单个活细胞内蛋白泛素化和去泛素化过程的时空信息获取新方法研究及应用

Ubiquitination and deubiquitination are very important biochemical processes to adjust proteins in the cell. They lead to the changes in protein structure, abnormal expression in space or time and inactivation, all of that are closely related with tumor growth, neuropathy, etc. However, at present, due to the lack of the effective in-situ analysis methods to study ubiquitination and deubiquitination processes, it hampers its basic research in this area and the developing of the related drugs. Aims of this project is to use microfluidic chips as cell culture and operating units, to develop single molecule fluorescence cross-correlation spectroscopy (FCCS) and fluorescence resonance energy transfer fluorescence correlation spectroscopy (FRET-FCS) as in situ analysis methods, to establish in situ analysis strategy to investigate ubiquitination and deubiquitination processes within single cell, and extract spatial-temporal information about the ubiquitination and deubiquitination processes. In the project, the ubiquitination and ubiquitination of tumor suppressor protein p53 in living cells are used as model. Non-natural amino acids (UAAs) are introduced into ubiquitin by the use of genetic codons technique; "On-off type" fluorescent probes are designed and synthesized; Ubiquitin is selectively labeled with the fluorescent probe using the biological orthogonal reaction between probes and UAAs of ubiquitin. The p53 is modified with green fluorescent protein by genetic engineering technique. FCCS and FRET-FCS is used to study the ubiquitination and deubiquitination processes. The project is expected to provide new methods for basic research of protein ubiquitination and the related drug screening.

蛋白泛素化和去泛素化是调节细胞内蛋白质的非常重要过程，其导致的蛋白结构改变、异常的时空表达以及活性失控与肿瘤生长、神经病变等密切相关。目前由于缺乏泛素化和去泛素化原位分析方法，严重阻碍了该领域基础研究及其相关药物的研发。本项目旨在以微流控芯片为在线细胞培养和操作系统，以单分子荧光互相关光谱（FCCS）和荧光共振能量转移-荧光相关光谱（FRET-FCS）为分析手段，建立单细胞内蛋白泛素化和去泛素化过程的原位实时分析方法，获取细胞内泛素化和去泛素化的时空信息。以抑癌蛋白p53的泛素化和去泛素化过程为模型；采用基因密码子拓展非天然氨基酸技术在泛素中引入非天然氨基酸；设计合成“开关型”荧光探针；利用生物正交反应对泛素进行选择性共价绿色荧光标记。采用基因重组技术将p53修饰红色荧光蛋白。采用FCCS和FRET-FCS技术原位研究p53泛素化和去泛素化过程，为泛素化的基础研究和相关药物筛选提供新方法。

活细胞内蛋白泛素化和去泛素化过程是调节细胞内蛋白质的非常重要过程，其导致的蛋白结构改变、异常的时空表达以及活性失控与肿瘤生长、神经病变等密切相关。但是目前可用于活细胞内蛋白泛素化和去泛素化过程原位分析的方法非常少。本项目通过从构建活细胞原位分析系统出发，采用单分子荧光自相关光谱、荧光互相关光谱（FCCS）和荧光共振能量转移-荧光相关光谱（FRET-FCS）等为分析技术，建立单细胞内蛋白泛素化和去泛素化过程的原位实时分析方法；并以p53等蛋白的泛素化和去泛素化过程为模型，对活细胞内p53等蛋白的泛素化和去泛素化过程以及药物对这两种过程的调控作用进行了细致的研究。这些研究不仅为调控蛋白的泛素化过程的药物研究提供了一种重要的活细胞原位分析手段，也为活细胞内蛋白泛素化和去泛素化过程研究提供了重要的科学工具。