Structure, dynamics and causality in protein interaction networks

问题和假设：蛋白质-蛋白质相互作用(PPI)对生物学的重要性反映在大规模研究它们的巨大努力中，包括我们自己的努力，这导致了第一次描述体内的PPI网络(Tarassov，等人)。《科学》，2008)。在过去的资助期间，我们开发并验证了一种新的方法来研究生化网络的动力学和确定蛋白质的功能，发现途径和变构的新机制。该方法是基于在大范围内测量同聚体蛋白质复合体(HPC)的动力学。 目的：我们的具体目标是：目标1.构建基于HPC的高质量、基于机制的信号转导和代谢通路，并分析HPC动力学的潜在机制。目的2.使用HPC分析和基于机制的HPC哨兵来预测特定营养限制下的信号和代谢网络拓扑。目的3.将HPC动力学推广到生物化学网络组织的多重扰动和预测。 方法：除了上述DHFR PCA菌株阵列外，我们还将使用质谱仪、GFP与蛋白质的融合来确定定位和丰度，以及一种新的红外荧光蛋白PCA来确定HPC的亚细胞定位变化和共聚焦显微镜来分析HPC变化的物理基础。这将导致一系列具有已知蛋白质复合体或丰度的潜在时空变化的HPC，我们将进一步应用一系列分析工具，主要基于现有的策略，来解释特定信号和代谢途径上的HPC动力学。 这项研究将通过以下方式促进知识的进步： 1-提供了一种直接探测体内生化网络动态的策略。 2-为确定基因功能的现有遗传和质谱学策略提供补充。 揭示动态生化网络组织的新原理和新机制。 4-揭示目前未知功能的基因的新功能。 5-有助于解开细胞机械形成和进化原理的谜团，我们在这方面已经取得了一些进展。

Problem and Hypothesis: The importance of protein-protein interactions (PPIs) to biology is reflected in the enormous efforts to study them on a large scale, including our own efforts, which resulted in a first description of a PPI network in vivo (Tarassov, et al. Science, 2008). In the past funding period we have developed and validated a novel approach to study dynamics of biochemical networks and to determine protein functions, discovery of pathways and a novel mechanism of allostery. The method is based on measuring dynamics of homomeric protein complexes (HPC) on a large scale. Objectives: Our specific aims are: Aim 1., to construct high-quality, mechanism-based sentinels of signal transduction and metabolic pathways based on HPC and analysis of underlying mechanisms of HPC dynamics. Aim 2. To use HPC analysis and mechanism-based HPC sentinels to predict signaling and metabolic network topology under specific nutrient limitation. Aim 3. To extend HPC dynamics to multiple perturbations and prediction of organization of biochemical networks in general. Methodologies: In addition to the DHFR PCA strain array described above, we will dissect the physical basis of changes in HPCs using mass spectroscopy, GFP fusions to the proteins to determine localization and abundance and a novel infrared fluorescent protein PCA to determine changes in subcellular localization of HPC and confocal microscopy. This will result in an array of HPCs with known underlying spatiotemporal changes in proteins complexes or abundance that we call HPC “sentinels” We will further apply a pipeline of analysis tools, largely based on existing strategies, to interpret HPC dynamics on specific signaling and metabolic pathways. This study will contribute to advancement of knowledge by: 1- Providing a strategy to directly probe the dynamics of biochemical networks in vivo. 2- Providing a complement to existing genetic and mass-spectroscopic strategies for determining gene function. 3 - Revealing new principles and mechanisms of dynamic biochemical network organization. 4 - Revealing novel functions of genes for which function is presently unknown. 5 - Contributing towards solving the mystery of the principles of formation and evolution of the cellular machinery, on which we have already made some progress.