Peptide Arrays for Understanding Histone Biochemistry

描述(由申请人提供)：本提案描述了一项研究计划，旨在开发和应用多肽阵列来阐明组蛋白脱乙酰酶(HDAC)活性的特异性和反应模式。这个过程在染色质建模和基因调控中起着重要作用，它是由酶和组蛋白底物网络介导的。有关酶-底物活性的生化研究非常有限，主要是因为缺乏监测和确定酶活性模式的方法。申请人已经开发了烷硫醇酸盐在金表面的自组装单分子层(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-飞行时间质谱仪(MALDI-TOF-MS)一起使用时，该平台提供了定量分析和无标记检测，该技术被称为SAMDI(MALDI-MS自组装单分子层)。这项拟议的工作将开发一种制备多肽阵列的方法，并使用这些阵列来表征HDAC酶的底物特异性，并通过在多肽底物中进行额外的翻译后修饰来表征组蛋白活性的调节。与公共健康相关的基因表达受组蛋白脱乙酰酶和组蛋白底物网络的调控，在这些网络中，偏离酶的表达可能导致几种癌症和衰老。有关酶-底物活性的生化研究非常有限，主要是因为缺乏监测和确定酶活性模式的方法。这项拟议的工作将开发一种制备多肽阵列的方法，并使用这些阵列来表征HDAC酶的底物特异性，并通过在多肽底物中进行额外的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.