TIFA/TRAF6/DNA2途径介导应激损伤中肾小管上皮细胞线粒体基因稳定性失衡的作用机制研究

Mitochondrial DNA (mtDNA) instability caused by stress factors inducing renal tubular cell (RTC) injury, which serves as a basis of the pathological change of acute kidney injury (AKI) and its regulating mechanism is unclear. Recently, DNA2 was identified as a key protein to maintain stability of nuclear genome as well as mtDNA. Our preliminary study indicated that DNA2 induced mtDNA instability due to its intracellular re-localization, which might be mediated by E3 ubiquitin ligase TRAF6 and its co-activator TIFA. The present study aims to further investigate the regulatory role and mechanism of TIFA/TRAF6/DNA2 pathway mediating RTC mtDNA instability under stress. The TIFA, TRAF6 and DNA2 expression manipulated cell models will be established and their biological function, including expression variations on gene or protein level, intracellular localization or migration, ubiquitin level change, etc, will be evaluated. Furthermore, the animal models of ischemia reperfusion AKI, septic AKI and drug toxicity AKI will be established on TRAF6 knock out mice, to confirm the mtDNA instability regulating mechanism in vivo. As the increasing prevalence of AKI, the results of this study will not only expand our understanding of the mechanisms of AKI, but also assist in the eventual development of new therapeutic targets for the disease.

应激致线粒体基因（mitochondrial DNA，mtDNA）稳定性失衡启动肾小管上皮细胞（Renal tubular cell, RTC）损伤是急性肾损伤（Acute kidney injury，AKI）的重要病理基础，机制未明。DNA2是新近鉴定的维持mtDNA稳定性的关键蛋白，课题组前期发现DNA2在AKI应激状态下发生细胞内再分布导致mtDNA稳定性丢失，且可能由E3泛素连接酶TRAF6及其协同蛋白TIFA介导。本研究拟进一步，建立TIFA、TRAF6及DNA2的RTC表达干预模型和敲除小鼠AKI模型，并通过蛋白质共表达、胞内迁移、泛素化修饰等的分析，评价蛋白质互作对mtDNA稳定性的调控，从分子、细胞及体内水平阐明介导应激损伤中RTC mtDNA稳定性失衡的TIFA/TRAF6/DNA2互作机制及其效应机制。本研究有助于深入理解AKI的病理机制，为疾病防治提供新线索。

应激致线粒体基因（mitochondrial DNA，mtDNA）稳定性失衡启动肾小管上皮细胞（Renal tubular cell, RTC）损伤是急性肾损伤（Acute kidney injury，AKI）的重要病理基础，机制未明。在本研究中，我们在细胞水平发现，在应激状态或上调TIFA可激活其协同蛋白E3泛素化连接酶TRAF6，结果发现细胞核内泛素化的DNA修复关键蛋白DNA2蛋白定量明显增加，而总体DNA2的表达以及线粒体内DNA2的蛋白表达未发生持续改变，下调TRAF6的表达可以逆转或阻断上述生物学效应。上述结果提示：E3泛素化连接酶TRAF6是应激状体下基因修复的重要调节因子，通过泛素化蛋白修饰DNA2，增加其稳定性并促进其核转位而发挥生物学效用。研究结果基本证实了本研究的科学假设，即TIFA/TRAF6/DNA2通过蛋白质互作介导了应激损伤中肾小管上皮细胞线粒体基因稳定性失衡，基于基因稳定性的角度为AKI的病理生理机制提供了重要线索。同时我们通过Dna2肾小管上皮细胞靶向敲除小鼠构建AKI模型，发现与野生型小鼠相比，Dna2-/-动物肾损伤病理表现更重，线粒体及mtDNA损伤也更重，且铁死亡相关标志物明显升高，这些结果以及我们在本研究中建立的细胞表达干预及基因修饰动物模型等，将为我们进一步的研究奠定坚实的基础。