Genome-wide Investigation of PDZ Domain Specificity

描述(由申请人提供)： 大多数接收和处理信号的真核蛋白质都是由相互作用和催化域的组合构成的。在过去十年确定的许多相互作用领域中，PDZ是最常遇到的领域之一。它们经常与其他相互作用模块一起被发现，并在指导受体酪氨酸激酶介导的信号的特异性、建立细胞极性、指导蛋白质运输和协调突触信号方面发挥作用。在PDZ基因敲除小鼠中观察到的严重神经元和发育表型，以及它们在亚瑟综合征和德杰林-索塔斯神经病等人类先天性疾病中的暗示，都突显了它们的重要性。PDZ功能的巨大多样性体现在它们的丰富性上；在小鼠基因组中编码了250多个PDZ。要了解它们各自的角色，首先必须以全面和相关的方式定义它们的识别属性。以前定义PDZ选择性的努力只集中在几个区域上，并且依赖于随机多肽的集合，而不是生理配体。为了提供对PDZ功能的全基因组理解，将使用蛋白质微阵列技术相对于大量基因组编码的配体来研究小鼠基因组中编码的每个PDZ的选择性。这项建议的目标是：(1)克隆、表达和纯化每个小鼠PDZ结构域；(2)筛选每个PDZ和每个PDZ之间的相互作用；(3)筛选每个PDZ和222个基因组编码的多肽配体，以确定PDZ和蛋白质之间的可能相互作用；(4)从生化角度研究预测相互作用的子集的生理相关性；以及(5)构建一个关系数据库，将蛋白质微阵列数据与已发表的PDZ结构域信息整合在一起。总而言之，这些努力应该为在全基因组水平上理解PDZ功能提供必要的基础，并有助于未来在PDZ功能出现问题时进行适当干预的努力。

DESCRIPTION (provided by applicant): Most eukaryotic proteins that receive and process signals are constructed from a combination of interaction and catalytic domains. Among the many interaction domains identified in the past decade, PDZ's are one of the most frequently encountered. They are often found in combination with other interaction modules and play a role in directing the specificity of receptor tyrosine kinase-mediated signaling, in establishing cell polarity, in directing protein trafficking, and in coordinating synaptic signaling. Their importance is underscored by the severe neuronal and developmental phenotypes observed in PDZ knockout mice and by their implication in human congenital diseases like Usher syndrome and Dejerine-Sottas neuropathy. The enormous diversity of PDZ function is manifest in their abundance; there are over 250 PDZ's encoded in the mouse genome. To understand their individual roles, it is necessary first to define their recognition properties in a comprehensive and relevant fashion. Previous efforts to define PDZ selectivity have focused on only a few domains and have relied on collections of randomized peptides, rather than on physiological ligands. In order to provide a genome-wide understanding of PDZ function, the selectivity of every PDZ encoded in the mouse genome will be investigated relative to a large collection of genomically-encoded ligands using protein microarray technology. The aims of this proposal are: (1) to clone, express, and purify every mouse PDZ domain; (2) to screen every PDZ with every PDZ to reveal putative PDZ-PDZ interactions; (3) to screen every PDZ with 222 genomically-encoded peptide ligands to identify putative PDZ-protein interactions; (4) to investigate the physiological relevance of a subset of predicted interactions biochemically; and (5) to construct a relational database that integrates protein microarray data with published information on PDZ domains. Collectively, these efforts should provide the necessary foundation to understand PDZ function on a genome-wide level and aid future efforts to intervene appropriately when PDZ function goes awry.