Biochemical and in Vivo Characterization of a Small, Membrane-Permeant, Caspase-Activatable Far-Red Fluorescent Peptide for Imaging Apoptosis

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• 作者：Kristin E. Bullok ; Dustin Maxwell ; Aparna H. Kesarwala ; Seth Gammon ; Julie L. Prior ; Margaret Snow ; Sam Stanley ; David Piwnica-Worms
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：April 3, 2007
• 年：2007
• 卷：46
• 期：13
• 页码：4055 - 4065
• 全文大小：671K
• 年卷期：v.46,no.13(April 3, 2007)
• ISSN：1520-4995

文摘

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 tomonitor cell death in cells and whole animals are desired. To specifically detect apoptosis in vivo, anovel cell-permeable activatable caspase substrate, TcapQ₆₄₇, was synthesized and K_m, k_cat, and K_i valueswere biochemically characterized. Specific cleavage of TcapQ₆₄₇ by effector caspases was demonstratedusing a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ₆₄₇with a k_cat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ₆₄₇cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents orC₆-ceramide, TcapQ₆₄₇ detected apoptosis in individual- and population-based fluorescent cell assays inan effector caspase inhibitor-specific manner. Further, only background fluorescence was observed incells incubated with dTcapQ₆₄₇, a noncleavable all D-amino acid control peptide. Finally, in vivo experimentsdemonstrated the utility of TcapQ₆₄₇ to detect parasite-induced apoptosis in human colon xenograft andliver abscess mouse models. Thus, TcapQ₆₄₇ represents a sensitive, effector caspase-specific far-red "smart"probe to noninvasively monitor apoptosis in vivo.