金属硫蛋白对衰老心肌中α-酮戊二酸脱氢酶硝化损伤的调控机制研究

氧化硝化损伤在心肌衰老过程中扮演重要的角色，非特异性抗氧化分子金属硫蛋白（MT）可显著抑制氧化硝化诱导的心肌功能退行性衰变，改善心肌功能。但MT对衰老心肌的作用机制尚不清楚。前期通过蛋白质组学技术对心肌MT特异高表达的衰老小鼠研究发现，MT可显著抑制衰老心肌中α-酮戊二酸脱氢酶（α-KGD）的硝化水平，而α-KGD又是体内能量代谢的关键酶，是维持心肌正常生理功能的必要因素之一，提示MT可能通过调控α-KGD的硝化水平，改善心肌的能量供应，从而减少心肌细胞的凋亡，发挥其对衰老心肌的保护作用。本课题旨在前期实验基础上进一步研究MT对衰老心肌中α-KGD硝基化修饰酪氨酸位点的调控，并通过体外表达、定点突变和动物体内转染检测各硝基化修饰位点对靶酶活性和亚细胞定位的影响，以期深入探索MT对衰老心肌保护机制与硝化损伤的关系。本项研究结果不仅能在理论上指导临床，而且有望开辟延缓心肌衰老方法的新篇章。

Oxidative and nitrative stress is a direct factor to cause myocardial damage, and plays a critical role in the occurrence of age-associated myocardial diseases. Metallothionein (MT) as a non-specific antioxidant, which significantly protects oxidative and nitrative stress associated myocardial dysfunction. However, the mechanism of signal transduction and network regulation of this process remains unknown. Previous proteomics studies on the MT-TG aged-mice (cardiac-specific overexpression of MT) showed that MT obviously inhibited age-induced 3-NT modifications of α-oxoglutarate dehydrogenase (α-KGD) in the heart. Moreover, α-KGD is a necessary component of α-oxoglutarate dehydrogenase complex, which is an important enzyme complex and plays an important role in numerous metabolic pathways in the organism. Therefore, it suggests that MT may regulate the nitration level of α-KGD to improve cardiac energy supply, reduce myocardial apoptosis and prevent age-associated myocardial dysfunction. The purpose of this project is to understand how MT regulates the nitration level of each tyrosine residue of α-KGD. Site-directed mutagenesis and in vitro recombinant protein expression systems will be introduced to identify the effects of each nitrated tyrosine residue on the activity ofα-KGD, which will be expected to explore the relationship between the protection mechanism of MT on age-associated myocardial diseases and nitrative damage. The achievement of this subject will not only provide a theoretical basis for clinical therapy but also exploit a new method for the diagnosis of age-associated myocardial diseases.