TCP1: ANALYSIS OF LYSINE MODIFICATION USING PROTEIN MICROARRAYS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 技术核心项目1(TCP-1)蛋白质微阵列：开发新的蛋白质微阵列技术，以识别蛋白质相互作用、小分子蛋白质相互作用、酶底物、结合的特异性和酶反应。这里要开发的技术的一个关键新方面是使用体外翻译系统作为 蛋白质生产，这可以显著增加产量。 我们建议研究分析赖氨酸修饰的最佳反应条件，确定酵母中不同(去)乙酰酶、甲基酶和(去)泛素酶的下游靶点，创建用于分析特定蛋白质乙酰化活性的E.Coil和人类线粒体蛋白质微阵列，并改进现有的蛋白质微阵列技术。这些研究有望提供通过不同类型的赖氨酸侧链上的翻译后修饰来调节蛋白质功能的全球图景，并进一步改善蛋白质微阵列技术的应用。具体来说，我们计划开展以下相互关联的活动： 1)制备酵母蛋白质组微阵列，系统地测试各种与赖氨酸修饰活性相关的生物检测的最佳检测条件。 2)构建一个包含大肠杆菌基因组中所有基因的表达文库，并对其编码的蛋白质进行纯化，建立大肠杆菌蛋白质组微阵列；利用这些大肠杆菌蛋白质组微阵列筛选新鉴定的E.coil的底物 乙酰基酶和脱乙酰基酶。 3)构建人线粒体蛋白芯片，以确定人Sirt3p脱乙酰基酶的相互作用伙伴，并确定相应的乙酰转移酶(S)。 4)进一步改进蛋白质微阵列技术，使之成为一种更快、更容易、更方便用户使用的方法，可以与其他检测方法相结合

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Technology Core Projects 1 (TCP-1) Protein microarrays: Developing new protein microarray technology to identify protein protein interactions, small moleculeprotein interactions, enzyme substrates, specificity of binding and enzymatic reactions. A key new aspect of the technology to be developed here is to use in vitro translation systems as the starting point for protein production, which can significantly increase yields. We propose to investigate the optimal reaction conditions for analyzing lysine modifications, to identify downstream targets of different (de)acetylases, methylases, and (de)ubiquitylases in yeast, to create E. coil and human mitochondrial protein microarrays for analyzing specific protein acetylation activities, and to improve current protein microarray technologies. These studies are expected to provide a global picture of regulating protein functions through different kinds of posttranslational modifications on lysine side chains, and to further improve the application of protein microarray technologies. Specifically, we plan the following interrelated activities: 1) To fabricate yeast proteome microarrays and systematically test the optimal assay conditions for different kinds of bioassays related to lysine modification activities. 2) To construct an expression library containing all genes in the genome of E. coli, and purify their encoded proteins to create an E. coli proteome microarray; use these E. coli proteome microarrays to screen for substrates of the newly identified E. coil acetylase and deacetylase. 3) To construct a prioritized human mitochondrial protein chip to identify the interacting partners of human Sirt3p deacetylase and identify the corresponding acetyltransferase(s). 4) To further improve the protein microarray technologies as a faster, easier and userfriendlier approach that can be coupled to other detection met