Membrane Targeting by Phosphoinositide Binding Proteins

描述(由申请人提供)： 许多参与细胞信号转导和膜转运的外周蛋白是针对特定细胞膜的，以响应磷脂酰肌醇(PI)磷酸化衍生物的时空动力学。然而，不同的PI介导膜靶向和激活其效应蛋白的机制才刚刚开始解开。基于我们以前对膜靶向域的结构和功能的研究，发现膜靶向域的大部分亚细胞靶向性可以通过控制它们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(Fab1p，YOTB，Vac1p，和EEA1)和Px(Phox)结构域的膜靶向性。这些研究表明，PI通过引起蛋白质构象和静电势的改变而特异性地诱导FYVE和PX结构域的膜穿透，这一过程对它们的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结合域，包括FYVE、PX和ENGIN(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。这项研究的具体目标如下：1)确定不同膜亲和力的FYVE结构域的体外和细胞膜靶向机制；2)确定不同PI特异性的Px结构域的体外和细胞膜靶向机制；3)确定两个不同的第10结构域的体外和细胞膜靶向机制。将使用的主要方法包括：(1)通过单层、沉淀、表面等离子体共振和荧光相关光谱测量来分析PI结合结构域与各种模型膜的相互作用；(2)定量分析转染到哺乳动物细胞中的荧光蛋白标记的N结合结构域及其突变体，以确定膜转位的速率和膜结合的解离常数。

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.