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Abstract 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. 

Abstract Protein posttranslational modification (PTM) is a biochemical mechanism benefitting cellular adaptation to dynamic intracellular and environmental conditions. Recently, several acylation marks have been identified as new protein PTMs occurring on specific lysine residues in mammalian cells: isobutyrylation, methacrylation, benzoylation, isonicotinylation, and lactylation. These acylation marks were initially discovered to occur on nucleosomal histones, but they potentially occur as prevalent biomarkers on non‐histone proteins as well. The existence of these PTMs is a downstream consequence of metabolism and demonstrates the intimate crosstalk between active cellular metabolites and regulation of protein function. Emerging evidence indicates that these acylation marks on histones affect DNA transcription and are functionally distinct from the well‐studied lysine acetylation. Herein, we discuss enzymatic regulation and metabolic etiology of these acylations, 'reader' proteins that recognize different acylations, and their possible physiological and pathological functions. Several of these modifications correlate with other well‐studied acylations and fine‐tune the regulation of gene expression. Overall, findings of these acylation marks reveal new molecular links between metabolism and epigenetics and open up many questions for future investigation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.