An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging.
An improved cell-penetrating, caspase-activatable, near-infrared fluorescent peptide for apoptosis imaging.
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DOI：
10.1021/bc800516n
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发表时间：
2009-04
影响因子：
4.7
通讯作者：
Piwnica-Worms, David
中科院分区：
文献类型：
--
作者：
Maxwell, Dustin;Chang, Qing;Zhang, Xu;Barnett, Edward M.;Piwnica-Worms, David
细胞凋亡是维持正常细胞稳态所必需的，细胞凋亡过程的失控与多种疾病的发生密切相关。此前，我们基于一种可激活的策略开发了一种穿透细胞的近红外荧光(NIF)探针，用于检测体内与凋亡相关的caspase活性。该探针由穿透细胞的TAT多肽与效应器识别序列(DEVD)连接组成，该序列两侧是荧光团-猝灭剂对(Alexa Fluor 647和QSY 21)。一旦暴露于效应半胱氨酸酶，识别序列被切割，导致荧光团-猝灭剂对分离和信号产生。在这里，我们介绍了第二代探针KcapQ的生化分析，它带有一个修改的细胞穿透肽序列(KKKRKV)。这种修饰导致了一种对效应器caspase酶更敏感的探针，在荧光团-猝灭剂对之间显示出出人意料的更高的猝灭效率，并且对细胞的潜在毒性较小。用重组caspase酶分析表明，该探针是针对效应caspase(caspase 3>；7>；6)的。对经阿霉素处理的HeLa细胞的凋亡分析显示，探针激活对凋亡细胞具有特异性。在大鼠视网膜神经元兴奋性毒性模型上，玻璃体内注射N-甲基-D-天冬氨酸(NMDA)诱导视网膜神经节细胞(RGCs)凋亡。使用临床适用方案玻璃体内注射KcapQ的NMDA预处理动物的眼球和视网膜平面图显示，与未经处理的对照动物相比，特定的和广泛分布的细胞相关荧光信号。NMDA处理的动物视网膜垂直切片的荧光显微镜图像证实，活化的探针主要定位于RGCs，并与TUNEL标记共同定位。因此，KcapQ代表了一种改进的效应器caspase可激活的NIF探针，用于增强对整个细胞和活体动物的凋亡的非侵入性分析。
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 (NIF) 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 co-localized with TUNEL labeling. Thus, KcapQ represents an improved effector caspase-activatable NIF probe for enhanced non-invasive analysis of apoptosis in whole cells and live animals.
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