整合素内吞进程中 Liprin-β1介导的蛋白质 间相互作用的结构与生化研究

STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF LIPRIN-β1 MEDIATED INTERACTIONS IN INTEGRIN ENDOCYTOSIS

易芃芃

11849369

硕士

生物学

魏志毅

2020-05-29

2020-07-08

哈尔滨工业大学

深圳

细胞膜上有许多用于行使一些特定功能的特化区域，例如由许多蛋白质在簇集的整合素黏附基础上形成的黏着斑。这些特化区域的动态变化与许多重要的细胞活动有关，如整合素介导的细胞黏附的周转，是细胞迁移的关键。而细胞迁移，对于发育、组织重塑等非常重要。在许多病理状态下都观察到细胞迁移的异常，如肿瘤细胞系的不正常迁移和侵袭。因此，研究这些细胞质膜特化区域及其调控背后的分子组装和机制尤为重要。整合素介导的黏着斑的周转，是由运动细胞尾部的内吞作用对其的拆解与回收，以及运动细胞前沿对其的重塑构成的。这些特化区域通常通过共享一个核心复合物来协调复杂的生物学功能，其中包括支架蛋白Liprin家族，主要分为Liprin-α和Liprin-β。Liprin-α是一种重要的蛋白，目前相关研究较多，它通过诱导微管靶向至细胞前沿，促进黏着斑的周转，对其周转起关键作用。然而，Liprin-β1作为Liprin家族中的一员，与Liprin-α共定位在黏着斑周围，它在黏着斑中的功能尚未得到充分的研究。根据实验室前期数据，Liprin-β1可能与一些和内吞作用相关的蛋白质Numb、RUFY1、SH3KBP1、HIP1有此前未报道过的相互作用。通过免疫共沉淀技术及蛋白免疫印迹法检测Liprin-β1与这些蛋白的相互作用，确认了Liprin-β1与其中两个蛋白Numb与RUFY1的相互作用，以及确定了Liprin-β1的N端区域参与和Numb、RUFY1的相互作用。此外，成功表达及纯化了Liprin-β1、Numb及RUFY1的相关截断片段，用快速蛋白液相色谱法、等温滴定微量量热法进一步确定了其相互作用区域：Liprin-β1_H与Numb_Nter，Liprin-β1_H、Liprin-β1_CC与RUFY1_Nter，并进行了Liprin-β1_H与Numb_Nter蛋白复合物的初步晶体筛选。同时利用快速蛋白液相色谱法与等温滴定微量量热法进一步确定Liprin-β1_H与Numb_Nter，Liprin-β1_H、Liprin-β1_CC与RUFY1_Nter的更小相互作用区域，以进行下一步的晶体筛选及x射线衍射。对Liprin-β1与Numb及RUFY1的复合物进行结构解析，以对其相互作用及相关调控有更深入的了解，进一步解释Liprin-β1介导的蛋白质间相互作用如何在整合素内吞中发挥作用。综上所述，本文首次发现并证明了Liprin-β1蛋白与内吞作用相关蛋白Numb及RUFY1之间的相互作用，并从结构与生化的角度进一步研究了Liprin-β1与Numb、RUFY1的相互作用机制。根据此研究结果，Liprin-β1与细胞内吞相关蛋白相互作用。Liprin-β1可能通过参与整合素蛋白的内吞作用，来调控整合素介导的黏着斑的动态变化。这一研究将为Liprin-β1在整合素内吞与黏着斑周转中的调控机制研究提供新的思路。

There are many specialized areas on the cell membrane that perform certain functions, such as the focal adhesion formed by many proteins on the basis of clustering integrin. The dynamics of these specialized regions are related to many important cellular processes, such as the turnover of integrin-mediated focal adhesions, which is critical to the cell migration. And cell migration is essential for tissue remodeling, wound healing, axon growth, and so on. Abnormalities in cell migration, such as abnormal migration and invasion of tumor cell lines, have been observed in many pathological states. Therefore, it is particularly important to investigate the molecular organization and mechanisms behind these plasma membrane specialized regions and their regulation. The turnover of integrin-mediated focal adhesions, is composed of disassembly and recycle of focal adhesions in the rear of migrating cells through endocytosis, and reformation of them on the leading edge of migrating cells. These specialized regions often share a core complex that coordinates complicated biological functions, including the scaffold protein Liprin family, which is mainly divided into Liprin-α and Liprin-β. Liprin-α is an important protein which has been widely studied. It plays a key role in the turnover of adhesions by inducing microtubules to target to the leading edge of migrating cells. However, as a member of Liprin family, Liprin-β1 colocalizes with Liprin-α around focal adhesions, its function in focal adhesions has not been well studied.Based on our preliminary data, it was found that Liprin-β1 may interact with some endocytosis related proteins Numb, RUFY1, SH3KBP1, and HIP1, which is unreported before. We firstly detected the interaction between Liprin-β1 and these proteins by Co-IP and Western Blot assay, and confirmed that Liprin-β1 indeed interacts with two of the endocytosis related proteins, Numb and RUFY1, and further confirmed that the N-terminal region of Liprin-β1 is involved in the interaction with Numb and RUFY1.And we successfully expressed and purified the relevant truncated fragments of Liprin-β1, Numb and RUFY1, and the interacting regions were further determined by Fast Protein Liquid Chromatography (FPLC) and Isothermal Titration Calorimeter (ITC) : Liprin-β1_H and Numb_Nter; Liprin-β1_H, Liprin-β1_CC and RUFY1_Nter, and preliminary crystal screening of Liprin-β1_H and Numb_Nter complex was carried out. At the same time, the smaller binding regions of Liprin-β1_H and Numb_Nter; Liprin-β1_H, Liprin-β1_CC and RUFY1_Nter are further detected by Fast Protein Liquid Chromatography (FPLC) and Isothermal Titration Calorimeter (ITC), to do further crystal screening and X-ray diffraction. The structure of the complexes of Liprin-β1 and Numb, RUFY1 will be analyzed to gain a better understanding of their interactions and related regulation and to further explain how Liprin-β1-mediated interactions play a role in integrin endocytosis.In summary, the interactions between Liprin-β1 and endocytosis related proteins Numb, RUFY1 were found and proved for the first time in this paper, and the interaction mechanism between Liprin-β1 and Numb, RUFY1 was further investigated. According to our results, Liprin-β1 interacts with endocytosis related proteins, and may regulate integrin-mediated focal adhesions dynamics by participating in the endocytosis of integrin. This study will provide a new idea for the investigation of the regulatory mechanism of Liprin-β1 in integrin endocytosis and focal adhesion turnover.

细胞迁移 整合素 Liprin-β1 内吞相关蛋白 蛋白质结晶

cell migration integrin Liprin-β1 endocytosis related proteins protein crystallization

中文

联合培养

南方科技大学

易芃芃. 整合素内吞进程中 Liprin-β1介导的蛋白质 间相互作用的结构与生化研究[D]. 深圳. 哈尔滨工业大学,2020.