TY - JOUR
T1 - Comparative studies of thiol-sensitive fluorogenic probes for HAT assays
AU - Gao, Tielong
AU - Yang, Chao
AU - Zheng, Yujun George
N1 - Funding Information:
Acknowledgments This work was partially supported by AHA grant 12GRNT12070056 and NIH grant R01GM086717.
PY - 2013/2
Y1 - 2013/2
N2 - Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compounds for the detection of the enzymatic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these molecules gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compounds for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaption to high-throughput screening of small molecule inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.
AB - Histone acetyltransferases (HATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. Recent studies showed that acetylation is widely distributed among cellular proteins, suggestive of diverse functions of HATs in cellular pathways. Nevertheless, currently available assays for HAT activity study are still quite limited. Here, we evaluated a series of thiol-sensitive fluorogenic compounds for the detection of the enzymatic activities of different HAT proteins. Upon conjugation to the thiol group of HSCoA, these molecules gain enhanced quantum yields and strong fluorescence, permitting facile quantitation of HAT activities. We investigated and compared the assay performances of these fluorogenic compounds for their capability as HAT activity reporters, including kinetics of reaction with HSCoA, influence on HAT activity, and fluorescence amplification factors. Our data suggest that CPM and coumarin maleic acid ester are excellent HAT probes owing to their fast reaction kinetics and dramatic fluorescence enhancement during the HAT reaction. Further, the microtiter plate measurements show that this fluorescent approach is robust and well suited for adaption to high-throughput screening of small molecule inhibitors of HATs, highlighting the value of this assay strategy in new drug discovery.
KW - Chromatin modification
KW - Epigenetics
KW - Fluorescent probe
KW - HAT
KW - Histone acetyltransferase
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U2 - 10.1007/s00216-012-6522-5
DO - 10.1007/s00216-012-6522-5
M3 - Article
C2 - 23138472
AN - SCOPUS:84873748672
SN - 1618-2642
VL - 405
SP - 1361
EP - 1371
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 4
ER -