Rational design of a genetically encoded infrared fluorescent protease reporter

 描述(申请人提供)：在几乎每一个主要的生物过程中，蛋白水解酶都扮演着基本的角色。蛋白分解系统的改变会导致许多疾病，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水解酶报告已经成功地在动物体内显示了水解酶活性。然而，这些记者在对发育中的胚胎进行成像方面存在局限性，因为很难将化学染料靶向特定的组织，而且注射化学染料可能会干扰发育。因此，基因编码的蛋白酶报告基因是首选的。此外，与基于荧光共振能量转移(FRET)的报告器相比，FRET信号较弱，需要图像处理才能获得FRET信号，因此，一种在蛋白酶激活后变为荧光的荧光性蛋白酶报告器将是理想的。这种基因编码的荧光酶报告将使我们能够以时空分辨率直接可视化完整动物的蛋白酶活性。在这里，我们的目标是：1)设计和改进基因编码的红外荧光蛋白水解酶报告。这种基因编码的蛋白水解酶报告在蛋白水解酶被激活时会变成荧光，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变化。细胞凋亡在果蝇胚胎发生中起重要作用。细胞凋亡的抑制会损害胚胎发育过程中的许多关键的形态发生运动，包括分裂、生殖带回缩、背部闭合和头部退化。然而，目前尚不清楚细胞凋亡是否在时空上与形态发生相关。在这里，我们将使用caspase报告符来可视化这些形态发生运动过程中细胞凋亡的时空动态。3)：研究果蝇和小鼠脑瘤发生过程中细胞凋亡的动态变化。在癌症的发展过程中，癌基因的激活会导致细胞过度增殖，从而触发细胞凋亡。避免细胞凋亡为肿瘤的快速发展扫清了这一障碍，也是癌症的标志之一。在癌症的发展过程中，这种细胞凋亡的动力学已经被提出。在这里，我们将使用caspase报告来描述果蝇和小鼠胶质母细胞瘤发展过程中细胞凋亡的动态变化。

 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.