Identifying Proteins Involved in DNA Damage Response

鉴定参与 DNA 损伤反应的蛋白质

• 批准号: 6917950
• 负责人: Guo-Min Li
• 金额: $ 7.37万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-05 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917950
• 关键词: DNA damage; DNA repair; adduct; apoptosis; benzopyrenes; biological signal transduction; cell growth regulation; chemical carcinogen; environment related neoplasm /cancer; environmental contamination; gene expression; hydrocarbons; mass spectrometry; molecular biology information system; phenanthrene; phosphorylation; protein quantitation /detection; protein structure function; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): DNA mismatch repair (MMR) plays an important role in maintaining genomic stability as defects in MMR lead to the development of cancer. In the past, the genome maintenance function of MMR had been attributed to its ability to correct mismatches. However, increasing evidence suggests that the MMR system also maintains genomic stability by promoting apoptosis in response to DNA damage induced by physical and chemical agents, including environmental chemical carcinogens. This response is known to be dependent on MutS and MutL homologs, and eliminates potentially carcinogenic cells from growing. The MMR-dependent apoptosis appears to involve a signaling network, which transmits a DNA damage signal to the apoptotic machinery to kill damaged cells. However, how the network works and what proteins are involved in this network are poorly understood. This study aims to identify and characterize proteins that participate in MMR-dependent apoptosis induced by environmental chemical carcinogens using a prote0mic approach. First, MMR-proficient and deficient cells will be treated with chemical carcinogens, and protein expression profiles before and after treatments from these cells will be analyzed by 2-dimensional gel electrophoresis. Proteins with differential expression will be subjected to mass spectromic analysis, and the resulting peptides will be used for database homology searching to reveal their identities. Second, given the involvement of protein phosphorylation or dephosphorylation in many signaling networks, we hypothesize that many proteins participating in MMR-dependent apoptosis may be phosphorylated or dephosphorylated. To test this hypothesis, proteins that are identified in this study and known previously to be involved in MMR-dependent apoptosis will be analyzed for their phosphorylation status by 1-dimensional or 2-dimensional gels combined with Western blot analysis, followed by mass spectrometry analysis. Data resulting from this project will formulate the basis for more detailed investigations (R01 applications) to fully understand the molecular mechanism by which the MMR system maintains genomic stability by promoting apoptosis induced by environmental chemical carcinogens. Because certain cancer chemotherapeutics, e.g., cisplatin and alkylating agents, can signal apoptosis in an MMR-dependent fashion, this study will also impact cancer treatment, particularly cancers caused by MMR defects.

描述（由申请人提供）：DNA 错配修复（MMR）在维持基因组稳定性方面发挥着重要作用，因为 MMR 缺陷会导致癌症的发展。过去，MMR的基因组维持功能被归因于其纠正错配的能力。然而，越来越多的证据表明，MMR 系统还通过促进细胞凋亡来响应物理和化学试剂（包括环境化学致癌物）诱导的 DNA 损伤，从而维持基因组稳定性。已知这种反应依赖于 MutS 和 MutL 同源物，并消除潜在致癌细胞的生长。 MMR 依赖性细胞凋亡似乎涉及信号网络，该网络将 DNA 损伤信号传递给细胞凋亡机制以杀死受损细胞。然而，人们对该网络如何工作以及该网络涉及哪些蛋白质知之甚少。本研究旨在利用蛋白质组学方法鉴定和表征参与环境化学致癌物诱导的 MMR 依赖性细胞凋亡的蛋白质。首先，用化学致癌剂处理MMR丰富和缺陷的细胞，并通过二维凝胶电泳分析这些细胞处理前后的蛋白质表达谱。具有差异表达的蛋白质将进行质谱分析，所得的肽将用于数据库同源性搜索以揭示其身份。其次，鉴于许多信号网络中蛋白质磷酸化或去磷酸化的参与，我们假设参与MMR依赖性细胞凋亡的许多蛋白质可能被磷酸化或去磷酸化。为了检验这一假设，将通过一维或二维凝胶结合蛋白质印迹分析，然后进行质谱分析，对本研究中鉴定出的且先前已知参与MMR依赖性细胞凋亡的蛋白质进行磷酸化状态分析。该项目产生的数据将为更详细的研究（R01应用）奠定基础，以充分了解MMR系统通过促进环境化学致癌物诱导的细胞凋亡来维持基因组稳定性的分子机制。由于某些癌症化疗药物（例如顺铂和烷化剂）可以以 MMR 依赖性方式发出细胞凋亡信号，因此这项研究也将影响癌症治疗，特别是由 MMR 缺陷引起的癌症。