Membrane Targeting by Phosphoinositide-Binding Proteins

描述(申请人提供)：磷脂酰肌醇的磷酸化衍生物，统称为磷脂酰肌醇(PI)，在参与细胞信号转导和囊泡运输的胞浆蛋白的膜募集和激活中发挥关键作用。大多数PI反应蛋白含有一个或多个能识别PI的模块化脂质结合域。其中一些PI结合域不仅能与PI结合，还能引起含PI膜的变形。尽管PI介导的膜靶向在健康和疾病中很重要，但对于PI通过与这些效应结构域的相互作用而不同地和特异性地介导不同的细胞功能的复杂机制仍然缺乏基本的了解。这项研究的主要目的是阐明不同的PI通过与三种主要的PF效应结构域，PX，THEN和BAR结构域的相互作用来调节细胞过程的机制。在过去的格栅时期，我们发现PI特异性地诱导许多效应结构域的膜穿透，从而调节细胞功能和含有这些结构域的蛋白质的调节。我们还开发了几种新的方法，可以对PI结合蛋白和膜变形蛋白进行大规模和深入的机制研究。在新的范式和新的方法下，我们将在下一个项目期追求三个具体目标。首先，基于哺乳动物Px结构域通过其不同的PI特异性和膜结合特性来介导不同的细胞过程的假设，我们将全面系统地测定Px结构域的膜结合特性，以阐明不同的Px结构域介导的细胞功能和调控的复杂机制。其次，我们将研究不同的PI诱导膜穿透、蛋白质自结合和膜变形的机制。最后，我们将确定几个杆状结构域和第十结构域如何不同地穿透和变形细胞膜，以了解细胞膜重塑过程中大量胞浆蛋白复杂和高度协调的动作的基础。该计划的一个长期目标是将从这些研究中学到的原理应用于开发新类型的治疗剂，这些治疗剂可以特异性地调节细胞信号和膜重塑所必需的PI结合蛋白的膜靶向和激活。在这些拟议的研究中使用的主要方法是广泛的生物物理、计算和结构技术以及荧光显微镜下的细胞成像。公共卫生相关性：许多人类疾病，包括癌症、糖尿病和炎症性疾病，已知与磷脂酰肌醇介导的细胞信号缺陷有关。因此，磷脂酰肌醇信号通路是药物开发的主要靶点，但这些通路的复杂性阻碍了药物发现的努力。因此，了解复杂的肌醇磷脂信号转导途径的作用机制将有助于开发新型的治疗药物，用于治疗因肌醇磷脂信号转导途径功能障碍引起的疾病。

DESCRIPTION (provided by applicant): Phosphorylated derivatives of phosphatidylinositol, collectively known as phosphoinositides (PIs), play a key role in the membrane recruitment and activation of cytosolic proteins involved in cell signaling and vesicle trafficking. The majority of PI-responsive proteins contain one or more modular lipid binding domains that specifically recognize PIs. Some of these PI-binding domains not only bind PIs but also induce the deformation of PI-containing membranes. Despite the importance of PI-mediated membrane targeting in health and disease, fundamental understanding of complex mechanisms by which PIs differentially and specifically mediate diverse cellular functions through interactions with these effector domains is still largely lacking. The primary objective of this proposed research is to elucidate the mechanisms by which various PIs mediate cellular processes through interactions with three major types pf effector domains, PX, ENTH, and BAR domains. During the past grating period, we discovered that PIs specifically induce the membrane penetration of many effector domains, thereby modulating the cellular functions and regulation of proteins harboring the domains. We have also developed several new methodologies that will allow for both large-scale and in-depth mechanistic studies of PI-binding and membrane-deforming proteins. With a new paradigm and new methodologies, we will pursue three specific aims in the next project period. First, based on the hypothesis that mammalian PX domains mediate diverse cellular processes through their divergent PI specificities and membrane binding properties, we will determine membrane binding properties of PX domains both comprehensively and systematically to elucidate the complex mechanisms of diverse PX domain-mediated cellular function and regulation. Second, we will investigate the mechanisms by which various PIs induce the membrane penetration, protein self-association, and membrane deformation by ENTH domains. Finally, we will determine how differently several BAR domains and ENTH domains penetrate and deform membranes to understand the basis of complex and highly orchestrated actions of a large number of cytosolic proteins during cellular membrane remodeling. A long-term objective of this program is to apply the principles learned from these studies to the development of new classes of therapeutic agents that can specifically modulate the membrane targeting and activation of PI-binding proteins essential for cell signaling and membrane remodeling. Principal methodologies to be used in these proposed studies are a wide range of biophysical, computational, and structural techniques as well as cell imaging by fluorescence microscopy. PUBLIC HEALTH RELEVANCE: Numerous human diseases, including cancer, diabetes, and inflammatory diseases, are known to be linked to defects in phosphoinositide-mediated cell signaling. Consequently, phosphoinositide signaling pathways are major targets for drug development but the complexity of these pathways has hampered the drug discovery effort. Therefore, understanding the mechanisms underlying complex phosphoinositide signaling pathways will greatly aid in developing new classes of therapeutic agents that can treat diseases caused by dysfunctional phosphoinositide signaling pathways.