Design of a Bioluminescent Assay Platform for Quantitative Measurement of Histone Acetyltransferase Enzymatic Activity.

Autor: Swain ND; Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, 30602, U.S.A., George Zheng Y; Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, 30602, U.S.A.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2024 Nov 22, pp. e202400692. Date of Electronic Publication: 2024 Nov 22.
DOI: 10.1002/cbic.202400692
Abstrakt: Protein acetylation and acylation are widespread post-translational modifications (PTMs) in eukaryotic and prokaryotic organisms. Histone acetyltransferase (HATs) enzymes catalyze the addition of short-chain acyl moieties to lysine residues on cellular proteins. Many HAT members are found to be dysregulated in human diseases, especially oncological processes. Screening potent and selective HAT inhibitors has promising application for therapeutic innovation. A biochemical assay for quantification of HAT activity utilizing luminescent output is highly desirable to improve upon limitations associated with the classic radiometric assay formats. Here we report the design of a bioluminescent technological platform for robust and sensitive quantification of HAT activity. This platform utilizes the metabolic enzyme acetyl-CoA synthetase 1 (ACS1) for a coupled reaction with firefly luciferase to generate luminescent signal relative to the HAT-catalyzed acetylation reaction. The biochemical assay was implemented in microtiter plate format and our results showed this assay sensitively detected catalytic activity of HAT enzyme p300, accurately measured its steady-state kinetic parameters of histone acetylation and measured the inhibitory potency of HAT inhibitor. This platform demonstrated excellent robustness, reproducibility, and signal-to-background ratios, with a screening window Z'=0.79. Our new bioluminescent design provides an alternative means for HAT enzymatic activity quantitation and HAT inhibitor screening.
(© 2024 Wiley-VCH GmbH.)
Databáze: MEDLINE
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