Wang Wenjuan, Li Ermin, Zou Jianqiu, Qu Chen, Ayala Juan, Wen Yuan, Islam Md Sadikul, Weintraub Neal L, Fulton David J R, Liang Qiangrong, Zhou Jiliang, Liu Jinbao, Li Jie, Sun Yi, Su Huabo
Vascular Biology Center (W.W., E.L., J. Zou, C.Q., J.A., Y.W., M.S.I., N.L.W., D.J.R.F., J. Li, H.S.), Medical College of Georgia, Augusta University.
Affiliated Cancer Hospital and Institute of Guangzhou Medical University, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, State Key Laboratory of Respiratory Disease, School of Basic Medical Sciences, Guangzhou Medical University, China (W.W., J. Liu).
Circ Res. 2024 Jul 19;135(3):e39-e56. doi: 10.1161/CIRCRESAHA.124.324285. Epub 2024 Jun 14.
Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart.
In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome.
RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, 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 the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. 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 (PTEN-induced kinase 1) in mitochondria.
These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.
通过线粒体自噬清除受损线粒体对细胞内稳态至关重要。除了帕金蛋白外，对于介导线粒体泛素化和更新的其他泛素连接酶知之甚少，尤其是在心脏等高代谢活性器官中。
在本研究中，我们结合了计算机分析和生化检测，以鉴定E3泛素连接酶Cullin-RING连接酶5（CRL5）作为线粒体泛素连接酶。我们构建了缺乏CRL5催化亚基RBX2（RING结构域蛋白2；也称为SAG [凋亡敏感基因]）的心肌细胞和小鼠，以了解RBX2缺失对线粒体泛素化、线粒体自噬和心脏功能的影响。我们还进行了蛋白质组学分析和RNA测序分析，以确定RBX2缺失对蛋白质组和转录组的影响。
RBX2和CRL5的2个核心成分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，增加了心肌细胞死亡，并对线粒体蛋白质组产生了全局性影响。在体内，成年小鼠心脏中该基因的缺失抑制了线粒体自噬活性，促使受损线粒体在心肌中积累，并扰乱了心肌代谢，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。RBX2在线粒体中的作用不依赖于帕金蛋白，帕金基因的缺失对RBX2缺陷心脏中心肌病的发生和发展没有影响。此外，RBX2控制线粒体中PINK1（PTEN诱导激酶1）的稳定性。
这些发现确定RBX2-CRL5是一种线粒体泛素连接酶，它以不依赖帕金蛋白、依赖PINK1的方式调节线粒体自噬和心脏内稳态。
