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Bioluminescence resonance energy transfer‐based detection of acetyl‐CoA
Author(s) -
Lieberman Whitney,
Jhulki Isita,
Meier Jordan
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r5412
Subject(s) - histone acetyltransferases , acetyltransferase , acetyltransferases , histone acetyltransferase , acetylation , biochemistry , acetyl coa , acyl coa , chemistry , epigenetics , histone , fluorophore , chromatin , enzyme , metabolite , biology , gene , fluorescence , physics , quantum mechanics
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.