Studying chromosome function using chemical biology

 描述(由申请人提供)：细胞分裂中的错误，即复制的DNA在两个子细胞之间分裂的过程，与疾病和发育缺陷有关。不适当的细胞分裂也被广泛应用于治疗癌症等疾病的治疗策略中。准确的染色体分离、适当的细胞周期进展和DNA损伤的修复依赖于关键蛋白质识别组蛋白的翻译后修饰，组蛋白组装染色质的基本单位，即核小体。用传统的方法很难全面地分析“读取”组蛋白上特定的翻译后修饰(或“标记”)以调节染色体生物学的蛋白质。我们的目标是通过设计新的基于化学的策略来填补这一知识空白，从而在特定的细胞环境中描述组蛋白标记的直接“读者”。S使用光交联剂，通过共价键，将动态的和弱的(通常是微摩尔的)蛋白质-蛋白质相互作用转化为稳定的缔合。这一策略与最先进的定量质谱学相结合，以确定特定组蛋白“标记”的“读者”。在完成项目期间，我们开发了这一“化学蛋白质组学”方法，命名为CLASPI(交叉连接辅助和基于SILAC的蛋白质识别)，并展示了它识别组蛋白“标记”的新“读取器”的能力，包括组蛋白H3甲基化和磷酸化。我们将以我们最近的出版物和初步数据为基础，重点确定两种不同的组蛋白磷酸化“标记”的“阅读器”，一个指示DNA损伤，并在细胞毒药物的长时间有丝分裂停止期间观察到；另一种在细胞分裂时与染色体相关，依赖于极光激酶的活性，极光激酶是细胞分裂的保守调节因子，也是抗癌药物的靶标。在细胞周期的不同阶段“读取”这些组蛋白“标记”的功能意义将通过高分辨率显微镜分析、关键蛋白被剔除的小鼠胚胎成纤维细胞以及作用于快速时间尺度上阻止其活性的化学抑制剂来检验。 负责在细胞中产生这些“记号”的激酶。该提案有三个目的：(1)鉴定能够“读取”组蛋白H_2AX上的磷酸化“标记”的蛋白质；(2)表征组蛋白H_2AX的磷酸化功能--“阅读器”；(3)描述活细胞中组蛋白翻译后修饰的“阅读器”。这项拟议的研究结合了化学和生物学的方法，以解开组蛋白标记是如何被蛋白质“解释”的，从而通过调节染色体分离、DNA损伤修复和细胞周期进程来确保稳定的基因组传播。这些研究还应该阐明细胞分裂的化学抑制剂如何杀死癌细胞。此外，对关键的翻译后修饰依赖的蛋白质-蛋白质相互作用的全面描述应该导致为治疗剂选择新的靶点。最后，我们开发的方法是通用的，可以广泛应用于剖析细胞中蛋白质-蛋白质相互作用的复杂和动态网络。

 DESCRIPTION (provided by applicant): Errors in cell division, the process during which replicated DNA is partitioned between two daughter cells, have been linked to diseases and developmental defects. Improper cell division has also been exploited in therapeutic strategies widely used to treat diseases, such as cancer. Accurate chromosome segregation, proper cell cycle progression and repair of DNA damage rely on key proteins recognizing post-translational modifications on histones, which assemble the basic units of chromatin known as nucleosomes. Comprehensively profiling proteins that `read' specific post-translational modifications (or `marks') on histones to regulate chromosome biology has been difficult using conventional approaches. Our goal is to fill this knowledge gap by devising new chemistry-based strategies to profile direct `readers' of histone `marks' in specific cellular contexts. Central to our approach s the conversion of dynamic and weak (typically micromolar) protein-protein interactions into stable associations, via covalent bonds, using photo-cross-linkers. This strategy is combined with state-of-the-art quantitative mass spectrometry to identify `readers' of specific histone `marks'. In the completed project period, we have developed this `chemical proteomics' approach, named CLASPI (cross-linking-assisted and SILAC-based protein identification), and demonstrated its ability to identify new `readers' of histone `marks', including histone H3 methylation and phosphorylation. We will build on our recent publications and preliminary data and will focus on identifying `readers' of two different histone phosphorylation `marks', one that indicates DNA damage and is observed during prolonged mitotic arrest with cytotoxic drugs, and another that associates with chromosomes in dividing cells and depends on the activity of Aurora kinase, a conserved regulator of cell division and a target of anti-cancer drugs. The functional significance of `reading' these histone `marks' at different stages of the cell cycle wil be examined using high-resolution microscopy assays, mouse embryonic fibroblasts with key proteins knocked out, and chemical inhibitors that act on fast time-scales to block the activity of kinases responsible for generating these `marks' in cells. The proposal has three aims: (i) To identify proteins that `read' a phosphorylation `mark' on histone H2AX, (ii) To characterize the functions of histone H2AX phosphorylation-`readers', and (iii) To profile `readers' of histone post-translational modifications in living cells. The proposed research combines chemistry and biology approaches to unravel how histone `marks' are `interpreted' by proteins to ensure stable genome propagation by regulating chromosome segregation, DNA damage repair and cell cycle progression. These studies should also shed light on how chemical inhibitors of cell division kill cancer cells. In addition, the comprehensive profiling of key post-translational modification-dependent protein-protein interactions should lead to the selection of new targets for therapeutic agents. Finally, the approaches we develop are general and can be broadly applied to dissect complex and dynamic networks of protein-protein interactions in cells.