Motif-based elucidation of protein modification codes

描述（由申请人提供）：保守组蛋白残基的翻译后修饰调节染色体生物学的许多方面。磷酸化、乙酰化、甲基化和去亚胺化的氨基酸簇产生了高度的组合复杂性，即所谓的“组蛋白密码”，它有可能指定大量不同的下游事件。对组蛋白密码的全面解释需要能够解读多种翻译后修饰对组蛋白相关蛋白结合和活性的贡献的方法。尤其不清楚的是组蛋白磷酸化调控作用的分子基础，特别是在有丝分裂过程中。作为一个模型系统，我们将研究haspin，一个在中期正常染色体排列所需的激酶。Haspin在体内有丝分裂过程中磷酸化核心组蛋白H3的新残基Thr-3。我们假设这种类型的磷酸化与其他修饰结合，在有丝分裂过程中在组蛋白上产生调节蛋白的结合位点。在Aim 1中，我们提出了一种新的、广泛适用的方法来筛选人类蛋白质组中与集群翻译后修饰结合的蛋白质。作为一个具体的例子，我们将分离与磷酸化组蛋白H3 （Thr-3）肽结合的蛋白质，其周围残基有或没有乙酰化、甲基化、去亚胺化和磷酸化的组合。在Aim 2中，我们提出了一种通用的方法来确定修饰特异性酶和结合蛋白识别的翻译后修饰模式。作为测试案例，我们将使用创新技术筛选一系列代表组蛋白H3的修饰肽库，以确定haspin活性的翻译后要求。总之，这些基于基序的方法将使我们能够通过组蛋白修饰来定义信号通路。最近的研究表明，除了磷酸化，许多细胞蛋白的甲基化和乙酰化，并证明了它们在转录因子活性、细胞运动、细胞内信号传导和运输、免疫突触形成和凋亡等多种过程中的重要性。虽然组蛋白是显著的例子，但蛋白质修饰代码的存在很可能是一种更普遍的现象。因此，确定这些标记的功能影响的方法的发展是生物医学所有领域未来工作的关键要求。

DESCRIPTION (provided by applicant): Post-translational modifications of conserved histone residues regulate many aspects of chromosome biology. Clusters of phosphorylated, acetylated, methylated and deiminated amino acids produce a high degree of combinatorial complexity, the so-called "histone code," that has the potential to specify a vast number of different downstream events. Full interpretation of the histone code requires approaches that can unscramble the contribution of multiple post-translational modifications to the binding and activity of histone-associated proteins. Particularly unclear is the molecular basis for the regulatory effect of histone phosphorylation, especially during mitosis. As a model system, we will study haspin, a kinase required for normal chromosome alignment at metaphase. Haspin phosphorylates a novel residue, Thr-3, of the core histone H3 during mitosis in vivo. We hypothesize that this type of phosphorylation, in combination with other modifications, generates binding sites on histones for regulatory proteins during mitosis. In Aim 1, we propose new and broadly applicable methodology to screen the human proteome for proteins that bind to clustered post-translational modifications. As a specific example, we will isolate proteins that bind to phospho-histone H3 (Thr-3) peptides with or without combinations of acetylated, methylated, deiminated and phosphorylated surrounding residues. In Aim 2, we propose a general means to determine the patterns of post-translational modification recognized by modification-specific enzymes and binding proteins. As a test case, we will use innovative technology to screen an array of modified peptide libraries representing histone H3 to determine the post-translational requirements for haspin activity. Together, these motif-based approaches will allow us to define pathways of signaling through histone modification. Recent studies have revealed, in addition to phosphorylation, the methylation and acetylation of many cellular proteins and demonstrated their importance in diverse processes including transcription factor activity, cell motility, intracellular signaling and trafficking, immune synapse formation and apoptosis. While histones are striking examples, it is likely that the existence of protein modification codes is a more general phenomenon. The development of methodology to determine the functional impact of these marks is therefore a critical requirement for future work in all areas of biomedicine.