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

• 批准号: 7917069
• 负责人: YA WANG
• 金额: $ 31万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-09 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917069
• 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

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（由申请人提供）：DNA损伤不仅是人类肿瘤发生的基本事件之一，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/OF 45），作为CHK 1的辅因子，存在于所有可分割的人类细胞系（在我们的实验室测试），以影响细胞对DNA损伤的反应。然而，自从2000年首次克隆MEPE/OF 45以来，据报道其作为SIBLING（小整合素结合配体N-连接糖蛋白）的成员之一的作用仅限于骨代谢。我们将在这项研究中验证我们的假设，即MEPE/OF 45通过两种机制影响细胞对CHK 1的DNA损伤的反应：1。 MEPE/OF 45与CHK 1相互作用并稳定CHK 1; 2. MEPE/OF 45在以下条件下发生降解： CHK 1的磷酸化。通过这种方式，MEPE/OF 45维持对DNA损伤的正常细胞反应。通过这两种机制，MEPE/OF 45在DNA损伤后保持CHK 1水平平衡，从而维持对DNA损伤的正常细胞反应。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/OF 45是否影响细胞对DNA损伤的反应取决于MEPE/OF 45与CHK 1的相互作用。B。确定MEPE/OF 45是否通过保护CHK 1免受泛素介导的降解来影响细胞对DNA损伤的反应。C.确定MEPE/OF 45是否影响细胞对DNA损伤的反应，是否参与CHK 1对MEPE/OF 45的磷酸化。我们将结合联合收割机、GST pull-down、定点突变、免疫沉淀、激酶分析、泛素化、磷酸化、质谱和晶体结构分析等方法，对MEPE/OF 45与CHK 1的相互作用位点、CHK 1的泛素化位点和降解决定子以及CHK 1对MEPE/OF 45的磷酸化位点进行鉴定。我们将通过观察CHK 1的半衰期、MEPE/OF 45来研究这些关键位点和关键结构域对细胞DNA损伤反应的影响。 用在关键位点或关键结构域突变的基因转染的MEPE/OF 45-/-或Chk 1条件性敲除细胞对DNA损伤的半衰期、存活敏感性。这些描述的研究将阐明MEPE/OF 45影响细胞对DNA损伤的反应的机制。我们相信，这些结果将增加一个功能的联系之间的基质细胞外蛋白和DNA损伤反应。因此，我们的研究结果不仅有利于癌症的预防和治疗，而且还将为研究基质细胞外蛋白如何维持正常细胞功能开辟一个新的领域。