PARK15/FBXO7 is dispensable for PINK1/Parkin-dependent mitophagy in iNeuron and HeLa cell systems

The protein kinase PINK1 and ubiquitin ligase Parkin promote removal of damaged mitochondria via a feed-forward mechanism involving ubiquitin (Ub) phosphorylation (pUb), Parkin activation, and ubiquitylation of mitochondrial outer membrane proteins to support recruitment of mitophagy receptors. The ubiquitin ligase substrate receptor FBXO7/PARK15 is mutated in an early-onset parkinsonian-pyramidal syndrome. Previous studies have proposed a role for FBXO7 in promoting Parkin-dependent mitophagy. Here, we systematically examine the involvement of FBXO7 in depolarization and mtUPR-dependent mitophagy in the well-established HeLa and induced-neurons cell systems. We find that FBXO7-/- cells have no demonstrable defect in: 1) kinetics of pUb accumulation, 2) pUb puncta on mitochondria by super-resolution imaging, 3) recruitment of Parkin and autophagy machinery to damaged mitochondria, 4) mitophagic flux, and 5) mitochondrial clearance as quantified by global proteomics. Moreover, global proteomics of neurogenesis in the absence of FBXO7 reveals no obvious alterations in mitochondria or other organelles. These results argue against a general role for FBXO7 in Parkin-dependent mitophagy and point to the need for additional studies to define how FBXO7 mutations promote parkinsonian-pyramidal syndrome.

蛋白激酶PINK1与泛素连接酶Parkin可通过涉及泛素（Ubiquitin, Ub）磷酸化（pUb）、Parkin激活以及线粒体外膜蛋白泛素化的前馈机制，促进受损线粒体的清除，进而支持线粒体自噬受体的招募。泛素连接酶底物受体FBXO7/PARK15发生突变时，可引发早发性帕金森-锥体束综合征。既往研究曾提出FBXO7在促进Parkin依赖性线粒体自噬过程中发挥作用。本文中，我们在成熟稳定的海拉细胞（HeLa）与诱导神经元细胞系中，系统探究了FBXO7在去极化及线粒体未折叠蛋白反应（mitochondrial unfolded protein response, mtUPR）依赖性线粒体自噬中的作用。我们发现，FBXO7-/-细胞在以下5个方面均未表现出可检测的缺陷：1）pUb积累的动力学过程；2）超分辨率成像下线粒体表面的pUb斑点；3）Parkin与自噬系统向受损线粒体的招募；4）线粒体自噬通量；5）经全局蛋白质组学定量分析的线粒体清除率。此外，对FBXO7缺失状态下神经发生过程的全局蛋白质组学分析显示，线粒体及其他细胞器未出现明显异常。上述结果表明，FBXO7并不参与一般性的Parkin依赖性线粒体自噬，同时提示需开展更多研究以明确FBXO7突变如何引发帕金森-锥体束综合征。