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，Wnt信号传导途径在胚胎发育以及成年和疾病过程中的细胞命运决定和模式化中起着至关重要的作用。 除了沿顶-基底轴沿着普遍存在的细胞极性之外，许多上皮组织和器官还在上皮平面内显示出被称为Wnt/平面细胞极性（PCP）的极化。 Wnt/PCP信号传导蛋白的核心，包括卷曲（Fz）受体和Dishevelled（Dsh;哺乳动物中的Dvl），参与果蝇翅膀中的组织发育。 随后在脊椎动物中的工作已经确定Wnt/PCP途径在进化上是保守的。 Dvl的质膜靶向是经典（β-连环蛋白）和Wnt/PCP信号传导中的关键步骤。 在哺乳动物中，Dvl以推定冗余功能的三种同种型（Dvl 1、Dvl 2和Dvl 3）存在，每种同种型由称为DIX、PDZ和DEP的三个保守结构域组成。 Dvl PDZ结构域介导与Fz受体的结合，而DEP结构域通过尚不清楚的机制促进Dvl靶向质膜。 Wnt/PCP途径的体内研究证实，Dvl 1与Fz受体的相互作用是pH和电荷依赖性的。 此外，Dvl 1 DEP结构域与阴离子磷脂，包括磷脂酸（PA）的弱相互作用，通过生化测定和诱变分析证明。 使用核磁共振（NMR）光谱，研究者已经鉴定并绘制了参与PA识别的Dvl 2 DEP结构域残基。 因此，该项目的具体目标1是表征Dvl 2 DEP结构域的脂质结合机制。 由于其表面存在大的碱性斑块，DEP结构域可能识别膜上的一对（或更多）脂质。 将使用在生理pH值范围内的NMR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的组合来定义Dvl 2 DEP结构域脂质结合性质。 由于Dvl PDZ与Fz受体弱结合，因此还将研究DEP结构域在这种相互作用中的贡献。 为了使用膜模拟物，他已经从NMR分析发现Dvl 2 DEP结构域在胆酸钠胶束中是稳定的。 因此，在特定目标2中，将测量DEP结构域对包埋在胶束中的PA（和特定目标1中鉴定的其他脂质配体）的亲和力谱，以开发用于预测亚细胞靶向的定量基础。 头基连接和疏水插入的协同作用将被定义，将脂质体结合特性。 DEP的膜渗透将用顺磁自旋标记和用淬灭剂通过色氨酸荧光分析来阐明。 膜协会的决定因素将使用表面等离子体共振动力学特征。 了解Dvl 2 DEP结构域识别磷脂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，产生功能特异性突变，并精确操纵Wnt/PCP途径。 说明：Wnt信号通路描述了几种不同的蛋白质网络的激活，其特征在于它们在胚胎发生中的作用，如轴形成，神经系统模式和细胞行为的协调，以及它们在癌症发展中的意义。 Wnt信号通路不同分支之间的平衡依赖于其参与蛋白的亚细胞定位。 通过在分子上定义Wnt通路中的蛋白质相互作用，研究者应该能够合理地操纵宿主蛋白质的亚细胞膜靶向。

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.