ERAP140 - POSTTRANSLATIONAL MODIFICATION IDENTIFICATION

描述(申请人提供)：利用遗传学、生物化学和质谱学的最新技术，我将重点研究最近发现的转录因子雌激素受体相关蛋白140(ERAP140)，以确定其在基因组调控中的特定作用，鉴定和表征这3rotein的翻译后修饰(PTM)，以评估它们在健康和疾病状态下对基因组调控的调节。我们的假设是，ERAP140的转录激活潜力及其在乳腺癌和三苯氧胺耐药乳腺癌中的作用，可能受到ERAP140的PTM状态的调节，目前尚未确定。我将重点介绍三种不同病理情况的肿瘤细胞株(MCF-7、T47D和MDA231)，它们表达非常高水平的ERAP140。利用染色质免疫沉淀(ChIP)、DNA启动子微阵列分析、同位素标记和蛋白质组学分析ERAP140及其PTM状态I将识别与其启动子特异性大分子转录复合体形成的改变模式的相关性。已确定的ERAP140 PTM位点的定点突变将在转录翻译分析和蛋白质相互作用分析中进行评估，以了解这些模式背后的作用机制。

DESCRIPTION (provided by applicant): Utilizing the latest techniques in genetics, biochemistry, and mass spectroscopy, I will focus on a recently discovered transcription factor, the estrogen receptor associated protein 140 (ERAP140) to determine its specific role in genomic regulation, identify and characterize posttranslational modifications (PTMs) of this 3rotein to evaluate their modulation of genomic regulation in healthy and diseased states. It is our hypothesis that the transcriptional activation potential of ERAP140 and its role in breast cancer, and tamoxifen resistant breast cancer, is likely modulated by, as yet, undetermined PTM states of ERAP140. I will focus on three 3reast tumor cell lines (MCF-7, T47D, and MDA231) with different pathologies expressing very high levels of ERAP140. Utilizing chromatin immunoprecipitation (CHIP), DNA promoter microarray analysis, isotopic labeling, and proteomics analysis of ERAP140 and its PTM state I will identify correlations with its altered pattern of promoter specific macromolecular transcription complex formation. Site-directed mutagenesis of the identified ERAP140 PTM sites will be evaluated in transcription translation assays and protein interaction assays for the mechanism of action behind these patterns.