TFEB硝基化修饰在高同型半胱氨酸血症促进血管老化中的分子机制研究

As the pathogenesis basis of multiple aging-related diseases, mechanisms involved in vascular aging are rather complicated. In our previous work, we found out that hyperhomocysteinemia (HHcy) rats represented the phenotype of vascular aging. Moreover, we also observed increased expression of aging makers and decreased autophagy level in homocysteine-treated human umbilical vein endothelial cells (HUVECs). Therefore, we assumed that HHcy may induce vascular aging via down-regulating autophagy. However, the underlying mechanisms still remain unclear. Transcription factor EB (TFEB) is considered as a mater regulator of autophagy-lysosome pathway. Post-translational modifications have major impact on TFEB activity. HHcy can inactivate proteins through nitration, meanwhile its impact on TFEB is still unknown. In this study, aiming at HHcy-induced aging phenotype and the role of TFEB nitration on autophagy, we will utilize HHcy rat model and cultured HUVECs to reveal the underlying mechanisms. Using Adenovirus tandem fluorescent mRFP (Red Fluorescent Protein)-GFP (Green Fluorescent Protein)-LC3, we would like to monitor autophagic flux. Through performing immunoprecipitation (IP), chromatin immune coprecipitation (ChIP), luciferase reporter assay, mass spectrometry and site-directed mutation, we would like to detect the regulation of HHcy on TFEB nitration. In this study, we aim to reveal the regulation mechanisms involved in vascular aging, and explore the possibility of alleviating HHcy-induced vascular aging via reducing TFEB nitration to elevate autophagy level.

血管老化是多种增龄相关性疾病的发病基础，发生机制极为复杂。我们前期研究发现HHcy模型大鼠出现明显血管老化表型，同时Hcy刺激的血管内皮细胞衰老相关蛋白表达增加，细胞自噬水平明显下降，推测HHcy可能通过下调自噬而促进血管老化，但具体机制不清。TFEB是调控自噬-溶酶体通路的重要转录因子，其转录活性受翻译后修饰的影响。HHcy可诱发硝化应激，使蛋白质发生硝基化修饰而失活，但对TFEB的影响尚不清楚。本研究将利用HHcy大鼠模型及体外细胞培养，以HHcy硝基化修饰TFEB对内皮细胞自噬水平的调节为切入点，利用LC3双标腺病毒检测自噬的动态变化；运用免疫沉淀、ChIP、双荧光素酶报告基因等结合质谱分析和体外基因定点突变技术，检测HHcy对TFEB硝基化修饰的调控情况。本研究旨在揭示血管老化的调控机制，探讨通过降低TFEB硝基化修饰以上调自噬相关基因表达、提高自噬水平，进而延缓血管老化的可能性。

血管老化是多种增龄相关心血管疾病的发病基础，发生机制极为复杂，目前对于造成血管老化的确切机制仍不十分清楚。自噬是体内一种高度保守的过程，能够通过将胞质中的大分子物质和一些损伤、衰老的细胞器通过溶酶体途径降解，实现能量的再循环，以维持细胞自身的稳定。已有大量研究表明，自噬和衰老具有十分密切的关系。高同型半胱氨酸血症是心血管疾病的独立危险因子。在本研究中，我们发现高同型半胱氨酸血症模型大鼠出现明显血管老化表型，同时同型半胱氨酸可使内皮细胞衰老相关蛋白表达增加，伴细胞自噬水平明显下降，表明高同型半胱氨酸血症可通过下调自噬而促进血管老化。TFEB是调控自噬-溶酶体通路的重要转录因子，其转录活性受蛋白质翻译后修饰的影响。高同型半胱氨酸血症可诱发硝化应激，使蛋白质发生硝基化修饰而失活。本研究发现同型半胱氨酸能够使TFEB发生硝基化修饰，影响TFEB的入核及转录活性，使自噬-溶酶体通路基因的转录下降；同时同型半胱氨酸也可能通过使TFEB的上游转录因子C/EBPβ发生硝基化修饰，使TFEB表达下调，最终影响自噬-溶酶体通路基因的转录。利用过氧亚硝基的清除剂FeMPyP削弱硝化应激，则可以抑制上述作用。本研究从蛋白质硝基化修饰的角度，揭示了硝化应激异常导致细胞自噬水平降低从而促进血管老化的调控机制，探讨了通过降低蛋白质硝基化修饰以上调自噬相关基因表达、提高自噬水平，进而延缓血管老化的可能性。本研究为临床上对高同型半胱氨酸血症所致血管老化的防治提供了新的思路和干预靶点。