An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging. Bioconjug Chem 2009 Apr;20(4):702-9
Date
04/01/2009Pubmed ID
19331388Pubmed Central ID
PMC2672423DOI
10.1021/bc800516nScopus ID
2-s2.0-65249177729 (requires institutional sign-in at Scopus site) 108 CitationsAbstract
Apoptosis is required for normal cellular homeostasis, and deregulation of the apoptotic process is implicated in various diseases. Previously, we developed a cell-penetrating near-infrared fluorescence (NIRF) probe based on an activatable strategy to detect apoptosis-associated caspase activity in vivo. This probe consisted of a cell-penetrating Tat peptide conjugated to an effector recognition sequence (DEVD) that was flanked by a fluorophore-quencher pair (Alexa Fluor 647 and QSY 21). Once exposed to effector caspases, the recognition sequence was cleaved, resulting in separation of the fluorophore-quencher pair and signal generation. Herein, we present biochemical analysis of a second generation probe, KcapQ, with a modified cell-penetrating peptide sequence (KKKRKV). This modification resulted in a probe that was more sensitive to effector caspase enzymes, displayed an unexpectedly higher quenching efficiency between the fluorophore-quencher pair, and was potentially less toxic to cells. Assays using recombinant caspase enzymes revealed that the probe was specific for effector caspases (caspase 3 > 7 > 6). Analysis of apoptosis in HeLa cells treated with doxorubicin showed probe activation specific to apoptotic cells. In a rat model of retinal neuronal excitotoxicity, intravitreal injection of N-methyl-d-aspartate (NMDA) induced apoptosis of retinal ganglion cells (RGCs). Eyecup and retinal flat-mount images of NMDA-pretreated animals injected intravitreally with KcapQ using a clinically applicable protocol showed specific and widely distributed cell-associated fluorescence signals compared to untreated control animals. Fluorescence microscopy images of vertical retinal sections from NMDA-pretreated animals confirmed that activated probe was predominantly localized to RGCs and colocalized with TUNEL labeling. Thus, KcapQ represents an improved effector caspase-activatable NIRF probe for enhanced noninvasive analysis of apoptosis in whole cells and live animals.
Author List
Maxwell D, Chang Q, Zhang X, Barnett EM, Piwnica-Worms DAuthor
Edward M. Barnett MD, PhD Professor in the Ophthalmology and Visual Sciences department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Apoptosis
Caspase Inhibitors
Caspases
Cell Survival
Cells
Enzyme Activation
Enzyme Inhibitors
Fluorescence
Fluorescent Dyes
HeLa Cells
Humans
Infrared Rays
Kinetics
Oligopeptides
Rats
Retinal Ganglion Cells
Spectrophotometry, Ultraviolet
