Analysis of signaling network by protein-interaction proteomics

为了了解依赖于蛋白质修饰的蛋白质-蛋白质相互作用的分子机制，主要的C激酶底物蛋白质Marcks的蛋白质晶体以钙调蛋白复合体的形式产生。另一个靶点是钙调素与神经元特异性NAP-22蛋白的N端肉豆蔻酸化结构域复合。前者的X射线结构揭示了Marcks-钙调蛋白相互作用所涉及的磷酸化依赖的蛋白质-蛋白质相互作用。后者的结构表明，肉豆蔻基部分直接参与了NAP-22-钙调蛋白的相互作用。第二个研究项目涉及细胞细胞器的蛋白质组学分析。建立了一种以抗体为基础的纯化方法，从大鼠肝脏中获得“纯净”的过氧化物体。阐明了包括几种新蛋白质在内的蛋白质组成。发现了一种新的过氧化物酶体特异性Lon蛋白水解酶，它是一种分子伴侣。对另一个细胞器--脂滴进行了类似的分析，发现其中一个小G蛋白Rab18定位于细胞器中。第三个研究项目是分析涉及蛋白质磷酸化和蛋白质相互作用网络的信号网络。作为模型系统，通过磷酸化蛋白质组分析分离酪氨酸磷酸化蛋白来分析EGF受体信号网络。大约150种蛋白质被鉴定出来，这些蛋白质要么是自身酪氨酸磷酸化的，要么是与磷酸化蛋白相互作用的蛋白质。其中约三分之一是新的蛋白质。通过分析相互作用蛋白和依赖于EGF的磷酸化来阐明这些蛋白的生理功能。其中几个被发现参与了EGF受体下调的调节。识别这些蛋白质和几个磷酸化位点的抗体被提出并用于分析EGF受体下游的信号网络。

To understand molecular mechanisms underlying protein modification-dependent protein-protein interactions, protein crystals of MARCKS, a major C kinase substrate protein, were produced as calmodulin complex. Another target was calmodulin complexed with the N-terminal myristoylated domain of neuron specific NAP-22 protein. The X-ray structure of the former complex revealed the phosphorylation-dependent protein-protein interaction involved in the MARCKS-calmodulin interaction. The latter structure demonstrated that the Myristoyl moiety in directly involved in the NAP-22-calmodulin interaction. The second research project deals with the proteomic analysis of cellular organelles. An antibody-based purification method was established to obtain ‘pure' peroxisomes from rat liver. Protein components including several novel proteins were elucidated. A novel peroxisome-specific Lon protease, a molecular chaperon, was discovered. Another organelle, lipid droplet, was analyzed similarly, and one of small G proteins, Rab 18, was found to be localized in the organelle. The third research project was to analyze signaling networks that involve protein phosphorylation and protein interaction networks. As a model system, the EGF receptor signaling networks were analyzed by isolating tyrosine-phosphorylated proteins by phospho-proteome analysis. About 150 proteins that were either tyrosine phosphorylated themselves or proteins interacting with phosphorylated proteins were identified. About one third of them were novel proteins. The physiological functions of these proteins were elucidated by analyzing interacting proteins and EGF-dependent phosphorylation. Several of them were found to be involved in the regulation of EGF receptor downregulation. Antibodies that recognize these proteins and several phosphorylation sites were raised and used to analyze the signaling networks downstream of the EGF receptor.