HDAC-protein interaction map, but it is not clear whether these interacting proteins are substrates or binding partners. The research in this proposal will bridge the gap between in vitro HDAC8 characterization and in vivo HDAC8 function and provide insight into in vivo HDAC8 substrate and binding partner interactions. I propose to (1) incorporate photo-active non-natural amino acids at different positions of HDAC8; (2) trap and analyze short and long-lived interacting partners and substrates with HDAC8 through mass spectrometry (in collaboration with Prof. B. Martin) and (3) validate substrates using an in vitro enzyme coupled assay. These experiments will facilitate identification of HDAC8 substrates. Development and application of these methods to other HDACs will lead to a better understanding of the HDAC interactome and development of HDAC-protein specific inhibitors.

 描述（由申请人提供）：赖氨酸乙酰化是一种可逆的翻译后修饰（PTM），它影响蛋白质功能，包括稳定性、定位以及与其他蛋白质和DNA的相互作用。乙酰化水平受组蛋白 N-乙酰转移酶 (HAT) 和组蛋白脱乙酰酶 (HDAC) 的相对活性调节。乙酰化赖氨酸侧链首先在组蛋白中被发现，它们被认为可以调节基因表达，改变核小体中 DNA 的可及性。然而，在哺乳动物蛋白质组中已鉴定出超过 9000 个乙酰化位点，并且这些蛋白质与从分解代谢到细胞稳态等多种细胞过程有关。异常乙酰化与 T 细胞淋巴瘤和多种癌症（包括胃癌、前列腺癌、结肠癌和乳腺癌）的发展有关。定义每种 HDAC 同工酶的底物库对于靶向药物开发和了解哪些途径受到每种酶抑制的影响至关重要。 HDAC8 是本提案的重点，已在体外进行了广泛的表征和研究，使 HDAC8 成为开发识别 HDAC 相互作用伙伴的工具的理想模型系统。已经测量了 HDAC8 对模拟潜在底物的乙酰化肽的活性；然而，经过验证的体内底物很少。最近的质谱分析已经得出了假定的体内 HDAC-蛋白质相互作用图，但尚不清楚这些相互作用的蛋白质是底物还是结合配偶体。本提案中的研究将弥合体外 HDAC8 表征和体内 HDAC8 功能之间的差距，并提供对体内 HDAC8 底物和结合伴侣相互作用的深入了解。我建议（1）在HDAC8的不同位置掺入光活性非天然氨基酸； (2) 通过质谱法（与 B. Martin 教授合作）用 HDAC8 捕获和分析短寿命和长寿命的相互作用伙伴和底物，以及 (3) 使用体外酶偶联测定验证底物。这些实验将有助于 HDAC8 底物的鉴定。这些方法的开发和应用到其他 HDAC 将有助于更好地了解 HDAC 相互作用组和 HDAC 蛋白特异性抑制剂的开发。