着色性干皮病基因XPD及其相关蛋白参与线粒体DNA氧化损伤修复的分子机制

Mitochondria as the “power house” in ATP production produce an increased level of reactive oxygen species (ROS), leading to a constant exposure of naked or unprotected mitochondrial DNA (mtDNA) to oxidative stress and subsequent accumulation of mtDNA damage and mutation. Thus, an efficient oxidative damage repair system is absolutely necessary for the maintenance of mitochondrial genome stability. Loss of function of XPD, the Xeroderma pigmentosum group D gene, can cause human disorders with common features of skin cancer predisposition and neural degeneration disorder. The later one is also the main phenotype of mitochondria dysfunction-related diseases. XPD-deficient cells are highly sensitive to oxidative stress, however, whether or not XPD protein can be located in mitochondria and participates in mtDNA oxidative damage repair are not well clear. Our preliminary data showed that XPD protein is located to inner membrane of mitochondria. XPD knock-down in U2OS cells largely compromise the mtDNA oxidative damage repair capacity. We further demonstrated that XPD is physically interact with TUFM, a mitochondrial Tu translation elongation factor. Knock down of TUFM expression also led to a significantly decreased capacity in mtDNA oxidative damage repair. This application is built on our preliminary observations and will elucidate the critical role of XPD and its associated proteins (such as TUFM) in mediating mtDNA oxidative damage repair, and further correlate with the dysregulated differentiation and senescence phenotypes in human neural stem cells. The data from this application will provide important insights into the molecular mechanism of XPD and its associated signalings in maintaining mitochondrial genome integrity, and form the basis for a better design of the precaution and intervention approaches in combatting the aging diseases related to compromised mitochondrial functions.

线粒体DNA不断受到线粒体内高水平氧自由基攻击而发生突变损伤，因此，高效的氧化损伤修复机制对维持线粒体基因组稳定性是必须的。XPD为着色性干皮病D型致病基因，其功能缺陷可导致以皮肤癌易感和神经退行性病变为主要特征的人类疾病综合征，后者也是线粒体疾病主要特征。XPD缺陷细胞对氧化损伤异常敏感，但XPD能否进入线粒体并参与线粒体DNA氧化损伤修复过程，目前还不清楚。我们前期结果证实XPD蛋白定位于线粒体内膜，其表达下调显著降低线粒体DNA氧化损伤修复能力；同时发现XPD与TUFM(线粒体Tu翻译延伸因子)相互作用，下调TUFM同样导致线粒体DNA氧化损伤修复能力的显著降低。本项目将重点阐明XPD及其互作蛋白（如TUFM）调控线粒体DNA氧化损伤修复，并进而影响神经干细胞分化老化过程的分子机制。所得结果将有助于阐明线粒体基因组稳定性维持的分子机理，为其异常调控相关疾病的预防和治疗提供重要理论依

线粒体DNA不断受到线粒体内高水平氧自由基攻击而发生突变损伤。因此，高效的氧化损伤修复机制对维持线粒体基因组稳定性是必须的。着色性干皮症D（XPD/ERCC2）编码ATP依赖的解旋酶，在细胞核转录及核苷酸切除修复（NER）中发挥必不可少的作用，但其是否参与线粒体DNA氧化损伤修复目前仍未知。我们前期结果发现XPD定位于线粒体内膜，其表达下调显著降低线粒体DNA氧化损伤修复能力，同时发现XPD与TUFM（线粒体Tu翻译延伸因子）相互作用，下调TUFM同样导致线粒体DNA氧化损伤修复能力的显著降低。本研究首先通过系列实验证明XPD在线粒体DNA的氧化性损伤修复机制中是一个关键因子，在通过促进有效率的线粒体DNA氧化性损伤修复进而保护线粒体基因组稳定性上具有重要作用。XPD形成两种复合物XPD-TUFM及XPD-MMS19，这两种复合物在有效修复线粒体DNA氧化损伤机制中发挥必不可少的作用。其次，我们还首次证实MMS19定位于线粒体内膜并参与线粒体DNA氧化性损伤修复。MMS19低表达线粒体DNA氧化损伤修复能力明显下降。通过免疫沉淀-质谱分析确定MMS19与线粒体ATP代谢相关的蛋白ANT2之前存在相互作用。在ANT2低表达的细胞中，线粒体DNA氧化损伤修复能力明显下降。以上研究表明，MMS19在维持线粒体基因组稳定性方面起着至关重要的作用。通过本项目实施，我们证实了XPD和MMS19在线粒体修复氧化损伤和维持维持线粒体功能稳态中的关键作用，相关结果有助于阐明线粒体基因组稳定性维持的分子机理，为其异常调控相关疾病的预防和治疗提供重要理论依据。