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

描述(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/OF45)作为CHK1的辅助因子，存在于所有可分化的人类细胞系中(在我们实验室测试)，以影响细胞对DNA损伤的反应。然而，自2000年MEPE/OF45首次被克隆以来，它作为小整合素结合配体N-连接糖蛋白兄弟的一员，一直被报道仅限于骨代谢。我们将在这项研究中验证我们的假设，即MEPE/OF45通过两种机制影响细胞对DNA损伤的反应。 MEPE/OF45与CHK1相互作用，稳定CHK1；2.MEPE/OF45在 CHK1的磷酸化。通过这种方式，MEPE/OF45维持了细胞对DNA损伤的正常反应。通过这两种机制，MEPE/OF45在DNA损伤后维持CHK1水平的平衡，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损伤的反应是否依赖于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而影响细胞对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下拉、定点突变、免疫共沉淀、激酶实验、泛素化、磷酸化、质谱学和晶体结构分析等方法，确定MEPE/OF45与CHK1的相互作用位点(S)、CHK1的泛素化位点(S)以及CHK1的降解和CHK1的磷酸化位点(S)。我们将通过观察CHK1的半衰期，MEPE/OF45来检测这些关键点(S)和关键域(S)对细胞DNA损伤反应的影响 转导关键点(S)或关键域(S)突变基因的MEPE/of45-/-或Chk1条件性敲除细胞的半衰期、存活敏感性。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将有助于癌症的预防和治疗，还将为研究细胞外基质蛋白如何维持正常的细胞功能开辟一个新的领域。

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.