组蛋白乙酰转移酶
乙酰转移酶
乙酰转移酶
组蛋白乙酰转移酶
乙酰化
生物化学
乙酰辅酶A
酰基辅酶A
化学
表观遗传学
组蛋白
荧光团
染色质
酶
代谢物
生物
基因
荧光
物理
量子力学
作者
Whitney K. Lieberman,Isita Jhulki,Jordan Meier
标识
DOI:10.1096/fasebj.2022.36.s1.r5412
摘要
CoA metabolites play a critical role in the epigenetic regulation of gene expression. Of particular significance is acetyl-CoA, a primary metabolite used by histone acetyltransferases to modify histone tails and therefore control chromatin state. In cancer cells, the dysregulation of acetyl-CoA can lead to the aberrant expression of genes controlling cell growth. One challenge in studying these acetyl-CoA dependent functions is the lack of optical methods to detect CoA metabolites. Towards that goal, we report a bioluminescence energy transfer (BRET)-based approach to detect acetyl-CoA. Our strategy is inspired by recent biophysical and chemoproteomic analyses of human acetyltransferases, which revealed that members of the N-terminal (NAA) acetyltransferase family preferentially bind acetyl-CoA over CoA. To explore whether we could develop an acetyl-CoA selective ligand-displacement assay, we first recombinantly expressed and purified a NAA-Nanoluc fusion protein. Next, we demonstrated its ability to generate a BRET signal when incubated with CoA-fluorophore conjugates. Finally, we evaluated whether NAA-Nanoluc BRET was altered by the addition of CoA metabolites, which revealed a direct relationship between the affinity of the CoA and optical signal. We report the first tunable indicator displacement assay for CoA metabolites and provide a new approach for studying the engagement of acetyltransferase enzymes by small molecule inhibitors.
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