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

基本信息

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
项目类别：
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

项目摘要

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基因毒剂和复制压力的挑战。损失基因组稳定性可能导致癌症、过早衰老和神经退行性疾病等病理状况。当基因组发生改变时，细胞协调一个统称为 DNA 的分子通路网络损伤反应 (DDR) 发出信号并促进 DNA 损伤的修复，停止细胞周期进程，直到基因组完整性得到恢复。多项证据表明基因组不稳定性有助于肿瘤发生、癌症进展和治疗耐药性的发展。遗传分析已确定参与 DNA 损伤反应的高、中、低外显率癌症易感基因和修复。尽管多年来已经进行了许多机械和遗传学研究，但系统性的研究分析在对不同类型的反应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可靠的方法，用于识别和复杂混合物中蛋白质的定量。而亲和纯化与质谱联用实验发现了复杂的蛋白质相互作用网络、蛋白质复合物的分析在染色质上组装更具挑战性，因为纯化方法效率低下，与质谱法有偏差或不兼容。然而，由于技术的发展，这种情况正在迅速发展蛋白质组学的进展。我建议对蛋白质景观进行全面且公正的定量和动力学分析在对不同基因毒性剂的反应过程中，它们组装在功能齐全的细胞核和染色体上，我称之为多尺度蛋白质组学的方法。具体来说，我将使用从脊椎动物非洲爪蟾卵与最先进的质谱分析相结合。这种无细胞系统允许在细胞系统中无法实现的实验操作，并允许前所未有的 DNA 损伤反应蛋白质组的表征。我假设蛋白质的特定子集在不同损伤条件下招募到染色质不仅决定了对 DNA 损伤的反应，还决定了还有冗余修复途径的使用，这应该有助于解释突变的发生癌症基因组中发现的修复形式。我将与戈蒂埃实验室的其他成员一起验证并对非洲爪蟾提取物和人类细胞中的发现进行功能表征。了解响应和修复 DNA 损伤的蛋白质网络如何发挥作用具有重要意义影响人类健康的潜力。通过识别有用的合成致死相互作用，可能会增强化疗药物的功效，以提高实验基因疗法的安全性和适用性。因此，我们预计我们的研究将为 DNA 损伤反应的调节提供新的见解，有助于更好地了解细胞如何在基因毒性作用下保持其基因组的稳定性侮辱。！