ULK1的O-GlcNAc修饰调控高葡萄糖诱导的自噬的机制研究

Unc-51 like autophagy activating kinase (ULK1) and Adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) are two important kinases regulating autophagy. Autophagy is activated when the cells are deprived of glucose or amino acids, or induced by Calcium signals upon glucagon stimulation. In these three situations, the interaction between ULK1 and AMPK is regulated by distinct mechanisms, and so are the post-translational modifications of ULK1. But whether O-GlcNAcylation plays a role in high glucose-induced autophagy is elusive. Our preliminary data revealed that (1) Under high glucose, ULK1 is O-GlcNAcylated, and HCD mass spectrometry unveiled a new O-GlcNAcylated peptide; (2) Under high glucose, ULK1 specifically dissociates from catalytic AMPKα2, but not AMPKα1; (3) Mass spectrometry analysis also uncovered several potential phosphorylation sites in the vicinity of the O-GlcNAcylated peptide. These sites not only conform to the consensus AMPK phosphorylation sequences, but also are abolished upon high glucose; (4) One potential O-GlcNAc site is mutated in melanoma. In this grant, we propose to further examine the underlying mechanism between autophagy and O-GlcNAc under high glucose. We will explore the possibility that O-GlcNAcylated ULK1 antagonizes AMPKα2-dependent phosphorylation, thus promoting the kinase activity of ULK1 and hence autophagy. We will also address whether the melanoma-associated mutant of ULK1 is defective in O-GlcNAc or phosphorylation, aiming to pave a way for therapeutic interventions of melanoma.

AMPK和ULK1是调控细胞自噬的重要激酶, 两者的互作在缺失葡萄糖, 缺失氨基酸或者胰高血糖素诱导的自噬中各不相同。蛋白质O-GlcNAc修饰与高糖诱导的自噬的关系尚不明确。我们初步研究发现:(1)高糖自噬时ULK1被O-GlcNAc糖基化,并质谱鉴定了位点 (2)高糖时ULK1特异与AMPKα2解离 (3)质谱表明糖基修饰附近有潜在的AMPK磷酸化位点,在高糖时消失 (4)糖基化位点中含黑色素瘤突变位点。本申请拟研究高糖自噬时ULK的O-GlcNAc如何拮抗与AMPKα2的互作以及拮抗AMPKα2对其磷酸化, 高糖时ULK1酶活是否激活而促进自噬, 黑色素瘤突变位点是否在该过程中出现异常。本项目不仅能进一步阐释O-GlcNAc对于高糖自噬的新调节机制, 辨明AMPKα1和2的区别, 且能揭示ULK1的O-GlcNAc与磷酸化如何通过自噬调控黑色素瘤, 为患者开发新的靶向治疗方法奠定基础

氧连接的N-乙酰葡糖胺（O-GlcNAc）是唯一发生在细胞内的参与信号转导的单糖修饰，一般发生于细胞质、细胞核以及线粒体中。O-GlcNAc 修饰发生在丝氨酸和苏氨酸上，因此可以与磷酸化之间形成阴阳关系，而参与信号转导过程。但由于其低丰度易水解性的特点导致技术上难以鉴定位点。人乳头瘤病毒（HPV）早期基因区中的E6和E7为致癌基因，可结合细胞的抑癌基因p53、pRb并使其失活，从而促使子宫颈癌、头颈部鳞状细胞癌（HNSCC）等的发生。本项目发现机体被HPV感染时自噬起始激酶ULK1发生新的糖基化（没有病毒感染时，ULK1发生糖基化于T635/T754），从而增强ULK1稳定性，提高自噬的水平。我们首先发现头颈癌细胞在HPV感染时，ULK1的O-GlcNAc水平会升高。进而通过监测ULK1 O-GlcNAc水平和ETD质谱分析，我们确定了新的O-GlcNAc位点S409/S410。我们继续通过生化和细胞实验测定，发现S409及其邻近的S410处的O-GlcNAcylation拮抗S423位点的磷酸化，抑制ULK1通过分子伴侣介导的自噬CMA通路的降解，从而稳定ULK1和增强自噬。进一步，我们通过TCGA数据显示，HPV阳性的HNSCC中ULK1的表达量更高，并与HNSCC患者的预后相关。综上所述，ULK1-Ser409/Ser410的O-GlcNAc糖基化会拮抗ULK1-S423位点的磷酸化，抑制ULK1通过CMA通路的降解而增加ULK1的稳定性，增强自噬，并提高HNSCC的预后。该项研究工作有助于阐明在动态的生物过程中，特别是在HPV感染的情况下，细胞内O-GlcNAc修饰大量增加，以调整生物系统以适应不断变化的外部和内部条件。提示自噬可以通过吞噬入侵的HPV颗粒来清除病毒颗粒，从而抑制HNSCC。课题负责人多次参加国际/国内学术会议做邀请/口头报告。本课题相关成果以通讯或共通讯作者发表JBC，Frontiers in Chemistry, Cell cycle等专业期刊７篇（其中JBC 2篇），另外参与发表５篇。现在还有一篇在JBC修回，有两篇在投稿中。