Studying chromosome function using chemical biology

利用化学生物学研究染色体功能

• 批准号: 8886346
• 负责人: TARUN M. KAPOOR
• 金额: $ 47.76万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886346
• 关键词: Address; Amber; Amino Acids; Antineoplastic Agents; Binding Proteins; Binding Sites; Biological Assay; Biology; Cell Culture Techniques; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Cycle Regulation; Cell Cycle Stage; Cell Death; Cell Division Process; Cell Line; Cell division; Cells; Chemicals; Chemistry; Chromatin; Chromosome Segregation; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytotoxic agent; DNA; DNA Damage; DNA Repair; Data; Defect; Development; Disease; Drug Targeting; Embryo; Ensure; Fibroblasts; Genes; Genome; Goals; Histone H2A; Histone H3; Histones; In Vitro; Knock-out; Knowledge; Lead; Life; Light; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediator of activation protein; Methodology; Methods; Methylation; Microscopy; Mitotic; Mus; Names; Nucleosomes; Phosphorylation; Phosphotransferases; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Binding; Proteins; Proteomics; Publications; Publishing; Reader; Reading; Regulation; Research; Resistance; Resolution; Role; Site; Stable Isotope Labeling; Taxane Compound; Therapeutic; Therapeutic Agents; Time; Ubiquitination; Work; aurora kinase; base; cancer cell; cancer therapy; cell killing; covalent bond; crosslink; daughter cell; human H2AX protein; improved; inhibitor/antagonist; insight; killings; kinase inhibitor; mutant; p53-binding protein 1; protein profiling; protein protein interaction; public health relevance; response; taxane; yeast two hybrid system

 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.

 描述（由申请人提供）：细胞分裂中的错误（复制的 DNA 在两个子细胞之间分配的过程）已与疾病和发育缺陷相关。不正确的细胞分裂也被广泛用于治疗癌症等疾病的治疗策略中。准确的染色体分离、正确的细胞周期进程和 DNA 损伤的修复依赖于识别组蛋白翻译后修饰的关键蛋白质，组蛋白组装称为核小体的染色质基本单位。使用传统方法来全面分析“读取”组蛋白上特定翻译后修饰（或“标记”）以调节染色体生物学的蛋白质一直很困难。我们的目标是通过设计新的基于化学的策略来描述特定细胞环境中组蛋白“标记”的直接“读者”，从而填补这一知识空白。我们方法的核心是使用光交联剂通过共价键将动态和弱（通常是微摩尔）蛋白质-蛋白质相互作用转化为稳定的关联。该策略与最先进的定量质谱法相结合，以识别特定组蛋白“标记”的“读者”。在项目完成期间，我们开发了这种“化学蛋白质组学”方法，名为 CLASPI（交联辅助和基于 SILAC 的蛋白质识别），并证明了其识别组蛋白“标记”新“读取器”的能力，包括组蛋白 H3 甲基化和磷酸化。我们将在最近的出版物和初步数据的基础上，重点识别两种不同组蛋白磷酸化“标记”的“读者”，一种表示 DNA 损伤，在细胞毒性药物长时间有丝分裂停滞期间观察到，另一种与分裂细胞中的染色体相关，取决于极光激酶的活性，极光激酶是细胞分裂的保守调节剂和抗癌靶点 药物。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检测、敲除关键蛋白的小鼠胚胎成纤维细胞以及在快速时间尺度上起作用以阻断组蛋白活性的化学抑制剂来检查。 负责在细胞中产生这些“标记”的激酶。该提案有三个目标：(i) 鉴定“读取”组蛋白 H2AX 上磷酸化“标记”的蛋白质，(ii) 表征组蛋白 H2AX 磷酸化“读取器”的功能，以及 (iii) 分析活细胞中组蛋白翻译后修饰的“读取器”。拟议的研究结合了化学和生物学方法，揭示了蛋白质如何“解释”组蛋白“标记”，以通过调节染色体分离、DNA损伤修复和细胞周期进程来确保基因组的稳定繁殖。这些研究还应该揭示细胞分裂的化学抑制剂如何杀死癌细胞。此外，对关键翻译后修饰依赖性蛋白质-蛋白质相互作用的全面分析应该有助于选择治疗药物的新靶点。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。