Structural basis of Dishevelled-2 membrane targeting in the Wnt/PCP signaling pat

描述（由申请人提供）：从果蝇到人类，在胚胎发育、成人和疾病过程中，Wnt信号通路在细胞命运决定和模式形成中起着至关重要的作用。除了沿顶基轴普遍存在的细胞极性外，许多上皮组织和器官也表现出上皮平面内的极化，称为Wnt/平面细胞极性（PCP）。Wnt/PCP信号蛋白的核心，包括卷曲（Fz）受体和disheveled (Dsh; Dvl)，参与果蝇翅膀的组织发育。随后在脊椎动物中的工作已经确定Wnt/PCP通路是进化保守的。质膜靶向Dvl是规范（¿-catenin）和Wnt/PCP信号传导的关键步骤。在哺乳动物中，Dvl以三种假定的冗余功能亚型（Dvl1、Dvl2和Dvl3）存在，每种亚型都由三个保守结构域组成，即DIX、PDZ和DEP。Dvl的PDZ结构域介导与Fz受体的结合，而DEP结构域则通过尚不清楚的机制促进Dvl靶向质膜。Wnt/PCP通路的体内研究证实，Dvl1与Fz受体的相互作用既依赖于pH值，也依赖于电荷。此外，生化分析和诱变分析表明，Dvl1 DEP结构域与阴离子磷脂（包括磷脂酸（PA））存在弱相互作用。利用核磁共振（NMR）波谱，研究者已经确定并绘制了参与PA识别的Dvl2 DEP结构域残基。因此，该项目的Specific Aim 1是表征Dvl2 DEP结构域的脂质结合机制。由于在其表面存在一个大的基本斑块，DEP结构域有可能在膜上识别一对（或更多）脂质。核磁共振滴定、脂质体结合测定、色氨酸荧光和表面等离子体共振分析在一系列生理pH值的组合将用于定义Dvl2 DEP结构域脂质结合特性。由于Dvl PDZ与Fz受体弱结合，DEP结构域在这种相互作用中的作用也将被研究。为了使用膜模拟物，他从核磁共振分析中发现Dvl2 DEP结构域在胆酸钠胶束中是稳定的。因此，在Specific Aim 2中，将测量嵌入胶束中的PA（以及在Specific Aim 1中鉴定的其他脂质配体）的DEP结构域的亲和谱，以建立预测亚细胞靶向的定量基础。头基团结扎和疏水插入的协同效应将被定义，脂质体结合特性也将被定义。DEP的膜渗透将通过顺磁自旋标记和色氨酸荧光猝灭剂分析来阐明。膜结合的决定因素将使用表面等离子体共振进行动力学表征。了解Dvl2 DEP结构域磷脂识别的结构基础将使我们能够获得机制见解，设计基于机制的抑制剂，创建功能特异性突变，并精确操纵Wnt/PCP途径。

DESCRIPTION (Provided by Applicant): From Drosophila to humans, the Wnt signaling pathway plays a crucial role in cell fate determination and patterning during embryonic development as well as in adults and in disease processes. In addition to the ubiquitous cell polarity along the apical-basal axis, many epithelial tissues and organs also display polarization within the plane of epithelium referred to as Wnt/planar cell polarity (PCP). A core of Wnt/PCP signaling proteins, including the Frizzled (Fz) receptor and Dishevelled (Dsh; Dvl in mammals), participate in tissue development in the Drosophila wing. Subsequent work in vertebrates has determined that the Wnt/PCP pathway is evolutionarily conserved. Plasma membrane targeting of Dvl is a critical step in both canonical (¿-catenin) and Wnt/PCP signaling. In mammals, Dvl exists in three isoforms (Dvl1, Dvl2, and Dvl3) of putative redundant function with each consisting of three conserved domains known as DIX, PDZ, and DEP. Whereas the Dvl PDZ domain mediates binding to the Fz receptor, the DEP domain facilitates targeting of Dvl to the plasma membrane by a yet unclear mechanism. In vivo studies of the Wnt/PCP pathway established that the interaction of Dvl1 with the Fz receptor is both pH- and charge-dependent. Furthermore, a weak interaction of Dvl1 DEP domain with anionic phospholipids, including phosphatidic acid (PA), was demonstrated by biochemical assays and mutagenesis analyses. Using nuclear magnetic resonance (NMR) spectroscopy, the investigator has identified and mapped the Dvl2 DEP domain residues involved in PA recognition. Thus, Specific Aim 1 of the project is to characterize the mechanism of lipid binding of the Dvl2 DEP domain. Due to the presence of a large basic patch on its surface, it is possible that the DEP domain recognizes a pair (or more) of lipids at the membrane. A combination of NMR titrations, liposome-binding assays, tryptophan fluorescence, and surface plasmon resonance analyses in a range of physiological pH values will be used to define the Dvl2 DEP domain lipid-binding properties. Since the Dvl PDZ weakly binds to the Fz receptor, the contribution of the DEP domain in this interaction will also be investigated. With the purpose of using membrane mimetics, he has found that the Dvl2 DEP domain is stable in sodium cholate micelles from NMR analysis. Consequently, in Specific Aim 2, the spectrum of affinities of the DEP domain for PA (and other lipids ligands identified in Specific Aim 1) embedded in micelles will be measured to develop a quantitative basis for predicting subcellular targeting. The cooperative effects of headgroup ligation and hydrophobic insertion will be defined, as will liposome-binding properties. Membrane penetration of DEP will be elucidated with paramagnetic spin labels and by tryptophan fluorescence analysis with quenchers. Determinants of membrane association will be kinetically characterized using surface plasmon resonance. Understanding the structural basis of phospholipid recognition by the Dvl2 DEP domain would enable us to derive mechanistic insights, design mechanism-based inhibitors, create functionally-specific mutations, and precisely manipulate the Wnt/PCP pathway. NARRATIVE: The Wnt signaling pathway describes the activation of several distinct networks of proteins characterized for their roles in embryogenesis such as axis formation, nervous system patterning, and coordination of cell behavior, as well as for their implications in cancer development. Equilibrium among the different branches of the Wnt signaling pathway depends upon the subcellular localization of its participant proteins. By molecularly defining protein interactions in the Wnt pathway, the investigator should be able to rationally manipulate subcellular membrane targeting of host proteins.