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发挥着重要作用，因为MMR中的缺陷会导致癌症的发展。在过去，MMR的基因组维持功能被归因于它纠正错配的能力。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，以应对包括环境化学致癌物在内的物理和化学因素引起的DNA损伤。已知这种反应依赖于MutS和MutL同系物，并消除了潜在的致癌细胞的生长。依赖MMR的细胞凋亡似乎涉及一个信号网络，该网络将DNA损伤信号传递给凋亡机制，以杀死受损的细胞。然而，这个网络是如何工作的，以及这个网络中涉及哪些蛋白质，人们对此知之甚少。本研究旨在用蛋白质组学方法鉴定和鉴定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，MMR熟练和缺陷的细胞将被化学致癌物处理，这些细胞处理前后的蛋白质表达谱将通过二维凝胶电泳进行分析。差异表达的蛋白质将接受质谱分析，得到的多肽将用于数据库同源性搜索，以揭示它们的身份。其次，考虑到蛋白质的磷酸化或去磷酸化参与了许多信号网络，我们假设许多参与MMR依赖的细胞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，将通过一维或二维凝胶结合蛋白质印迹分析来分析在本研究中确定的、先前已知参与MMR依赖的细胞凋亡的蛋白质的磷酸化状态，然后进行质谱分析。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，如顺铂和烷化剂，可以MMR依赖的方式发出细胞凋亡信号，这项研究也将影响癌症的治疗，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.