肌球蛋白Myo9b C-端区域Rho GAP活性自抑制及调控的结构机制研究

Cell migration is a fundamental activity of cells and is critical for a variety of biological processes, including embryonic development, wound healing and cancer metastasis. The dynamic reorganization of actin filaments during cell migration is rigorously controlled by the Rho family of small GTPases. Myo9b is a motorized Rho GAP that plays a prominent role in regulating cell migration. Myo9b is predominantly localized at the leading edge for controlling the distribution and activity of RhoA. The C-terminal AID-C1-RhoGAP region of Myo9b adopts an autoinhibited state and can directly bind to the PIP lipid membranes. However, the molecular mechanisms underlying the AID-C1-RhoGAP autoinhibition and the recognition of the PIP lipid remain unclear. In this proposal, we focus on the structural studies of the C-terminal AID-C1-RhoGAP of Myo9b and the mechanism of the PIP lipid recognition by this region. By using molecular biology, structural biology and cell biology, we will work on the autoinhibited conformation of AID-C1-RhoGAP and the structural and functional dissection of the AID-C1-RhoGAP/PIP lipid complex. The outcome of this proposal will reveal the molecular mechanisms for the AID-C1-RhoGAP autoinhibition and its PIP lipid recognition, which may shed light on the understanding of the fundamental role of Myo9b in controlling cell migration.

细胞迁移是细胞的基本生命活动，在胚胎发育、伤口愈合、癌症转移等过程中发挥重要生物学功能。细胞迁移过程中微丝骨架的动态重组受到Rho家族小GTP酶的调控。肌球蛋白Myo9b是调控细胞迁移的重要Rho GAP蛋白，定位于细胞迁移前端调控RhoA的活性分布。研究发现，Myo9b C-端AID-C1-RhoGAP区域的Rho GAP活性处于自抑制状态，其还可以与PIP脂质直接结合，但该区域自抑制及其与PIP脂质结合的结构机制目前都不清楚。本项目选择Myo9b AID-C1-RhoGAP为主要研究对象，将综合运用分子生物学、结构生物学以及细胞生物学，对该区域进行结构与功能研究，重点研究其自抑制状态的整体结构及PIP脂质复合物的结构和功能。通过本项目的研究，将揭示Myo9b C-端区域Rho GAP活性自抑制的结构特征及其特异结合PIP脂质的分子机制，有利于进一步认识Myo9b调控细胞迁移的功能机理。

细胞迁移是细胞的基本运动行为，在众多生物学过程中都起重要功能作用。细胞迁移相关微丝骨架的动态重组受到Rho家族小GTP酶的精密调控。肌球蛋白Myo9b是细胞迁移的重要调控蛋白，定位于细胞迁移前端并调控RhoA活性的动态分布。Myo9b C-端区域的活性处于自抑制状态，但该区域自抑制的分子机制一直不清楚。综合运用分子生物学、结构生物学以及细胞生物学等方法，本项目对Myo9b C-端含有RhoGAP结构域的区域开展相关结构与功能研究。首先解析得到突变体RhoGAP-A1845N/RhoA复合物的晶体结构，验证该突变对相互作用的增强效果。其次解析得到RhoGAP-HD区域的晶体结构，发现HD结构域结合RhoGAP结构域活性中心并抑制其活性。研究显示，C-端整体区域在体外和细胞内都发生液-液相分离现象，其正负电荷富集区域是驱动液-液相分离现象发生的主要因素。此外，RhoA与C-端整体区域相互作用，并协同促进该区域的液-液相分离过程。通过本项目的研究，主要揭示Myo9b C-端区域的结构特征及其活性自抑制的分子机制，新发现的液-液相分离现象有利于进一步理解Myo9b细胞迁移调控相关生物学功能。