Rational design of a genetically encoded infrared fluorescent protease reporter

 描述（由申请人提供）：蛋白酶在几乎所有主要的生物过程中发挥着重要作用。蛋白水解系统的变化导致许多疾病，包括癌症、神经退行性疾病和心血管疾病。几种基于化学染料的蛋白酶报告基因已经成功地在动物中成像蛋白酶活性。然而，这些报告者在成像发育中的胚胎方面具有局限性，因为难以将化学染料靶向特定组织，并且化学染料的注射可能干扰发育。因此，优选遗传编码的蛋白酶报告基因。此外，与基于荧光共振能量转移（FRET）的报告物相比，基于FRET的报告物遭受弱信号并且需要图像处理以获得FRET信号，在蛋白酶活化时变成荧光的荧光蛋白酶报告物将是理想的。这样的基因编码的荧光蛋白酶报告将使我们能够直接可视化蛋白酶活性与完整的动物时空分辨率。本论文的主要目的是：1）设计和改进基因编码的红外荧光蛋白酶报告基因。这种基因编码的蛋白酶报告基因在蛋白酶激活后变得发荧光，并且不需要外源辅因子。2）：观察胚胎形态发生过程中细胞凋亡的时空动态。细胞凋亡在果蝇胚胎发生过程中起着重要作用。细胞凋亡的抑制损害胚胎发育过程中的许多重要的形态发生运动，包括分割，胚带回缩，背闭合和头部退化。然而，目前尚不清楚凋亡是否是时空相关的形态发生。在这里，我们将使用半胱天冬酶报告可视化这些形态发生运动过程中的凋亡的时空动态。3）：研究果蝇和小鼠脑肿瘤发生过程中细胞凋亡的动态变化。在癌症发展过程中，癌基因激活导致细胞过度增殖，从而引发细胞凋亡。逃避细胞凋亡清除了这一障碍，使肿瘤快速发展，这是癌症的标志之一。先前已经提出了癌症发展过程中细胞凋亡的这种动力学。在这里，我们将使用半胱天冬酶报告的特点在果蝇和小鼠胶质母细胞瘤的发展过程中的细胞凋亡的动力学。

 DESCRIPTION (provided by applicant): Proteases play fundamental roles in almost every major biological process. Changes to proteolytic systems lead to many diseases including cancer, neurodegenerative and cardiovascular diseases. Several chemical dye-based protease reporters have been successful in imaging protease activity in animals. However, these reporters have limitations in imaging developing embryos since it is difficult to target chemical dyes to specific tissues and injection of chemical dyes may perturb development. A genetically encoded protease reporter is thus preferred. Furthermore, compared to fluorescence resonance energy transfer (FRET)-based reporters that suffers from weak signals and require image processing to obtain FRET signal, a fluorogenic protease reporter that becomes fluorescent upon protease activation will be ideal. Such genetically encoded fluorogenic protease reporters will enable us to directly visualize protease activity with spatiotemporal resolution in intact animals. Here we aim to: 1) Design and improve genetically encoded infrared fluorescent protease reporters. Such genetically encoded protease reporters become fluorescent upon protease activation and require no exogenous cofactor. 2): To visualize spatiotemporal dynamics of apoptosis during embryonic morphogenesis. Apoptosis plays an essential role in embryogenesis of Drosophila. Inhibition of apoptosis impairs many critical morphogenetic movements during embryo development, including segmentation, germ band retraction, dorsal closure and head involution. However, it is not clear whether apoptosis is spatiotemporally correlated to morphogenesis. Here, we will use a caspase reporter to visualize spatiotemporal dynamics of apoptosis during these morphogenetic movements. 3): To characterize dynamics of apoptosis during brain tumor development in Drosophila and mice. During cancer development, oncogene activation leads to cell overprolieration, which triggers apoptosis. Evading apoptosis clears this barrier for rapid tumor development, and is one of the hallmarks of cancer. Such dynamics of apoptosis during cancer development has previously been proposed. Here we will use the caspase reporter to characterize dynamics of apoptosis during glioblastoma development in Drosophila and mice.