Membrane Targeting by Phosphoinositide Binding Proteins

描述（由申请人提供）： 参与细胞信号传导和膜运输的许多外周蛋白质响应于磷脂酰肌醇（phosphoinositides; PI）的磷酸化衍生物的时空动力学而靶向特定的细胞膜。然而，不同PI介导其效应蛋白的膜靶向和激活的机制才刚刚开始解开。基于我们先前对膜靶向结构域的结构-功能研究，该研究揭示了膜靶向结构域的亚细胞靶向作用可以通过控制其与体外模型膜结合的生物物理原理来解释，我们研究了最近发现的PI结合结构域FYVE（Fab 1 p，YOTB，Vac 1 p和EEA 1）和PX（Phox）结构域的膜靶向作用。这些研究表明，PI通过引起蛋白质构象和静电势的变化特异性地诱导FYVE和PX结构域的膜渗透，并且该过程对其膜结合和细胞活性是必不可少的。这项研究的主要目的是将这些研究扩展到广泛的PI结合结构域，包括FYVE，PX和ENTH（Epsin N-末端同源）结构域，并充分阐明各种PI诱导其体外和细胞膜靶向的机制。本研究的具体目的如下：1）确定具有不同膜亲和力的各种FYVE结构域的体外和细胞膜靶向机制; 2）确定具有不同PI特异性的PX结构域的体外和细胞膜靶向机制; 3）确定两种不同ENTH结构域的体外和细胞膜靶向机制。主要方法包括：（1）通过单层膜、沉降、表面等离子体共振和荧光相关光谱测量，对PI结合结构域与各种模型膜的相互作用进行生物物理分析;（2）荧光蛋白标记的N-结合结构域及其突变体转染到哺乳动物细胞中，以确定膜转位的速率和结合结构域的解离常数。膜结合

DESCRIPTION (provided by applicant): Many peripheral proteins involved in cell signaling and membrane trafficking are targeted to specific cell membranes in response to the spatiotemporal dynamics of phosphorylated derivatives of phosphatidylinositiol (phosphoinositides; PI). However, the mechanisms by which different PIs mediate the membrane targeting and activation of their effector proteins are only beginning to unravel. Based on our previous structure-function studies on membrane targeting domains, which revealed that much of subcellular targeting by membrane targeting domains can be accounted for by the biophysical principles that govern their binding to model membranes in vitro, we have investigated the membrane targeting by recently discovered PI-binding domains, FYVE (Fab1p, YOTB, Vac1p, and EEA1) and PX (Phox) domains. These studies have shown that PIs specifically induce the membrane penetration of FYVE and PX domains by causing changes in protein conformation and electrostatic potential and that this process is essential for their membrane binding and cellular activities. The primary objective of this proposed research is to extend these studies to a broad range of PI-binding domains, including FYVE, PX, and ENTH (Epsin N-Terminal Homology) domains, and to fully elucidate the mechanisms by which various PIs induce their in vitro and cellular membrane targeting. Specific aims for this proposed research are as follows: 1) Determination of the in vitro and cellular membrane targeting mechanisms of various FYVE domains with different membrane affinities; 2) Determination of the in vitro and cellular membrane targeting mechanisms of PX domains with different PI specificities; 3) Determination of the in vitro and cellular membrane targeting mechanisms of two distinct ENTH domains. The principal methodologies to be used include: (1) the biophysical analysis of interactions of PI-binding domains with various model membranes by monolayer, sedimentation, surface plasmon resonance, and fluorescence correlation spectroscopy measurements; (2) the quantitative analysis of fluorescent protein-tagged N-binding domains and their mutants transfected into mammalian cells to determine the rates of membrane translocation and the dissociation constants for membrane binding.