HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking
| Autor: | Brent R. Martin, Jeffrey E. Lopez, Jaimeen D. Majmudar, Carol A. Fierke, Sarah E. Haynes |
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| Rok vydání: | 2017 |
| Předmět: |
Cell Extracts
Proteomics 0301 basic medicine Phenylalanine Lysine Biochemistry Isozyme Article Histone Deacetylases Catalysis Substrate Specificity Benzophenones 03 medical and health sciences Colloid and Surface Chemistry Catalytic Domain Humans chemistry.chemical_classification biology Chemistry Reproducibility of Results Active site Acetylation HDAC8 General Chemistry Photochemical Processes Amino acid Repressor Proteins Cross-Linking Reagents 030104 developmental biology Enzyme biology.protein Histone deacetylase |
| Zdroj: | Journal of the American Chemical Society. 139:16222-16227 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/jacs.7b07603 |
| Popis: | The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues, however, the specificity and substrate profile of each enzyme remains largely unknown. Due to transient enzyme-substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold-changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photo-reactive, non-natural amino acid, p-benzoyl-L-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical-crosslinking approach identified novel HDAC8 specific substrates with greater catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery. |
| Databáze: | OpenAIRE |
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