表征肿瘤微环境中受体膜蛋白动态修饰与相互作用的化学蛋白质组学分析新方法

Following the guideline of “dynamic characteristics of chemical modification of biomacromolecules”, this project intends to large-scale characterize the dynamic tyrosine phosphorylation of membrane receptors and their protein complexes, and then systematically decipher the intercellular signaling pathways. Tumor microenvironment has drawn tremendous attention in the cancer research field in recent years. The intercellular signaling in tumor microenvironment are generally mediated by the formation of protein complexes between membrane receptors and ligand proteins secreted from neighboring cells and the subsequent dynamic tyrosine phosphorylation. However, these post-translational modification-dependent protein complexes are dynamically regulated with weak binding affinity, which is hard to be characterized by conventional approaches. This project is aiming to develop a new chemical proteomic strategy with a focus on pancreatic tumor microenvironment. First, the activated and modified membrane receptors will be identified by high-sensitive phosphotyrosine proteome analysis. By combining the metabolic labeling of membrane receptors with photoreactive monosaccharides and molecular cloning approaches, we expect to achieve the chemical and biological labeling of the membrane receptors on the living cells and the high-selective profiling of the secreted proteins binding with membrane receptors. Through the bioorthogonal chemical activation of related tyrosine kinases, we expect to characterize the downstream signaling pathways mediated by the selected membrane receptors. We envision the developed strategy will provide great support for the discovery of innovative anticancer drug targets and cancer biomarkers.

本项目以研究“生物大分子化学修饰的动态特性”为导向，规模化表征受体膜蛋白的动态酪氨酸磷酸化修饰及其复合物，进而实现对细胞间信号转导通路的系统解析。肿瘤微环境是近年来癌症研究领域的热点方向。肿瘤微环境的细胞间信号转导一般是由受体膜蛋白与相邻细胞来源的分泌蛋白结合形成复合物并触发动态的酪氨酸磷酸化修饰来介导。这些翻译后修饰依赖的蛋白质复合物具有结合力弱和动态变化的特征，很难用常规的方法进行研究。本项目拟以胰腺癌肿瘤微环境为研究对象，发展新的化学蛋白质组学研究策略：发展高灵敏度酪氨酸磷酸化蛋白质组学技术鉴定激活并修饰的受体膜蛋白；结合含有光反应基团的单糖代谢标记和分子克隆技术，实现活细胞上受体膜蛋白的化学生物标记，从而高选择性地鉴定与其结合的分泌蛋白；通过相关酪氨酸激酶的生物正交化学激活，表征特定的受体膜蛋白下游的信号转导通路。该策略的发展将为创新抗癌药物靶点和肿瘤生物标志物的发现提供帮助。

对酪氨酸磷酸化修饰介导的蛋白质复合物动态变化的研究对解析肿瘤微环境的细胞间信号转导通路具有重要意义。本项目以发生酪氨酸磷酸化激活的受体膜蛋白为研究切入点，发展了一系列新的化学蛋白质组学研究策略，实现了对相关受体膜蛋白复合物、受体酪氨酸激酶底物的规模化鉴定:发展了基于APEX近程标记技术的功能蛋白质复合物时空分析方法，实现了分钟级分辨率的APEX2近程标记蛋白质组学分析；发展了受体膜蛋白酪氨酸磷酸化介导的复合物的高通量蛋白质组学分析方法，极大提高蛋白复合物的鉴定效率；通过生物正交化学激活，首次实现了在活细胞中对酪氨酸激酶底物的系统鉴定，确定了269个SRC激酶的潜在底物，并验证了3个新的SRC底物。本项目将为创新肿瘤生物标志物与抗癌药物靶点的发现提供线索。

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