蛋白质类泛素化修饰的单分子检测与成像分析研究

Covalent modification of proteins by a group of ubiquitin-like proteins (Ubls), i.e., Ubl modification, has been implicated in a wide range of normal biological processes, and also in disease-related abnormalities, such as cancer. However, the functional analysis of Ubl modifications is often hampered by the low amount and heterogeneity of specific modified proteins in the cell. To overcome these problems, we will introduce the newly developed protein tags, such as SNAP tag, CLIP tag and Halo tag, into Ubl modifications and construct genetically-encoded protein tag-based biosensors to convert invisible modification event into fluorescent dye or nanomaterial. The combination of biosensor-mediated translation with total internal reflection fluorescence (TIRF) imaging will result in the measurement of Ubl modifications at the single-molecule level, while the employment of laser scanning confocal microscope will lead to in situ imaging of Ubl modifications in the cell. Furthermore, we will measure the change of Ubl modifications in respond to different stimuli, and explore their spatio-temporal distribution within the cell. We believe that this study would provide new insights into Ubl modifications as well as their roles in tumorigenesis.

蛋白质类泛素化修饰是由类泛素蛋白所介导的一类蛋白质翻译后修饰行为，参与调控几乎所有细胞通路和过程；异常类泛素化修饰会导致包括癌症在内多种疾病的发生。虽然类泛素化修饰广泛调节各种生物学过程，但是其修饰产物具有化学计量值低、微观结构不均一的特点, 这极大妨碍了对类泛素化修饰行为及其生物学功能的全面认识。本项目针对上述难点开展研究工作，将多种蛋白标签引入类泛素化修饰研究中来，构建新型生物传感器将“不可见”的类泛素化修饰行为转换为可被测定的荧光分子，进一步发展可对类泛素化修饰进行单分子检测和原位成像的方法，并对修饰产物的异质性以及时空分布特征进行分析，从而加深对类泛素修饰行为及其在癌症发生过程中作用的认识。

蛋白质类泛素化修饰是细胞精确调控胞内蛋白质结构、功能以及空间分布的一种重要方式，虽然蛋白质类泛素化修饰发生于细胞的全部生命过程，但是类泛素化修饰产物具有丰度低、异质性的特点。为实现蛋白质类泛素化修饰的高灵敏检测和分析，我们引入SNAP/CLIP标签以及四半胱氨酸标签标记技术，将SNAP/CLIP以及四半胱氨酸标签添加到类泛素化修饰蛋白上，借助这些标签与底物之间的特异和高效作用，将蛋白质类泛素化修饰行为转化为可被测定的荧光信号。并进一步结合全内反射荧光成像手段，实现对蛋白质类泛素化修饰的单分子成像和检测。通过对获得荧光点数量和强度的进一步分析，进行蛋白质类泛素化修饰的量化分析。利用发展的单分子检测技术，我们比较了不同生理条件下肿瘤抑制因子p53、RAN GTPase酶激活蛋白1以及核点蛋白Sp100上发生小类泛素化修饰的水平。本项目的实施，将能为蛋白质类泛素化修饰检测提供一种新的技术手段，并有望加深对蛋白质类泛素化修饰行为及其在肿瘤等疾病发生中作用的认识。