参与细胞内吞体代谢调控的重要蛋白质复合物的结构和功能研究

The transport of cellular substances primarily refers to the exchange of substances (such as proteins, nucleic acids, fats, and carbohydrates) between the cell and the environment. Endocytosis is a major process by which the cell absorbs extracellular materials by engulfing them, and thus plays crucial roles in the regulation of many cell biological processes, including cell development and apotosis, immune response, neurotransmission, intercellular communication, signal transduction, and cellular homeostasis. It is also closely related to the occurrence and development of various diseases. In the endocytic pathway of eukaryotic cells, early endosomes located in the periphery of the cell are responsible for internalizing molecules from the plasma membrane and recycling them back to the surface. Continuous fusion of vesicles or early endosomes leads to the formation of late endosomes. Late endosomes sort the internalized materials prior to their delivery to lysosomes where they are degraded into simple compounds. Many proteins are involved in endocytosis and among them small GTPases Rab5 and Rab7 play important roles in regulating intracellular membrane trafficking. Rab5 and its GEF Rabex5 and effector Rabaptin5 regulate the fusion of vesicles and the formation of early endosomes; Rab7 and its GEF and effectors regulate the maturation of late endosomes; Mon1 and Ccz1 modulate the transition of early to late endosomes by interacting differently with Rab5 and Rab7. In this project, we will carry out structural and functional studies of these important proteins and protein complexes to elucidate the molecular basis of the biological functions of individual proteins and protein complexes and the protein-protein interaction, and the molecular mechanisms of their functional roles in endocytosis using a combination of structural biology, biochemistry, molecular biology, and cell biology. This study may also help to understand the relevance of dysfunction of the endocytic pathway and particularly these proteins to the development of human disease processes.

细胞物质运输主要指细胞与环境间的物质交换，内吞作用是细胞外的大分子物质进入细胞的主要方式，在细胞的营养吸收、免疫调节、信号转导、神经传递、细胞发育和凋亡等过程中发挥非常重要的作用，与很多疾病的发生发展也密切相关。内吞作用由最初形成的早期内吞体通过不断融合转变为晚期内吞体，然后与溶酶体融合后完成降解和消化。小G蛋白Rab5与其GEF蛋白Rabex5和效应蛋白Rabaptin5形成的复合物调控早期内吞体的融合；Rab7与其GEF和效应蛋白形成的复合物促进晚期内吞体的成熟；Mon1和Ccz1形成的复合物通过与Rab5和Rab7的不同结合，调控早期内吞体到晚期内吞体的转变。本项目拟运用结构生物学、生物化学、分子生物学和细胞生物学等方法，研究这些重要蛋白质复合物的结构和功能，阐明它们各组分蛋白的功能和相互作用关系、在内吞体代谢过程中的调控作用和分子机制，探讨它们的功能失调与疾病发生发展的关系。

内吞作用在细胞的营养吸收、免疫调节、信号转导、神经传递、细胞发育和凋亡等过程中发挥非常重要的作用，与很多疾病的发生发展也密切相关。内吞体代谢是由最初形成的早期内吞体通过不断融合转变为晚期内吞体，然后与溶酶体融合后完成降解和消化的过程。. 本项目以内吞体代谢过程中发挥重要功能的Rab5-Rabex5-Rabaptin5、Rab9A-RUTBC2、Arl1-BIG1、CASTOR1和Ego1-Ego2-Ego3等蛋白质复合物为研究对象，运用结构生物学、生物化学、分子生物学和细胞生物学等方法开展这些复合物的结构、功能和分子机制研究。主要研究成果包括解析了Rabex5-Rabaptin5和Rab5-Rabex5-Rabaptin5三元复合物的晶体结构，结合生物物理和生物化学的分析结果，阐明了Rabex5蛋白GEF 活性调控的分子机制；测定了小G蛋白Rab9A与效应蛋白RUTBC2复合物的晶体结构，阐明了Rab9A特异性识别RUTBC2的分子基础；发现Arl1结合在具有Arf1 GEF活性的BIG1蛋白的N端DCB结构域，进而通过Arl1 N端的高尔基体定位修饰招募BIG1，并进一步激活下游Arf1蛋白，调控下游胞内运输和内吞体代谢过程；测定了CASTOR1与arginine复合物的晶体结构，阐明了CASTOR1特异性结合arginine、感应氨基酸水平的分子基础；测定了酵母Ego1-Ego2-Ego3三元复合物的结构，揭示了Ego1-Ego2-Ego3三元复合物的组装机制以及募集下游小G蛋白复合物Gtr1-Gtr2在酵母囊泡上定位的分子机制。其它研究成果还包括测定了S6K1、DNMT2、FANCM-FAPP24和WORP-DNA等重要蛋白质或蛋白质复合物的结构，并开展了相关的功能研究，阐明了它们参与细胞生命活动的功能及其分子机制。总计解析了13个蛋白质和蛋白质复合物的晶体结构，并将这些蛋白质结构信息储存于国际PDB数据库；在国际高水平学术刊物上发表了标注该项目支持的研究论文10篇。这些研究工作阐明了这些重要蛋白质复合物的结构和功能，揭示了它们各组分蛋白的功能和相互作用关系，以及在内吞体代谢过程中的调控作用和分子机制，为进一步研究它们的功能失调与疾病发生发展的关系奠定了良好的基础。