由机械力引发的细丝蛋白-平滑肌蛋白间相互作用及其分子机制研究

由机械力引发的细丝蛋白-平滑肌蛋白相互作用是由申请人发现的一种血管平滑肌细胞内平滑肌蛋白与细丝蛋白间的新型作用模式，也是细胞生物学研究领域的最新热点之一。机械力传导机理在控制细胞迁移、生长、分化以及组织修复和再生中扮演非常重要的角色。本项目通过研究该最新发现的细丝蛋白A和平滑肌蛋白间的相互作用，探索和解析由机械力引发和传导的该类细胞信号通路的生物学机理。机械力与生化信号的转化对研究重力、肌肉收缩、血压以及动脉硬化和哮喘等疾病都具有非常重要的意义。本项目的具体研究内容包括：1）解析细丝蛋白A-平滑肌蛋白复合体的三维结构；2）应用基因突变技术研究平滑肌蛋白与细丝蛋白A的相互作用对肌动蛋白网的体外物理化学性质的影响；3）研究细丝蛋白A与平滑肌蛋白相互作用的有无对细胞生长、迁移以及细胞收缩等的影响；4）探究心血管疾病中基因变异与细丝蛋白/平滑肌蛋白之间的关联性以及对平滑肌细胞功能的影响。

The long-term goal of my research is to understand the molecular mechanism of cellular mechanotransduction, a conversion of mechanical forces, such as gravity, muscle contraction, blood flow, and pressure, into biochemical signals. Mechanotransduction plays a crucial role in tissue repair and regeneration by controlling cell migration, growth, and differentiation. In this project we elucidate the role of a novel force-dependent interaction between filamin A (FLNA) and smoothelin (SMTN) in vascular smooth muscle cells (VSMCs). Vertebrate FLNA is actin-crosslinking protein that interact with multiple partners. We and others have shown that mechanical force regulates one such interaction – with integrins – by opening a cryptic binding site on FLNA. To identify other such associations, we employed mass spectrometry-based proteomics followed by in silico screening to identify additional proteins that contain the FLN binding site. We found that SMTNs (A and B) bind selectively to the open form of the FLNA integrin-binding domain. SMTN-A is abundant in visceral smooth muscle and is essential for the contractility of intestinal smooth muscle. SMTN-B is expressed in vascular smooth muscle cells, and loss of SMTN-B results in increased mean arterial pressures. Since FLNA mutations also cause cardiovascular defects, we hypothesize that the FLNA-SMTN interaction plays a critical role in VSMC contraction and mechanics, and that alterations in this interaction contribute to disease, including atherosclerosis, hypertension, and asthma. We will: 1) biochemically characterize the FLNA-SMTN interaction to obtain a high resolution structure of the complex using nuclear magnetic resonance spectroscopy or X-ray crystallography; 2) determine the mechanical properties of FLNA-actin networks in the presence of SMTN with or without mechanical force mediated by myosin contraction in vitro; 3) investigate the role of the FLNA-SMTN interaction in living cell mechanics, signaling, and behaviors such as contraction, growth, migration; 4) examine an effect of disease-causing mutations in FLNA and SMTN on their interaction and expression levels, and on VSMC function. In summary, determining the role of FLNA-SMTN interactions in normal and aberrant VSMC mechanics in cardiovascular diseases may pave the way to new therapeutics for cardiovascular and lung disease.