INJURY-INDUCED PHOSPHORYLATION SITES IN HEPATOCYTE NUCLEAR FACTOR-4 (HNF-4)

肝细胞核因子 4 (HNF-4) 中损伤诱导的磷酸化位点

• 批准号: 7369324
• 负责人: PETER A BURKE
• 金额: $ 0.4万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369324
• 关键词: INJURY; INDUCED; PHOSPHORYLATION; SITES; HEPATOCYTE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Following serious trauma the body mounts several distinct and specific injury responses. Many of these, for example specific immune responses, or wound or bone repair, can take several days or weeks to reach levels sufficient to establish reversal of a specific condition. In addition, a more nonspecific response, referred to as the Acute Phase Response (APR), is mounted within the first 24 hours following injury. The APR is common to a wide range of traumas, and although changes to the normal physiology of many tissues are observed, significant changes to the liver phenotype occur due to the massive induction of protective proteins by the liver. Whilst the APR is designed for survival, for some critically ill patients it is likely that severe or prolonged activation with its interruption of normal homeostatic function contributes to organ failure and death. Evidence suggests that the APR?s repression of steady-state liver function is a byproduct of its mode of induction. It has also been suggested to be a highly regulated process which shares pathways important to early cell type development, and with some signals associated with the proliferative response. Previous work suggests that injury induced regulation of differentiated genes may be mediated through phosphorylation of liver-specific transcription factors, particularly the Hepatic Nuclear Factor-4 (HNF-4). An improved understanding of the mechanisms regulating this process would have therapeutic importance in support of the APR. The primary aim of this project is the identification and localization of phosphorylation sites in HNF-4 for control and injury-induced models using mass spectrometric techniques. HNF-4 proteins were isolated from control and injury induced male rat liver nuclear cell extracts by immunoprecipitation (IP). Isolated proteins were separated by 1D-SDS-PAGE and subjected to in-gel tryptic digestion. MALDI-TOF mass spectrometry was performed using a Bruker Reflex IV mass spectrometer. Capillary LC-MS/MS studies were performed using a Waters CapLC system interfaced with an Applied Biosystems Q-Star Pulsar I QoTOF MS. CapLC separations were performed on a Waters AtlantisTM C18 100¿m x 150 mm NanoEase column, employing a 50 min gradient of 5-90% acetonitrile, 0.1% formic acid. Data were analysed using Mascot (Matrix Science) and Aldente (SwissProt) database search engines. Based upon previous results obtained from Western blots performed with a HNF-4 specific antibody it had been suggested that a successful IP of HNF-4 had been achieved. Early mass spectrometric characterization using MALDI and capLC-MS/MS, however, was unable to detect any HNF-4 in the sample provided,and it was determined that an alternative antibody was required, with improved specificity for IP applications. The new antibody has improved results but further investigation is required to complete identification of the isolated proteins. Once HNF-4 has been successfully isolated and identified, future directions include identification, localization, and comparison of the phosphorylation sites in both control and injury models.

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。严重的创伤后，身体会安装几种不同的特定伤害反应。其中许多，例如特定的免疫复杂或胜利或骨修复，可能需要几天或几周才能达到足以建立特定状况的逆转水平。此外，在受伤后的前24小时内，更非特异性的响应（称为急性相响应（APR））安装。 APR对广泛的创伤是常见的，尽管观察到许多组织的正常生理学的变化，但由于肝脏对保护蛋白的大量诱导，肝脏表型的显着变化。虽然APR是为生存而设计的，但对于某些重症患者而言，正常稳态功能的中断可能会导致器官衰竭和死亡，因此可能严重或延长激活。证据表明，稳态肝功能的APR反射是其诱导方式的副产品。还建议它是一个高度调节的过程，具有对早期细胞类型发育重要的途径，并且与增殖反应有关的一些信号。先前的工作表明，损伤诱导的分化基因的调节可以通过肝特异性转录因子的磷酸化，尤其是肝核因子-4（HNF-4）来介导。对治疗这一过程的机制的改进理解将具有支持APR的治疗意义。该项目的主要目的是使用质谱技术鉴定和定位HNF-4中的磷酸化位点，用于对照和损伤诱导的模型。通过免疫沉淀（IP）从对照和损伤诱导的雄性大鼠肝核细胞提取物中分离出HNF-4蛋白。通过1D-SDS-PAGE分离分离的蛋白质，并进行凝胶胰蛋白酶消化。使用Bruker反射IV质谱仪进行MALDI-TOF质谱仪。毛细管LC-MS/MS研究是使用与Applied Biosystems Q-Star Pulsar I Qotof MS进行的Waters CAPLC系统进行的。在水域上进行CAPLC分离，使用5-90％乙腈，0.1％正式酸的50分钟梯度，使用50分钟的梯度。使用吉祥物（Matrix Science）和Aldente（SwissProt）数据库搜索引擎分析数据。根据先前从使用HNF-4特异性抗体进行的蛋白质印迹获得的结果，已经提出已经成功实现了HNF-4的IP。但是，使用MALDI和CAPLC-MS/MS的早期质谱表征无法检测到所提供的样品中的任何HNF-4，并且确定需要一种替代抗体，并且对IP应用的特异性提高了。新抗体有改善的结果，但是需要进一步研究以完成分离的蛋白质。一旦成功隔离和鉴定了HNF-4，将来的方向包括对照模型和损伤​​模型中磷酸化位点的识别，定位和比较。