Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.
PLoS One. 2011 Mar 8;6(3):e16746. doi: 10.1371/journal.pone.0016746.
PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H(2)O(2) resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.
PINK1 和 Parkin 突变导致常染色体隐性帕金森病 (PD)。在果蝇和 SH-SY5Y 细胞中，Parkin 被 PINK1 募集到受损的线粒体，在那里它泛素化 Mitofusins，从而促进线粒体分裂和线粒体自噬。在这里，我们研究了内源性 PINK1 和 Parkin 突变对线粒体融合和裂变因子的泛素化以及线粒体网络结构的影响。用线粒体膜电位 (Δψ) 抑制剂或 H2O2 处理对照成纤维细胞导致 Mfn1/2 的泛素化，但不导致 OPA1 或 Fis1 的泛素化。通过 PINK1/Parkin 途径的 Mitofusins 泛素化在处理后 1 小时内观察到。在 Δψ 和泛素蛋白酶体系统 (UPS) 的联合抑制下，未检测到 Mitofusins 的泛素化。关于形态变化，我们观察到在线粒体应激下，PD 患者细胞中线粒体分支增加的趋势。在 PD 患者来源的细胞中，我们首次证明 PINK1 和 Parkin 突变会损害 Mitofusins 的泛素化。在 UPS 抑制剂存在的情况下，泛素化的 Mitofusin 被 UPS 去泛素化而不是降解，这表明 UPS 参与了 Mitofusin 的降解。
