ERAP140 - POSTTRANSLATIONAL MODIFICATION IDENTIFICATION

描述（由申请人提供）：我将利用遗传学，生物化学和质谱的最新技术，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节中的特定作用，鉴定和表征该蛋白的翻译后修饰（PTMs），以评估其在健康和患病状态下的基因组调节。我们的假设是，ERAP140的转录激活电位及其在乳腺癌和他莫昔芬耐药乳腺癌中的作用可能是由尚未确定的ERAP140的PTM状态调节的。我将重点研究三种不同病理的乳腺癌细胞系（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ERAP140及其PTM状态的蛋白质组学分析，我将确定与启动子特异性大分子转录复合物形成模式改变的相关性。鉴定的ERAP140 PTM位点的定点诱变将在转录翻译试验和蛋白质相互作用试验中进行评估，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.