Identifying Proteins Involved in DNA Damage Response

描述（申请人提供）：DNA错配修复（DNA mismatch repair， MMR）在维持基因组稳定性方面起着重要作用，MMR缺陷导致癌症的发生。在过去，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应对物理和化学试剂（包括环境化学致癌物）诱导的DNA损伤，从而维持基因组的稳定性。已知这种反应依赖于MutS和MutL同源物，并消除了潜在的致癌细胞的生长。mmr依赖性细胞凋亡似乎涉及一个信号网络，该信号网络将DNA损伤信号传递给凋亡机制以杀死受损细胞。然而，这个网络是如何工作的，以及这个网络中涉及什么蛋白质，人们知之甚少。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，用化学致癌物处理精通核磁共振和缺陷的细胞，并通过二维凝胶电泳分析这些细胞治疗前后的蛋白质表达谱。对差异表达的蛋白进行质谱分析，得到的多肽将用于数据库同源性检索以揭示其身份。其次，考虑到许多信号网络中蛋白质磷酸化或去磷酸化的参与，我们假设许多参与mmr依赖性凋亡的蛋白质可能被磷酸化或去磷酸化。为了验证这一假设，我们将通过一维或二维凝胶结合Western blot分析，分析在本研究中鉴定出的、先前已知与mmr依赖性细胞凋亡有关的蛋白质的磷酸化状态，然后进行质谱分析。本项目获得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，如顺铂和烷基化剂，可以以MMR依赖的方式发出细胞凋亡信号，因此本研究也将影响癌症治疗，特别是由MMR缺陷引起的癌症。

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.