Biochemical and in vivo characterization of a small, membrane-permeant, caspase-activatable far-red fluorescent peptide for imaging apoptosis.
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, TcapQ647, was synthesized and Km, kcat, and Ki values were biochemically characterized. Specific cleavage of TcapQ647 by effector caspases was demonstrated using a panel of purified recombinant enzyme assays. Of note, caspase 3 was shown to cleave TcapQ647 with a kcat 7-fold greater than caspase 7 and 16-fold greater than caspase 6. No evidence of TcapQ647 cleavage by initiator caspases was observed. In KB 3-1 or Jurkat cells treated with cytotoxic agents or C6-ceramide, TcapQ647 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 dTcapQ647, a noncleavable all d-amino acid control peptide. Finally, in vivo experiments demonstrated the utility of TcapQ647 to detect parasite-induced apoptosis in human colon xenograft and liver abscess mouse models. Thus, TcapQ647 represents a sensitive, effector caspase-specific far-red "smart" probe to noninvasively monitor apoptosis in vivo.
Bullok
Kristin E
KE
Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University Medical School, St. Louis, Missouri 63110, USA.
Maxwell
Dustin
D
Kesarwala
Aparna H
AH
Gammon
Seth
S
Prior
Julie L
JL
Snow
Margaret
M
Stanley
Sam
S
Piwnica-Worms
David
D
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D019169:Jurkat Cells; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D051379:Mice; D009368:Neoplasm Transplantation; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014183:Transplantation, Heterologous
D005456:Fluorescent Dyes; D010455:Peptides; C519706:TcapQ647 peptide; C067297:8-aminohexylamino cAMP; D000242:Cyclic AMP; D020169:Caspases