类泛素蛋白FAT10抑制心肌细胞自噬的机制研究

Autophagy is important in the process of myocardial infarction (MI). Our previous published work demonstrated that during MI, the expression of FAT10 was upregulated. Overexpression of FAT10 protected cardial myocytes from death. In this work, we found that autophagy was also increased during MI. Moreover, FAT10 inhibited phagophore elongation which further repressed autophagy, however, detailed mechamisms were unknown. Reportedly, Sirt1 targeted deacetylation of LC3 promoted autophagy. Additionally, in MI both rats model and hypoxia/oxygenation treated NRMCs, FAT10 was found to reduce sirt1 expression. Most importantly, our previous data found that Sirt1 was the down molecular of FAT10 which was confirmed by GST-pull down in our previous work. Thus, we speculated that FAT10 regulated autophagy via sirt1-deacetylated LC3. We aimed to explore the relationship between FAT10 and sirt1-LC3 in a model of MI in FAT10-/- mice. The results will reply theoretical basis for future cardial myocytes protection in clinical application.

细胞自噬在心肌梗死(MI)中起关键作用。本团队研究成果显示，MI时，类泛素蛋白FAT10升高，并抑制心肌细胞凋亡，对心肌具有保护作用。进一步研究发现，FAT10通过抑制MI大鼠自噬体膜的延伸，从而抑制心肌细胞过度自噬。已有报道表明：sirt1诱导LC3去乙酰化可促进自噬体膜的延伸。但FAT10调节自噬体膜延伸的具体机制尚不清楚。本课题基因芯片结果发现，sirt1可能是FAT10底物，且FAT10可与sirt1结合，降低sirt1表达。故我们的假设是：FAT10可能通过调控sirt1表达，影响LC3去乙酰化进而调节自噬。我们拟通过大鼠及心脏FAT10-/-小鼠MI模型及原代乳鼠心肌细胞缺氧复氧，研究FAT10对Sirt1调节的作用及具体机制，并明确FAT10通过sirt1-LC3通路影响自噬。研究结果将为临床寻找新的心肌梗死治疗靶点提供实验依据。

自噬在缺血性心肌损伤中起有害作用。去乙酰化酶SIRT1是一种成熟的自噬调节因子，可以被泛素样蛋白SUMO1修饰。我们之前的工作表明，另一种泛素样蛋白FAT10通过稳定小窝蛋白3蛋白发挥对心肌缺血的保护作用;然而，FAT10通过SIRT1对自噬的影响尚不清楚。在本研究中，我们构建了FAT10基因敲除大鼠模型，以评估FAT10在自噬过程中的作用。通过体内和体外实验证实，FAT10抑制自噬可以保护心脏免受缺血性心肌损伤。在机制上，FAT10主要参与了自噬小体形成过程的调控。FAT10通过调节SIRT1的降解来影响自噬，导致SIRT1核易位减少，并通过其c端甘氨酸残基抑制SIRT1的活性。值得注意的是，FAT10与SUMO1在SIRT1的K734修饰位点竞争，进一步降低了LC3去乙酰化，抑制了自噬。我们的研究结果表明，FAT10可以通过其末端甘氨酸与SUMO1竞争SIRT1 Lys734结合位点，促进SIRT1降解，降低LC3乙酰化水平，抑制自噬体膜延伸，从而抑制心肌细胞的自噬，继而减少MI面积，最终改善心功能。本研究首次揭示李两个泛素蛋白FAT10与SUMO1可对同一底物蛋白SIRT1发生竞争拮抗作用。这是FAT10作为心脏保护器调控自噬的一种新机制。