A new role of MEPE/OF45 as a co-factor of CHK1 for DNA damage response

MEPE/OF45 作为 CHK1 辅助因子在 DNA 损伤反应中的新作用

基本信息

批准号：
7678912
负责人：
YA WANG
金额：
$ 27.9万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-21 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7678912
关键词：
Affect Amino Acid Sequence Amino Acids Antibodies Baculoviruses Biological Assay C-terminal CHES1 gene Camptothecin Cell Line Cell Nucleus Cell physiology Cells Cessation of life Complex Crystallization Cytoplasm DNA Damage Data Equilibrium Event Extracellular Matrix Extracellular Matrix Degradation Extracellular Matrix Proteins Figs - dietary Genes Glycoproteins Goals Half-Life Human Human Cell Line Immunofluorescence Immunologic Immunoprecipitation Integrin Binding Ionizing radiation Knock-out Laboratories Ligands Link Location Lysine MEPE gene Mass Spectrum Analysis Mediating Mus Mutate Nature Normal Cell Osteoblasts Osteocytes Pathway interactions Peptide Sequence Determination Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Prevention Proteins Proteolysis Rattus Recovery Renal Tissue Reporting Role Salivary Glands Site Site-Directed Mutagenesis Small Interfering RNA Structure System Testing Therapeutic Time Transfection Ubiquitin Ubiquitination Universities biological adaptation to stress bone bone metabolism cancer prevention cancer therapy cell type field study killings member mineralization multicatalytic endopeptidase complex mutant prevent response tumor tumorigenesis ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): DNA damage is not only one of the essential events for human tumorigenesis but also one of the major therapeutic aims in human cancer treatment. Our recent data showed for the first time that Matrix extracellular phosphoglycoprotein/osteoblast/osteocyte factor 45 (MEPE/OF45), as a co-factor of CHK1, presents in all dividable human cell lines (tested in our laboratory) to affect cellular response to DNA damage. Since MEPE/OF45 was first cloned in 2000, however, its role as one member of SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein), has been reported to be limited to bone metabolism. We will test our hypothesis in this study that MEPE/OF45 affects cellular response to DNA damage through CHK1 by two mechanisms: 1. MEPE/OF45 interacts with CHK1 and stabilizes CHK1; 2. MEPE/OF45 undergoes degradation after phosphorylation by CHK1. In this way MEPE/OF45 maintains normal cellular response to DNA damage. Through these two mechanisms, MEPE/OF45 keeps the CHK1 level balanced following DNA damage and thus maintains normal cellular response to DNA damage. Our preliminary data strongly supports our hypothesis. Three specific aims will be performed: A. Determine whether MEPE/OF45 affecting cell response to DNA damage depends on the interaction of MEPE/OF45 with CHK1. B. Determine whether MEPE/OF45 affecting cell response to DNA damage is through protecting CHK1 from the ubiquitin-mediated degradation. C. Determine whether MEPE/OF45 affecting cell response to DNA damage is involved in the phosphorylation of MEPE/OF45 by CHK1. We will combine approaches of GST pull-down, site-directed mutagenesis, immunoprecipitation, kinase assay, ubiquitination, phosphorylation, Mass-Spectrum and crystal structure analysis to identify the interaction site(s) between MEPE/OF45 and CHK1, the ubiquitination site(s) as well as degron of CHK1 and the phosphorylation site(s) of MEPE/OF45 by CHK1. We will examine the effects of these key site(s) and key domain(s) on cellular DNA damage response by observing CHK1 half-life, MEPE/OF45 half-life, survival sensitivity of MEPE/OF45-/- or Chk1 conditional knock out cells transfected with the gene mutated at the key site(s) or key domain(s) to DNA damage. These described studies will elucidate the mechanism by which MEPE/OF45 affects cellular response to DNA damage. We believe that these results will add a functional link between matrix extracellular protein and DNA damage response. Therefore, our results not will only benefit cancer prevention and cancer treatment but will also open a new field for studying how matrix extracellular protein to maintain normal cell function.

描述（申请人提供）：DNA损伤不仅是人类肿瘤发生的重要事件之一，也是人类癌症治疗的主要治疗目标之一。我们最近的数据首次表明，基质细胞外磷酸糖蛋白/成骨细胞/骨细胞因子 45 (MEPE/OF45) 作为 CHK1 的辅助因子，存在于所有可分裂的人类细胞系中（在我们实验室进行了测试），以影响细胞对DNA 损伤。然而，自 2000 年首次克隆 MEPE/OF45 以来，据报道其作为 SIBLING（小整合素结合配体 N 连接糖蛋白）成员之一的作用仅限于骨代谢。我们将在本研究中检验我们的假设，即 MEPE/OF45 通过 CHK1 通过两种机制影响细胞对 DNA 损伤的反应： 1. MEPE/OF45 与 CHK1 相互作用并稳定 CHK1； 2. MEPE/OF45 经历降解后 CHK1 磷酸化。通过这种方式，MEPE/OF45 维持细胞对 DNA 损伤的正常反应。通过这两种机制，MEPE/OF45 在 DNA 损伤后保持 CHK1 水平平衡，从而维持细胞对 DNA 损伤的正常反应。我们的初步数据有力地支持了我们的假设。将实现三个具体目标： A. 确定 MEPE/OF45 影响细胞对 DNA 损伤的反应是否取决于 MEPE/OF45 与 CHK1 的相互作用。 B. 确定 MEPE/OF45 是否通过保护 CHK1 免受泛素介导的降解来影响细胞对 DNA 损伤的反应。 C. 确定影响细胞对 DNA 损伤反应的 MEPE/OF45 是否参与 CHK1 对 MEPE/OF45 的磷酸化。我们将结合 GST Pull-down、定点突变、免疫沉淀、激酶测定、泛素化、磷酸化、质谱和晶体结构分析等方法来鉴定 MEPE/OF45 和 CHK1（泛素化位点）之间的相互作用位点（ s) 以及 CHK1 的降解决定子和 CHK1 对 MEPE/OF45 的磷酸化位点。我们将通过观察 CHK1 半衰期、MEPE/OF45 来检查这些关键位点和关键域对细胞 DNA 损伤反应的影响转染关键位点或关键结构域突变基因的 MEPE/OF45-/- 或 Chk1 条件敲除细胞的半衰期、生存敏感性对 DNA 损伤的影响。这些研究将阐明 MEPE/OF45 影响细胞对 DNA 损伤反应的机制。我们相信这些结果将增加基质细胞外蛋白和 DNA 损伤反应之间的功能联系。因此，我们的研究结果不仅有利于癌症的预防和治疗，也将为研究基质细胞外蛋白如何维持正常细胞功能开辟新的领域。