Peptide Arrays for Understanding Histone Biochemistry

描述（由申请人提供）：该提案描述了一个研究项目，旨在开发和应用肽阵列来阐明组蛋白去乙酰化酶（HDAC）活性的特异性和反应性模式。这一过程在染色质建模和基因调控中起着重要作用，是由酶和组蛋白底物网络介导的。相关酶-底物活性的生化研究非常有限，主要是由于缺乏监测和定义酶活性模式的方法。申请人开发了金上烷硫酸酯的自组装单层（sam）作为生物芯片的平台，当与传统的基质辅助激光解吸电离飞行时间质谱（MALDI-ToF-MS）一起使用时，提供定量分析和无标记检测，这种技术称为SAMDI （MALDI-MS的自组装单层）。这项工作将开发一种制备肽阵列的途径，并使用这些阵列来表征HDAC酶的底物特异性，并通过肽底物中额外的翻译后修饰来表征组蛋白活性的调节。基因表达受组蛋白去乙酰化酶和组蛋白底物网络的调控，其中的偏差可导致几种癌症和衰老。相关酶-底物活性的生化研究非常有限，主要是由于缺乏监测和定义酶活性模式的方法。这项工作将开发一种制备肽阵列的途径，并使用这些阵列来表征HDAC酶的底物特异性，并通过肽底物中的额外翻译后修饰来表征组蛋白活性的调节。

DESCRIPTION (provided by applicant): This proposal describes a research program that aims to develop and apply peptide arrays to elucidate specificity and reactivity patterns of histone deacetylase (HDAC) activity. This process, which plays a fundamental role in chromatin modeling and gene regulation, is mediated by a network of enzymes and histone substrates. Biochemical studies of the relevant enzyme-substrate activities are very limited, owing primarily to a lack of methods to monitor and define patterns of enzyme activity. The applicants have developed self-assembled monolayers (SAMs) of alkanethiolates on gold as a platform for biochips that provide for quantitative assays and label-free detection when used with conventional matrix assisted laser desorption ionization -time of flight mass spectrometry (MALDI-ToF-MS), a technique termed SAMDI (self-assembled monolayers for MALDI-MS). The proposed work will develop a route to prepare peptide arrays and use these arrays to characterize the substrate specificities of HDAC enzymes and characterize the regulation of histone activity by additional post-translational modifications in the peptide substrate. PUBLIC HEALTH RELEVANCE Gene expression is regulated by a network of histone deacetylase enzymes and histone substrates in which deviations can lead towards several cancers and aging. Biochemical studies of the relevant enzyme-substrate activities are very limited, owing primarily to a lack of methods to monitor and define patterns of enzyme activity. The proposed work will develop a route to prepare peptide arrays and use these arrays to characterize the substrate specificities of HDAC enzymes and characterize the regulation of histone activity by additional posttranslational modifications in the peptide substrate.