Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.
Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.
Department of Biology and Chemistry, City University of Hong Kong , 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.|Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong , Shenzhen 518057, People's Republic of China.
Yusheng
Y
Xie
College of Biotechnology and Bioengineering, Zhejiang University of Technology , Hangzhou 310014, People's Republic of China.
Jingyan
J
Ge
Department of Biology and Chemistry, City University of Hong Kong , 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.|Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong , Shenzhen 518057, People's Republic of China.
Haipeng
H
Lei
Department of Chemistry, National University of Singapore , Singapore 119077.
Bo
B
Peng
Department of Biology and Chemistry, City University of Hong Kong , 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.|Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong , Shenzhen 518057, People's Republic of China.
Huatang
H
Zhang
Department of Chemistry, National University of Singapore , Singapore 119077.
Danyang
D
Wang
Department of Chemistry, National University of Singapore , Singapore 119077.
Sijun
S
Pan
Department of Biology and Chemistry, City University of Hong Kong , 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.|Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong , Shenzhen 518057, People's Republic of China.
Ganchao
G
Chen
Department of Physiology, University of Hong Kong , Pok Fu Lam, Hong Kong, People's Republic of China.
Lanfang
L
Chen
Department of Physiology, University of Hong Kong , Pok Fu Lam, Hong Kong, People's Republic of China.
Yi
Y
Wang
Department of Physiology, University of Hong Kong , Pok Fu Lam, Hong Kong, People's Republic of China.
Quan
Q
Hao
Department of Chemistry, National University of Singapore , Singapore 119077.
Shao Q
SQ
Yao
Department of Biology and Chemistry, City University of Hong Kong , 83 Tat Chee Avenue, Kowloon, Hong Kong, People's Republic of China.|Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong , Shenzhen 518057, People's Republic of China.
Hongyan
H
Sun
D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006655:Histone Deacetylases / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D040901:Proteomics
D005456:Fluorescent Dyes; D006655:Histone Deacetylases