Measuring and Predicting PDZ Domain-Peptide Interactions on a Genome-wide Scale

在众多的蛋白质相互作用结构域中，PDZ结构域是最常见的结构域之一 遇到了。它们经常与其他交互模块组合在一起，并扮演一个 在建立和维持细胞极性、指导蛋白质运输以及在 协调突触信号。它们的重要性被严重的神经元和 PDZ结构域敲除小鼠的发育表型观察及其在临床中的意义 人类先天疾病。PDZ区域功能巨大多样性体现在它们的 丰度：小鼠基因组中至少有246个PDZ结构域编码。要理解 它们各自的角色，首先要从广义和广义上定义它们的识别属性 生物相关的时尚，并构建准确预测它们相互作用的模型 在哺乳动物蛋白质组中。这里，一种实验和计算相结合的方法 用于检测、测量、预测和干扰PDZ结构域介导的蛋白质。 并调查它们之间的生物学相关性。在目标1中，提出了一项战略， 使用蛋白质微阵列和高通量荧光偏振来训练统计 在蛋白质组范围内预测PDZ结构域肽相互作用的模型。实验 最初将专注于小鼠的PDZ结构域，但随后将扩展到包括人类 域也是如此。由这些努力产生的实验数据和预测模型 对研究含有PDZ结构域的蛋白质的生物界来说应该是有价值的。 在目标2和目标3中，将对新的相互作用的生物学相关性进行更深入的研究 细节。初步实验发现了PDZ之间的几种新的相互作用 域名和-连环蛋白。在目标2中，提出了实验来检验以下假设 连环蛋白在形成或维持紧密连接方面起着重要作用。初步实验 还发现了PDZ结构域和多药之间的许多新的相互作用 抗性蛋白。在目标3中，提出了实验来确定PDZ结构域如何包含 蛋白质调节细胞表面的表达、亚细胞定位和功能。 这些毒品运输者中。了解多药耐药蛋白是如何调控的可能 提供了新的方法来对抗常见的耐药机制。总体而言，我们希望 我们的高通量实验所强调的新的交互作用和 由这些数据产生的预测模型将被生物界广泛采用 作为研究PDZ结构域介导的蛋白质相互作用的有力资源

Among the myriad protein interaction domains, PDZ domains are one of the most frequently encountered. They are often found in combination with other interaction modules and play an important role in establishing and maintaining 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 domain knockout mice and by their implication in human congenital diseases. The enormous diversity of PDZ domain function is manifest in their abundance: there are at least 246 PDZ domains encoded in the mouse genome. To understand their individual roles, it is necessary first to define their recognition properties in a broad and biologically relevant fashion and to construct models that accurately predict their interactions across the mammalian proteome. Here, a combined experimental and computational approach is presented to detect, measure, predict, and perturb PDZ domain-mediated protein¿protein interactions and to investigate their biological relevance. In Aim 1, a strategy is presented that uses protein microarrays and high-throughput fluorescence polarization to train a statistical model that predicts PDZ domain¿peptide interactions on a proteome-wide scale. Experiments will initially focus on mouse PDZ domains, but will subsequently be extended to include human domains as well. Both the experimental data and predictive model arising from these efforts should prove valuable to the biological community that studies PDZ domain-containing proteins. In Aims 2 and 3, the biological relevance of novel interactions will be investigated in greater detail. Preliminary experiments have uncovered several novel interactions between PDZ domains and β-catenin. In Aim 2, experiments are proposed to test the hypothesis that β- catenin plays a functional role in forming or maintaining tight junctions. Preliminary experiments have also uncovered numerous novel interactions between PDZ domains and multidrug resistance proteins. In Aim 3, experiments are proposed to determine how PDZ domaincontaining proteins modulate the cell-surface expression, subcellular localization, and function of these drug transporters. Understanding how multidrug resistance proteins are regulated may provide new ways to counteract common mechanisms of drug resistance. Overall, it is our hope that both the novel interactions highlighted by our high-throughput experiments and the predictive models resulting from these data will be broadly adopted by the biological community as a powerful resource to study PDZ domain-mediated protein¿protein interactions