TAB2的O-GlcNAcylation糖基化修饰及其在肝脏炎症恶性转化中的功能研究

Hepatocellular carcinoma (HCC) is one of the most common malignant carcinoma. 90% of the primary HCC is caused by inflammatory transformation called “inflammation-fibrosis-cancer axis (IFC)”. The activation of TAK1-TABs complex and its downstream NF-κB and MAPK signaling pathways affect the IFC process and liver cancer progression. O-GlcNAcylation, similar to phosphorylation, is one of the most important post-translational modifications dynamicly controlling proteins function. Our previous studies have demonstrated that there are multiple O-GlcNAcylation modification sites, including a TSSTSSS cluster, on TAB2. Wherase, the biological function of TAB2 O-GlcNAcylation, especially its role in inflammation-fibrosis-cancer axis, remains under investigation. In this study, we will focus on the O-GlcNAcylation modification of TAB2 , trying to figure out the effect of TAB2 O-GlcNAcylation on TAK1-TABs protein-protein interaction and downstream signaling such as NFκB activity, and explore the biological role of this modification in liver cells inflammation-related transformation. This project will deepen the understanding of molecular mechanism of hepatic inflammatory transformation and HCC progression.

肝癌是最常见的恶性肿瘤，90%的原发性肝癌的形成与肝脏细胞“炎-癌转化“相关。TAK1-TABs 复合物介导了促炎细胞因子TNF-α等对下游NF-κB和MAPKs等信号通路的激活，是影响肝脏炎症反应以及原发性肝癌发生的关键分子。O-GlcNAcylation糖基化是类似磷酸化的重要翻译后修饰方式，可快速动态调节蛋白活性和功能。我们前期研究发现，TAB2含多个O-GlcNAcylation位点，其中包括一段密集的丝/苏氨酸修饰簇，但其具体生物学功能尚未明确。本项目拟进一步明确TAB2的关键性O-GlcNAc修饰位点；并利用分子、细胞、动物等多种体内外模型，深入探讨肝脏细胞在炎症微环境下如何通过O-GlcNAcylation修饰对TAK1-TABs复合物活性及NF-κB等下游通路产生影响，及其在肝脏“炎-癌转化"中的功能。本项目可加深对炎症相关原发性肝癌发生机制的理论认识，具有重要的科学价值。

O-GlcNAcylation糖基化修饰是细胞应对营养应激的重要翻译后调控方式。TAK1-TABs复合物是调控细胞内炎症反应应激的关键性蛋白复合体，但是人们对其O-GlcNAcylation修饰研究知之甚少。本项目从TAB2的O-GlcNAcylation化修饰现象的发现出发，首先通过抗体验证、Cpnag J 糖苷酶水解、 Gal T标记 、 体外OGT Labeling等实验证实TAB2是OGT的直接底物。接着，我们通过开发新的化学酶标记结合ETD质谱的糖基化鉴定技术方法，鉴定到了多个含有TAB2的O-GlcNAcylation糖基化位点的肽段（GTSSLSQQTPR, TSSTSSSVNSQTLNR以及 VVVTQPNTK 等)。接着，我们进一步从分子水平明确了T348/S349/S350这三个连续的O-GlcNAcylation位点簇是其关键的糖基化位点，参与了IL-1β介导的下游炎症反应。高糖环境可以促进内源性的TAB2的O-GlcNAcylation，促进下游的包括NFAT在内的炎症信号通路的活化。本项目探讨了TAB2的O-GlcNAcylation修饰的位点及在炎症信号通路中的作用，是肝脏中高糖环境下的炎症反应及炎-癌转化的分子机制的初步探索。