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结构域是最常见的蛋白质相互作用结构域之一。 遇到的。它们通常与其他交互模块结合使用， 在建立和维持细胞极性，指导蛋白质运输， 协调突触信号。它们的重要性被严重的神经元和 在PDZ结构域敲除小鼠中观察到的发育表型以及它们在 人类先天性疾病PDZ结构域功能的巨大多样性表现在它们的 丰度：在小鼠基因组中编码至少246个PDZ结构域。了解 它们各自的作用，首先有必要在广泛的范围内定义它们的识别特性， 生物相关的时尚，并构建模型，准确地预测他们的相互作用 在哺乳动物蛋白质组中。在这里，结合实验和计算的方法， 用于检测、测量、预测和干扰PDZ结构域介导的蛋白质 相互作用，并研究其生物相关性。在目标1中，提出了一种战略， 使用蛋白质微阵列和高通量荧光偏振来训练统计 该模型预测PDZ结构域肽在蛋白质组范围内的相互作用。实验 最初将集中在小鼠PDZ结构域，但随后将扩展到包括人类 域也。这些努力所产生的实验数据和预测模型 应该证明对研究含PDZ结构域蛋白的生物界有价值。 在目标2和3中，将在更大范围内研究新型相互作用的生物学相关性。 详细初步实验发现了PDZ之间的几种新型相互作用 结构域和β-连环蛋白。β在目标2中，提出了实验来检验假设，即-β 连环蛋白在形成或维持紧密连接中起功能性作用。初步实验 还发现了PDZ结构域与多药耐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表达、亚细胞定位和功能 这些药物转运体的了解多药耐药蛋白是如何调节的， 提供新的方法来对抗常见的耐药性机制。总的来说，我们希望 我们的高通量实验所强调的新的相互作用和 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与蛋白质相互作用的强大资源

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