期刊
BIOCHEMISTRY
卷 46, 期 13, 页码 4055-4065出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi061959n
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资金
- NCI NIH HHS [R01 CA82841, P50 CA94056] Funding Source: Medline
- NIAID NIH HHS [R01 AI30084] Funding Source: Medline
Apoptosis is an important process involved in diverse developmental pathways, homeostasis, and response to therapy for a variety of diseases. Thus, noninvasive methods to study regulation and to monitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, a novel cell-permeable activatable caspase substrate, TcapQ(647), was synthesized and K-m, k(cat), and K-i values were biochemically characterized. Specific cleavage of TcapQ(647) by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ(647) with a k(cat) 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ(647) cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C-6-ceramide, TcapQ(647) detected apoptosis in individual- and population-based fluorescent cell assays in an effector caspase inhibitor-specific manner. Further, only background fluorescence was observed in cells incubated with dTcapQ(647), a noncleavable all D-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ(647) to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ(647) represents a sensitive, effector caspase-specific far-red smart probe to noninvasively monitor apoptosis in vivo.
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