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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 在严重创伤后，身体会产生几种不同和特定的损伤反应。其中许多因素，例如特定的免疫反应，或伤口或骨骼修复，可能需要几天或几周的时间才能达到足以逆转特定情况的水平。此外，一种更非特异性的反应，称为急性时相反应(APR)，是在受伤后的头24小时内启动的。APR在各种创伤中很常见，虽然观察到许多组织的正常生理发生变化，但由于肝脏大量诱导保护蛋白，肝脏表型发生了显著变化。虽然APR是为生存而设计的，但对于一些危重患者来说，严重或长期的激活及其正常内稳功能的中断可能会导致器官衰竭和死亡。证据表明，APR？S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控的过程，共享对早期细胞类型发育至关重要的通路，并与一些与增殖反应相关的信号。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的转录因子，特别是肝细胞核因子-4(HNF-4)的磷酸化来介导的。对调节这一过程的机制的更好理解将对支持APR具有重要的治疗意义。该项目的主要目的是利用质谱学技术鉴定和定位HNF-4中用于控制和损伤诱导模型的磷酸化位点。 用免疫沉淀法从对照组和损伤诱导的雄性大鼠肝核细胞提取液中分离HNF-4蛋白。分离蛋白用1D-SDS-PAGE分离，凝胶内胰酶消化。使用Bruker Reflex IV质谱仪进行MALDI-TOF质谱分析。毛细管LC-MS/MS研究使用Waters CapLC系统与应用生物系统Q-Star Pulsar I QoTOF MS相连接。CapLC分离在Waters AtlantisTM C18 C18 100？m x 150 mm NanoEase柱上进行，采用5-90%乙腈-0.1%甲酸的50min梯度洗脱。使用Mascot(Matrix Science)和Aldente(SwissProt)数据库搜索引擎对数据进行分析。

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.