Nuclear and cytosolic thioredoxin redox state

描述：(改编自申请者摘要)：活性氧物种(ROS)和内毒素(LPS)诱导的细胞因子反应与酒精性肝病的发病机制有关。ROS信号中的一个关键问题是当后者被氧化剂抑制时，氧化剂信号如何影响转录激活。如果核氧化还原不受细胞质氧化还原的控制，就可能发生这种情况。然而，普遍的文献表明，核GSH池类似于细胞质池，因为核孔对小分子化合物具有渗透性。我建议使用硫氧还蛋白(TRX)来研究核内的氧化还原。我已经建立了使用一种新的蛋白质印迹技术来测量TrX氧化还原的方法。我的假设是，在生理和毒理刺激诱导的氧化条件下，核Trx的氧化还原状态将不同于细胞质Trx。我将通过测量人类单核细胞系(THP-1)对分化剂(VitD3和佛波酯)和脂多糖的反应，检测细胞核和细胞质Trx的氧化还原以及细胞GSH/GSSG的氧化还原来验证这一假设。我将确定分离的核是否具有独立调节Trx氧化还原状态的能力，以及核Trx氧化还原状态是否影响依赖于NF-kB的基因表达。这项研究应该会为我未来的职业生涯提供一个重要的知识进步和坚实的基础。

DESCRIPTION: (Adapted from the Applicant's Abstract): Reactive oxygen species (ROS) and endotoxin (LPS)-induced cytokine responses have been implicated in the pathogenesis of alcoholic liver disease. A key question in ROS signaling is how an oxidant signal can effect transcriptional activation when the latter is inhibited by oxidants. This can occur if the nuclear redox is controlled independently of the cytoplasmic redox. However, the prevailing literature indicates that the nuclear GSH pool is similar to the cytoplasmic pool because nuclear pores are permeable to small molecular weight compounds. I propose to use thioredoxin (Trx) to study redox in the nucleus. I have established methods to measure the redox of Trx using a novel Western blot technique. My hypothesis is that the redox state of nuclear Trx will differ from cytoplasmic Trx under oxidative conditions induced by physiologic and toxicologic stimuli. I will test this hypothesis by measuring redox of nuclear and cytoplasmic Trx and redox of cellular GSH/GSSG in a human monocyte cell line (THP-1) in response to differentiating agents (Vit D3 and phorbol ester) and LPS. I will determine whether isolated nuclei have the capacity to independently regulate Trx redox state and whether nuclear Trx redox state affects NF-kB-dependent gene expression. The research should provide an important advance in knowledge and also a solid foundation for my future career.