Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell-specific quantification of protein abundance changes, we sought to apply an enzyme-activated cellular fluorescence system in vivo by delivering ester-masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES-1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.

中文翻译：

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秀丽隐杆线虫中的内源水解酶的体内递送和被掩盖的荧光水解酶底物的激活

经常通过在体内用绿色荧光蛋白（GFP）标记分子来研究蛋白质的表达和定位，但是蛋白质水平的细微变化并不容易被检测到。为了开发一种灵敏的体内方法来放大荧光信号并允许对蛋白质丰度变化进行细胞特异性定量分析，我们试图通过将酯类掩蔽的荧光团递送至秀丽隐杆线虫来在体内应用酶激活的细胞荧光系统。表达猪肝酯酶（PLE）的神经元。为了帮助摄取到感觉神经元膜中，我们合成了两种新颖的具有长烃尾的荧光水解酶底物。重组PLE在体外激活了这些荧光团。体内激活发生在感觉神经元中，而肠道溶酶体的有效激活可通过成像和微孔板定量，部分归因于肠道酯酶1（GES-1）活性。这些数据证明了生物正交水解酶及其荧光底物有望作为体内神经元成像工具，并用于表征内源秀丽隐杆线虫水解酶底物特异性。