Multiscale proteomics studies of DNA repair and genomic stability

DNA 修复和基因组稳定性的多尺度蛋白质组学研究

• 批准号: 10229517
• 负责人: Tomas Aparicio Casado
• 金额: $ 16.24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-05 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10229517
• 关键词: Affinity Chromatography; Cancer Patient; Cancer-Predisposing Gene; Cell Cycle Progression; Cell Nucleus; Cell-Free System; Cells; Chromatin; Chromosomes; Complex Mixtures; DNA; DNA Damage; DNA Repair; DNA Sequence Alteration; DNA lesion; Defect; Development; Genetic study; Genome; Genome Stability; Genomic Instability; Health; Human; Kinetics; Knowledge; Laboratories; Lead; Light; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Molecular; Mutagens; Nerve Degeneration; Pathologic; Pathway Analysis; Pathway interactions; Penetrance; Pharmaceutical Preparations; Premature aging syndrome; Protein Analysis; Proteins; Proteome; Proteomics; Public Health; Radiation therapy; Regulation; Research; Resistance; Resolution; Safety; Signal Transduction; System; Work; Xenopus; Xenopus laevis; base; cancer genome; cancer therapy; chemotherapy; design; egg; experimental study; gene therapy; genetic analysis; genome integrity; genotoxicity; improved; insight; member; novel; protein complex; recruit; repaired; replication stress; response; therapy development; tumor; tumor progression; tumorigenesis

The integrity of the genome is continuously challenged by genotoxic agents and replication stress. Loss of genome stability can lead to pathological conditions such as cancer, premature aging, and neurodegeneration. Upon genomic alterations, cells coordinate a network of molecular pathways collectively known as the DNA damage response (DDR) that signals and promotes repair of DNA lesions, halting cell cycle progression until genome integrity is restored. Several lines of evidence indicate that genomic instability contributes to oncogenesis, cancer progression, and development of therapy resistance. Genetic analyses have identified high-, moderate-, and low-penetrance cancer susceptibility genes that are involved in DNA damage response and repair. Although many mechanistic and genetic studies have been performed over the years, a systematic analysis of the protein changes taking place on the chromosomes during the response to different types of genome perturbations is still lacking. High-resolution mass spectrometry-based proteomics is a robust method for the identification and quantification of proteins from complex mixtures. While affinity purification combined with mass spectrometry experiments have led to the discovery of intricate protein interaction networks, analyses of protein complexes assembled on chromatin have been much more challenging because purification methods are inefficient, biased or not compatible with mass spectrometry. However, this is rapidly evolving due to technological advances in proteomics. I propose to perform a comprehensive and unbiased quantitative and kinetic analysis of the protein landscapes assembled on fully functional nuclei and chromosomes during the response to different genotoxic agents, an approach that I call multiscale proteomics. Specifically, I will employ the cell-free extracts derived from the vertebrate Xenopus laevis eggs combined with state-of-the-art mass spectrometry analyses. This cell-free system allows experimental manipulations that cannot be achieved in cell systems and permits unprecedented characterization of the DNA damage response proteomes. I hypothesize that specific subsets of proteins recruited to chromatin under different damage conditions dictate not only the response to DNA damage, but also the usage of redundant repair pathways, which should shed some light on the occurrence of mutagenic forms of repair found in cancer genomes. Together with other members of the Gautier laboratory, I will validate and functionally characterize the findings in both Xenopus extracts and in human cells. Understanding how the protein networks that respond to and repair DNA damage work holds considerable potential to impact human health. From identifying useful synthetic lethal interactions that might enhance the efficacy of chemotherapy drugs to improving the safety and applicability of experimental gene therapies. Thus, we anticipate our studies will provide new insights on the regulation of the DNA damage responses, contributing to a better understanding of how the cells maintain the stability of their genomes upon genotoxic insults. !

基因组的完整性不断受到遗传毒性因子和复制应激的挑战。损失 基因组稳定性可导致病理状况，例如癌症、过早衰老和神经变性。 在基因组改变后，细胞协调分子通路网络，这些分子通路统称为DNA 损伤反应（DDR），发出信号并促进DNA损伤修复，停止细胞周期进程， 基因组完整性得以恢复。一些证据表明，基因组的不稳定性有助于 肿瘤发生、癌症进展和治疗抗性的发展。基因分析表明 参与DNA损伤反应的高、中、低突变率癌症易感基因 和修复。尽管多年来已经进行了许多机制和遗传学研究，但系统性的 分析在对不同类型的免疫应答期间染色体上发生的蛋白质变化， 基因组扰动仍然缺乏。 基于高分辨率质谱的蛋白质组学是一种稳健的鉴定方法， 从复杂混合物中定量蛋白质。而亲和纯化结合质谱 实验发现了复杂的蛋白质相互作用网络，分析了蛋白质复合物， 在染色质上组装的方法更具挑战性，因为纯化方法效率低， 有偏差或与质谱法不兼容。然而，由于技术的发展， 蛋白质组学的进展 我建议对蛋白质景观进行全面和公正的定量和动力学分析 在对不同遗传毒性剂的反应过程中， 我称之为多尺度蛋白质组学。具体地说，我将使用来自于 脊椎动物非洲爪蟾卵结合最先进的质谱分析。该无细胞 系统允许在细胞系统中无法实现的实验操作，并且允许前所未有的 DNA损伤反应蛋白质组的表征。我假设特定的蛋白质亚群 在不同的损伤条件下，被招募到染色质的细胞不仅决定了对DNA损伤的反应， 还使用了冗余修复途径，这应该对诱变性的发生有一定的启示。 在癌症基因组中发现的修复形式。与戈蒂埃实验室的其他成员一起，我将验证 并在功能上表征爪蟾提取物和人类细胞中的发现。 了解蛋白质网络如何响应和修复DNA损伤的工作具有相当大的意义。 可能影响人类健康。从识别有用的合成致命的相互作用，可能会提高 化疗药物的有效性，以提高实验性基因治疗的安全性和适用性。因此，在本发明中， 我们期望我们的研究将为DNA损伤反应的调节提供新的见解， 有助于更好地理解细胞在遗传毒性时如何保持其基因组的稳定性， 侮辱。 !