Identifying Proteins Involved in DNA Damage Response

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起着重要作用，因为MMR缺陷会导致癌症的发生。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维持基因组的稳定性，以响应由物理和化学试剂（包括环境化学致癌物）诱导的DNA损伤。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依赖的细胞凋亡似乎涉及一个信号网络，该信号网络将DNA损伤信号传递到凋亡机制以杀死受损细胞。然而，该网络如何工作以及该网络中涉及哪些蛋白质还知之甚少。本研究旨在通过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，将用化学致癌物处理MMR-熟练的和缺陷的细胞，并通过二维凝胶电泳分析这些细胞在处理之前和之后的蛋白质表达谱。具有差异表达的蛋白质将进行质谱分析，所得肽将用于数据库同源性搜索以揭示其身份。第二，鉴于蛋白磷酸化或去磷酸化参与许多信号网络，我们假设，许多蛋白参与MMR依赖性细胞凋亡可能是磷酸化或去磷酸化。为了验证这一假设，将通过一维或二维凝胶结合Western印迹分析，然后进行质谱分析，分析本研究中鉴定的和先前已知参与MMR依赖性细胞凋亡的蛋白质的磷酸化状态。该项目产生的数据将为更详细的研究（R 01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。因为某些癌症化疗药物，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，这项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.