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

描述(申请人提供)：从果蝇到人类，Wnt信号通路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形成中发挥着关键作用。除了根尖-基底轴上普遍存在的细胞极化外，许多上皮组织和器官还在上皮平面内表现出极化，称为Wnt/平面细胞极性(PCP)。Wnt/PCP信号蛋白的核心，包括FrizzledFz(Fz)受体和disheveled(Dsh；哺乳动物中的Dvl)，参与果蝇翅膀的组织发育。在脊椎动物中的后续工作确定了Wnt/PCP途径在进化上是保守的。DVL的质膜靶向在规范的(连环蛋白)和Wnt/PCP信号转导中都是一个关键步骤。在哺乳动物中，DVL以三种可能的冗余功能亚型(Dvl1、Dvl2和Dvl3)存在，每种亚型都包含三个保守的结构域，称为DIX、PDZ和DEP。DVL PDZ结构域介导与FZ受体的结合，而DEP结构域通过一种尚不清楚的机制促进DVL与质膜的靶向。对Wnt/PCP途径的体内研究证实，Dvl1与Fz受体的相互作用既是pH依赖的，也是电荷依赖的。此外，生化分析和诱变分析表明，Dvl1 DEP结构域与包括磷脂酸(PA)在内的阴离子磷脂具有弱相互作用。利用核磁共振(NMR)光谱，研究人员已经识别并定位了参与PA识别的Dvl2 DEP结构域残基。因此，该项目的具体目标1是表征Dvl2 DEP结构域的脂结合机制。由于其表面存在一个大的基本补丁，DEP结构域可能识别膜上的一对(或更多)脂类。结合核磁共振滴定、脂质体结合分析、色氨酸荧光和表面等离子体共振分析，在一系列生理pH值范围内，将被用来定义Dvl2DEP结构域脂结合特性。由于DVL PDZ与FZ受体弱结合，因此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，在特定目标2中，将测量嵌入胶束中的PA(以及特定目标1中确定的其他脂类配体)的DEP结构域的亲和力光谱，以开发预测亚细胞靶向的定量基础。将定义头基连接和疏水插入的协同效应，以及脂质体结合特性。DEP的膜穿透性将通过顺磁自旋标记和带有猝灭剂的色氨酸荧光分析来阐明。膜结合的决定因素将使用表面等离子激元共振进行动力学表征。了解Dvl2 DEP结构域识别磷脂的结构基础将使我们能够推导出机制上的见解，设计基于机制的抑制剂，创造功能特异性的突变，并精确地操纵Wnt/PCP途径。 叙述：Wnt信号通路描述了几个不同的蛋白质网络的激活，其特征是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取决于其参与蛋白的亚细胞定位。通过从分子上定义Wnt途径中的蛋白质相互作用，研究人员应该能够合理地操纵宿主蛋白的亚细胞膜靶向。

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.