铁调节蛋白IRP2的去泛素化酶鉴定及其在帕金森病黑质铁聚集中的作用研究

Iron regulatory protein 2 is a key factor in regulating the brian iron homeostasis. It had been found that IRP2-/- mice displayed neurodegenerative symptoms and nigral iron deposition. In the previous study, we also found downregulation of IRP2 expression in the substantia nigra of Parkinson's disease (PD) transgenic mice. However, the underlying mechanism is largly unkonwn. Ubiquitination is a major pathway in regulating protein stability. Even though two ubiquitin ligases that promote IRP2 degradation have been found, the deubiquitinase(DUB) of IRP2 has not been reported. We found that OTUD3 could specifically remove the ubiquitination of IRP2, which up-regualted its level. Protein levels of OTUD3 in PD transgenic mice were decreased. In order to further study the molecular mechanism of OTUD3 on regulating IRP2 and its role in the nigral iron deposition in PD, we frist study the interaction between OTUD3 and IRP2 from the molecular and cellular levels. Then, we would elucidate the effects of the stability of IRP2 regulated by OTUD3 on dopaminergic iron metabolism in PD. We also used PD transgenic mice and OTUD3 knockout mice model to study the role of OTUD3 and IRP2 ineraction in nigral iron deposition in PD. Taken together, this study would find the first DUB of IRP2, and would provide a new mechanism for the nigral iron deposition in PD.

铁调节蛋白IRP2是调控脑铁稳态的关键因素。研究发现IRP2-/-小鼠出现神经退行性病变和黑质铁聚集，我们在前期工作中，观察到帕金森病（PD）小鼠黑质IRP2下调，但机制不明。蛋白质的泛素化修饰是调节其稳定性的主要途径，目前已发现两个IRP2的泛素连接酶，但其去泛素化酶未见报道。我们对人源DUB家族进行无偏性筛选，发现OTUD3可特异性去除IRP2的泛素化，上调IRP2的表达，PD小鼠OTUD3下调。为了进一步研究OTUD3调控IRP2的分子机制，及在PD黑质铁聚集中的作用，本项目拟在分子和细胞水平，研究OTUD3和IRP2的相互作用，明确OTUD3调控IRP2稳定性在细胞铁代谢及PD中的作用；在整体水平，利用PD转基因和OTUD3-/-小鼠，研究OTUD3和IRP2的相互作用在PD黑质铁聚集的作用。本项目有望鉴定出IRP2的第一个去泛素化酶OTUD3，并为揭示PD黑质铁聚集提供新机制。

帕金森病（Parkinson’s disease, PD）病人早期铁选择性沉积在黑质，铁代谢调控异常是导致PD中多巴胺能神经元损伤的一个关键因素。IRP2是调控脑铁稳态的一个关键因素。目前已发现两个IRP2的E3泛素连接酶，但其DUBs未见报道。本研究筛选到IRP2的去泛素化酶OTU结构域蛋白3 (OTUD3)，在细胞质中与IRP2相互作用，去除IRP2的泛素化并以不依赖铁的方式稳定IRP2蛋白。敲除OTUD3导致铁代谢紊乱，OTUD3敲除小鼠出现PD样的铁积累、运动障碍和黑质纹状体多巴胺能神经退行性变。在表达人A53T α-突触核蛋白突变体的转基因PD小鼠中检测到OTUD3蛋白水平下降。在散发性PD病例或对照组中发现OTUD3存在5个单核苷酸多态性突变。综上，OTUD3是IRP2的一个去泛素化酶，并在PD的黑质铁稳态的调节中发挥关键作用。