GSTp磷酸化入核抑制LPS诱导的HMGB1出核释放的分子机制研究

HMGB1 translocation from the nucleus to the cytoplasm and release is a key factor of infectious death.Posttranslational modifications is important in HMGB1 nuclear translocation and release into the extracellular space,which is a potential therapeutic target of inflammatory deseases.GSTp plays an important role in oxidation-reduction balance in cells.But the effect of GSTp in nucleus is unclear.We have found that GSTp attenuates the severe inflammatory respons and reduces the lethality of endotoxic shock.Our recent study ddemonstrated that GSTp could be phosphorylated and transported into nucleus after LPS stimulation.GSTp interacted with nuclear HMGB1to prevent its translocation from nucleus to cytoplasm and inhibited release of pro-inflammatory cytokines. In this project,we will investigate the effect of GSTp on preventing HMGB1 nuclear tranlocation and related mechanisms.The researches include: 1.Indentification of the protein domains of HMGB1 for GSTp interation.2.The effects of GSTp on LPS-induced HMGB1 phosphorylation,oxidation/reduction,acetylation and nuclear translocation. 3.The molecular mechanisms of GSTp phosphorylation and transportation into the nucleus.4, The effect of GSTp on HMGB1 upstream kinase PKC.The present project is important for realizing the regulation for HMGB1 release and effect of GSTp on regulating inflammatory response.

HMGB1出核释放是感染后致死的关键因素。HMGB1蛋白的翻译后调控在其出核释放中的作用是治疗炎症性疾病策略新的关注点。GSTp对细胞的氧化还原平衡十分重要，但GSTp在细胞核中的作用尚不清楚。申请者发现GSTp蛋白对LPS引起的过度炎症反应及动物死亡有明显的抑制作用。申请者近期的研究显示，LPS刺激巨噬细胞时，GSTp被磷酸化并入核，在核中与HMGB1结合，抑制HMGB1出核，减少LPS导致的炎症因子产生。因此本项拟对GSTp抑制HMGB1出核及释放的作用和机制进行研究，主要研究内容：确定GSTp对LPS所致HMGB1出核的抑制作用；明确HMGB1分子上与GSTp结合的功能区；阐明GSTp被磷酸化入核的分子机制以及GSTp对HMGB1磷酸化和上游激酶PKC的影响；观察GSTp对HMGB1其它翻译后修饰的影响。本项目对深入了解HMGB1的释放调控以及GSTp的入核及抗炎作用具有重要意义。

GSTp作为一个重要的二型代谢酶在维持细胞应激条件下的稳态具有重要作用，当LPS刺激单核巨噬细胞后，GSTp可以被LPS激活的PKC磷酸化，通过质谱的检测结果表明，其磷酸化位点主要在蛋白的丝氨酸184位，该磷酸化的结果将导致GSTp的入核，入核后的GSTp将与核内的HMGB1结合，该结合将进一步阻止HMGB1的磷酸化，并阻止了帮助HMGB1出核释放的蛋白CRM1与之结合，进而有效地控制了LPS引起的晚期炎症因子的出核和胞外释放，抑制了LPS或CLP模型中小鼠的死亡率。该研究结果对今后脓毒症的发生/发展的理解及控制具有重要意义。.相关的研究结果发表在2018Frontiers in immunology 上。