Department of Ophthalmology, Eye Diseases and Optometry Institute, Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases, College of Optometry, Peking University Health Science Center, Peking University People's Hospital Beijing, China.
Beijing Key Laboratory of DNA Damage Response and College of Life Science, Capital Normal University, Beijing, China.
Invest Ophthalmol Vis Sci. 2022 Sep 1;63(10):7. doi: 10.1167/iovs.63.10.7.
Age-related macular degeneration (AMD) is currently the leading cause of blindness worldwide. Previously, we identified ubiquitin-protein ligase E3D (UBE3D) as an AMD-associated protein for East Asian populations, and here we further demonstrate that UBE3D could be associated with DNA damage response.
The established I-SceI-inducible GFP reporter system was used to explore the effect of UBE3D on homologous recombination. Immunoprecipitation-mass spectrometry (MS) was used to explore potential UBE3D-interacting proteins and validated with coimmunoprecipitation assays and the pulldown assays. Micrococcal nuclease (MNase) assays were used to investigate the function of UBE3D on heterochromatin de-condensation upon DNA damage. An aged mouse model of blue light-induced eye damage was constructed, and electroretinography (ERG) and optical coherence tomography (OCT) were performed to compare the differences between wild-type and UBE3D+/- mice.
First, we show that GFP-UBE3D is recruited to damage sites by PCNA, through a PCNA-interacting protein (PIP) box. Furthermore, UBE3D interacts with KAP1 via R377R378 and oxidation of the AMD-associated V379M mutation abolishes KAP1-UBE3D binding. By MNase assays, UBE3D depletion reduces the chromatin relaxation levels upon DNA damage. In addition, UBE3D depletion renders less KAP1 recruitment. Compared with wild type, blue light induces less damage in UBE3D+/- mice as measured by ERG and OCT, consistent with our biochemical results.
Hence, we propose that one potential mechanism that UBE3D-V379M contributes to AMD pathogenesis might be via defective DNA damage repair linked with oxidative stress and our results offered a potential direction for the treatment of AMD.
年龄相关性黄斑变性（AMD）是目前全球致盲的主要原因。此前，我们发现泛素蛋白连接酶 E3D（UBE3D）是东亚人群 AMD 的相关蛋白，在此基础上，我们进一步证明 UBE3D 可能与 DNA 损伤反应有关。
使用已建立的 I-SceI 诱导 GFP 报告系统来研究 UBE3D 对同源重组的影响。免疫沉淀-质谱（MS）用于探索潜在的 UBE3D 相互作用蛋白，并通过共免疫沉淀和下拉实验进行验证。微球菌核酸酶（MNase）实验用于研究 UBE3D 在 DNA 损伤时对异染色质解凝聚的作用。构建了蓝光诱导的年龄相关性眼病小鼠模型，进行视网膜电图（ERG）和光相干断层扫描（OCT）检查，比较野生型和 UBE3D+/- 小鼠之间的差异。
首先，我们发现 GFP-UBE3D 通过 PCNA 相互作用蛋白（PIP）盒募集到损伤部位。此外，UBE3D 通过 R377R378 与 KAP1 相互作用，AMD 相关的 V379M 突变的氧化会破坏 KAP1-UBE3D 结合。通过 MNase 实验，UBE3D 耗竭降低了 DNA 损伤后的染色质松弛水平。此外，UBE3D 耗竭导致 KAP1 募集减少。与野生型相比，ERG 和 OCT 测量的蓝光诱导的 UBE3D+/- 小鼠损伤较小，与我们的生化结果一致。
因此，我们提出 UBE3D-V379M 导致 AMD 发病机制的一个潜在机制可能是通过与氧化应激相关的 DNA 损伤修复缺陷，我们的结果为 AMD 的治疗提供了一个潜在的方向。
