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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在严重的创伤之后，身体会产生几种不同的和特定的损伤反应。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扭转特定疾病的水平。此外，一种非特异性反应，称为急性期反应（APR），在损伤后的第一个24小时内发生。APR在广泛的创伤中是常见的，尽管观察到许多组织的正常生理变化，但由于肝脏大量诱导保护蛋白，肝脏表型发生显著变化。虽然APR是为生存而设计的，但对于一些危重患者来说，严重或长时间的激活与正常的体内平衡功能的中断可能会导致器官衰竭和死亡。有证据表明，APR？S对稳态肝功能的抑制是其诱导方式的副产品。它也被认为是一个高度调控的过程，共享早期细胞类型发育的重要途径，以及与增殖反应相关的一些信号。先前的研究表明，损伤诱导的分化基因调控可能是通过肝脏特异性转录因子的磷酸化介导的，特别是肝核因子-4 （HNF-4）。进一步了解这一过程的调控机制将对支持apr具有重要的治疗意义。本项目的主要目的是利用质谱技术识别和定位HNF-4的磷酸化位点，用于控制和损伤诱导模型。采用免疫沉淀法（IP）从对照和损伤雄性大鼠肝核细胞提取物中分离出HNF-4蛋白。分离蛋白经1D-SDS-PAGE分离，凝胶内胰消化。MALDI-TOF质谱分析采用布鲁克反射IV质谱仪。毛细管LC-MS/MS研究使用Waters CapLC系统与Applied Biosystems Q-Star Pulsar I QoTOF MS接口进行，CapLC分离在Waters AtlantisTM C18 100¿m x 150 mm NanoEase柱上进行，梯度为5-90%乙腈，0.1%甲酸，梯度为50 min。使用Mascot （Matrix Science）和Aldente （SwissProt）数据库搜索引擎分析数据。根据先前使用HNF-4特异性抗体进行Western blots获得的结果，表明已经成功地实现了HNF-4的IP。然而，使用MALDI和capLC-MS/MS进行的早期质谱表征无法在提供的样品中检测到任何HNF-4，因此确定需要一种替代抗体，以提高IP应用的特异性。新抗体改善了结果，但需要进一步的研究来完成分离蛋白的鉴定。一旦成功分离和鉴定出HNF-4，未来的方向包括鉴定、定位和对照和损伤模型中磷酸化位点的比较。

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.