Membrane Targeting by Phosphoinositide-Binding Proteins

描述（由申请人提供）：磷脂酰肌醇的磷酸化衍生物，统称为磷酸肌醇（PI），在细胞信号传导和囊泡运输中涉及的胞质蛋白的膜募集和活化中发挥关键作用。大多数PI响应蛋白含有一个或多个特异性识别PI的模块化脂质结合结构域。这些PI结合结构域中的一些不仅结合PI，而且还诱导含PI的膜的变形。尽管PI介导的膜靶向在健康和疾病中的重要性，但对PI通过与这些效应结构域的相互作用而差异性和特异性地介导多种细胞功能的复杂机制的基本理解仍然很缺乏。这项研究的主要目的是阐明各种PI通过与三种主要类型的效应域PX、ENTH和BAR域相互作用来介导细胞过程的机制。在过去的光栅期间，我们发现PI特异性地诱导许多效应器结构域的膜穿透，从而调节细胞功能和含有结构域的蛋白质的调节。我们还开发了几种新的方法，将允许PI结合和膜变形蛋白的大规模和深入的机制研究。在新的模式和新的方法下，我们将在下一个项目期间实现三个具体目标。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介导不同的细胞过程的假设，我们将全面和系统地确定PX结构域的膜结合特性，以阐明不同PX结构域介导的细胞功能和调节的复杂机制。其次，我们将研究各种PI通过ENTH结构域诱导膜穿透、蛋白自缔合和膜变形的机制。最后，我们将确定几个BAR域和ENTH域如何不同地穿透和变形膜，以了解细胞膜重塑过程中大量胞质蛋白的复杂和高度协调的作用的基础。该计划的长期目标是将从这些研究中学到的原理应用于开发新类别的治疗剂，这些治疗剂可以特异性调节细胞信号传导和膜重塑所必需的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，以及荧光显微镜细胞成像。公共卫生相关性：已知许多人类疾病，包括癌症、糖尿病和炎症性疾病，都与磷酸肌醇介导的细胞信号传导缺陷有关。因此，磷酸肌醇信号通路是药物开发的主要目标，但这些通路的复杂性阻碍了药物发现的努力。因此，了解复杂的磷脂酰肌醇信号通路的机制将大大有助于开发新的治疗药物，可以治疗由功能失调的磷脂酰肌醇信号通路引起的疾病。

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.