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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。严重创伤后，身体会产生几种不同的特定损伤反应。其中许多，例如特异性免疫反应，或伤口或骨骼修复，可能需要几天或几周才能达到足以逆转特定病症的水平。此外，一种更非特异性的反应，称为急性期反应（APR），在受伤后的第一个24小时内发生。APR是常见于广泛的创伤，虽然观察到许多组织的正常生理学变化，但由于肝脏大量诱导保护性蛋白，肝脏表型发生显著变化。虽然APR是为生存而设计的，但对于一些重症患者，严重或长期激活并中断正常的体内平衡功能可能会导致器官衰竭和死亡。有证据表明，该公司？对稳态肝功能的抑制是其诱导模式的副产品。它也被认为是一个高度调节的过程，共享对早期细胞类型发育重要的途径，并具有与增殖反应相关的一些信号。以前的工作表明，损伤诱导的分化基因的调节可能是通过肝脏特异性转录因子，特别是肝核因子-4（HNF-4）的磷酸化介导的。一个更好的了解调节这一过程的机制将有治疗的重要性，在支持的APR。该项目的主要目的是识别和定位的磷酸化位点HNF-4的控制和损伤诱导的模型，使用质谱技术。 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HNF-4蛋白。通过1D-SDS-PAGE分离的蛋白质，并进行凝胶内胰蛋白酶消化。使用Bruker Reflex IV质谱仪进行MALDI-TOF质谱。使用与Applied Biosystems Q-Star Pulsar I QoTOF MS连接的沃茨CapLC系统进行毛细管LC-MS/MS研究。在沃茨CapitisTM C18 100 μ m × 150 mm NanoEase柱上进行CapLC分离，使用5-90%乙腈、0.1%甲酸的50 min梯度。使用Mascot（Matrix Science）和Aldente（SwissProt）数据库搜索引擎分析数据。 基于先前从用HNF-4特异性抗体进行的蛋白质印迹获得的结果，已经表明已经实现了HNF-4的成功IP。然而，使用MALDI和capLC-MS/MS的早期质谱表征无法在提供的样品中检测到任何HNF-4，并且确定需要具有针对IP应用的改进的特异性的替代抗体。新抗体的结果有所改善，但需要进一步研究才能完成分离蛋白的鉴定。一旦HNF-4已被成功分离和鉴定，未来的方向包括识别，定位，并在控制和损伤模型中的磷酸化位点的比较。

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.