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.
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.
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