Wang Wenjuan, Li Ermin, Zou Jianqiu, Qu Chen, Ayala Juan, Wen Yuan, Islam Md Sadikul, Weintraub Neal L, Fulton David J, Liang Qiangrong, Zhou Jiliang, Liu Jinbao, Li Jie, Sun Yi, Su Huabo
bioRxiv. 2024 Jul 2:2024.02.24.581168. doi: 10.1101/2024.02.24.581168.
Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. , deletion of the gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.
通过线粒体自噬清除受损线粒体对细胞内稳态至关重要。虽然泛素（Ub）连接酶帕金森蛋白（PARKIN）在线粒体自噬中的作用已得到广泛研究，但越来越多的证据表明，在心脏等高代谢活性器官中存在不依赖PARKIN的线粒体自噬。在此，我们确定了Cullin-RING泛素连接酶5（CRL5）在心肌细胞基础线粒体更新中的关键作用。CRL5是一种多亚基泛素连接酶，由催化性的RING盒蛋白RBX2（也称为SAG）、支架蛋白Cullin 5（CUL5）和一个底物识别受体组成。对线粒体外膜相互作用蛋白质组的分析揭示了CRL与线粒体之间的紧密关联。亚细胞分级分离、免疫染色和免疫金电子显微镜证实，CRL5的两个核心成分RBX2和Cul5定位于线粒体。RBX2的缺失抑制了线粒体泛素化和更新，损害了线粒体膜电位和呼吸作用，并增加了心肌细胞的死亡。此外，在成年小鼠心脏中删除该基因会抑制线粒体自噬活性，引发心肌中受损线粒体的积累，并扰乱心肌代谢，导致扩张型心肌病和心力衰竭的快速发展。同样，在发育中的心脏中敲除RBX2会导致扩张型心肌病和心力衰竭。值得注意的是，RBX2在线粒体中的作用不依赖于PARKIN，并且PARKIN基因缺失对RBX2缺陷心脏中心肌病的发生和进展没有影响。此外，RBX2控制线粒体中PINK1的稳定性。蛋白质组学和生化分析进一步揭示了RBX2缺陷对线粒体蛋白质组的整体影响，并确定了几种线粒体蛋白为其假定底物。这些发现确定RBX2-CRL5是一种线粒体泛素连接酶，它在生理条件下以不依赖PARKIN、依赖PINK1的方式控制线粒体自噬，从而调节心脏内稳态。
