Mitochondrial quality control in the heart

项目总结 成年心肌细胞(CMS)严重依赖线粒体的正常功能来提供燃料。 用于收缩(ATP)和调节其他基本的细胞过程。线粒体的健康由一种 选择性的自噬形式，称为有丝分裂吞噬，去除泛素(Ub)标记的受损线粒体。 我们目前对有丝分裂的理解是基于两个关键蛋白的作用，即Ub连接酶Parkin和 激酶PINK1，它标志着功能障碍的线粒体通过磷酸化的Ub链进行降解。 令人惊讶的是，切除帕金并没有影响年轻和年轻患者的体内平衡有丝分裂或心功能。 衰老小鼠，提示存在尚未确定的帕金非依赖性控制机制 细胞质雄性不育的线粒体周转率。鉴于成人心脏中强烈的有丝分裂活动和 在各种心脏疾病中丝裂原吞噬功能受损，越来越需要确定新的、非帕金依赖的 心脏中有丝分裂的调节者。在这项提案的初步数据中，我们记录了一个新的机制，将 卡尔林-环Ub连接酶5(CRL5)与心脏线粒体质量控制。CRL5是一种多蛋白质复合体 由环盒蛋白RBX2、支架蛋白Culllin 5(Cul5)、接头蛋白Elongin B/C和 各种底物受体蛋白。CRL5的激活需要Cul5的内化，这一过程 将小的Ub样蛋白NEDD8与靶蛋白结合。通过控制其蛋白质的周转 底物，CRL5参与了几个生物过程和人类疾病，但在心脏中的作用不是 目前还不得而知。抑制代谢(调节CRL5活性的上游信号)，受到强有力的抑制 线粒体泛素化和有丝分裂，以及缺乏代谢的小鼠由于 线粒体吞噬功能受损和线粒体功能障碍。蛋白质组学分析发现RBX2和Cul5是相关的 线粒体处于动态平衡；它们向线粒体的募集随着线粒体的增加而上调 损坏。RBX2是线粒体泛素化和周转所必需的，它在线粒体中的作用是 独立于帕金，但由卡尔5重述。此外，成年小鼠中RBX2基因的缺失引发了 损伤的线粒体堆积，导致心力衰竭。这些发现支持我们的中心假设 RBX2-CRL5 Ub连接酶介导线粒体泛素化和有丝分裂调节心肌细胞 在动态平衡和压力下的功能和生存。提出了三个目标。目标1将定义 RBX2-CRL5在正常健康小鼠心脏线粒体周转中的作用。目标2 将通过识别CRL5的底物受体来确定CRL5介导的有丝分裂吞噬的分子基础， 线粒体中的底物和支架蛋白以及CRL5和PINK1之间的相互作用。 目的3将测试调节RBX2以改善有丝分裂和心脏损伤的可行性。 超负荷的心脏。这项研究具有重要意义，因为它将确定CRL5是一种线粒体Ub连接酶，可以维持 线粒体的完整性，从而为心力衰竭的治疗提供了潜在的治疗靶点。

PROJECT SUMMARY Adult cardiomyocytes (CMs) are critically dependent on the proper function of mitochondria to supply the fuel for contraction (ATP) and to regulate other essential cellular processes. Mitochondrial health is maintained by a selective form of autophagy, termed mitophagy, which removes Ubiquitin (Ub)-labeled damaged mitochondria. Our current understanding of mitophagy is based on the actions of two key proteins, the Ub ligase Parkin and kinase PINK1, which mark dysfunctional mitochondria for degradation via a phosphorylated Ub chain. Surprisingly, ablation of Parkin failed to impact homeostatic mitophagy or cardiac function in both young and ageing mice, suggesting the existence of yet to be identified, Parkin-independent mechanisms control mitochondrial turnover in CMs. Given robust mitophagic activity in adult hearts and the deleterious impact of impaired mitophagy in various cardiac diseases, there is a growing need to identify novel, Parkin-independent regulators of mitophagy in the heart. In preliminary data for this proposal, we document a new mechanism linking Cullin-Ring Ub ligase 5 (CRL5) with mitochondrial quality control in the heart. CRL5 is a multi-protein complex comprised of the RING box protein RBX2, scaffold protein Culllin 5 (Cul5), adaptor proteins Elongin B/C, and various substrate receptor proteins. Activation of CRL5 requires the neddylation of Cul5, a process that conjugates the small Ub-like protein, NEDD8 to target proteins. By controlling the turnover of its protein substrates, CRL5 participates in several biological processes and human diseases, but a role in the heart is not yet known. Inhibition of neddylation (upstream signaling that governs CRL5 activity), robustly suppressed mitochondrial ubiquitination and mitophagy, and mice deficient in neddylation develop heart failure due to impaired mitophagy and mitochondrial dysfunction. Proteomics analysis identifies RBX2 and Cul5 are associated with mitochondria in homeostasis; and their recruitment to mitochondria is upregulated following mitochondria damage. RBX2 is required for mitochondrial ubiquitination and turnover, and its actions in mitochondria are independent of Parkin but are recapitulated by Cul5. Moreover, deletion of RBX2 in adult mice provokes accumulation of damaged mitochondria and leads to heart failure. These findings support our central hypothesis that RBX2-CRL5 Ub ligase mediates mitochondrial ubiquitination and mitophagy to regulate cardiac myocyte function and survival in homeostasis and under stress. Three aims are proposed. Aim 1 will define the role of RBX2-CRL5 in mitochondrial turnover in the healthy normal heart using RBX2- and Cul5- deficient mice. Aim 2 will identify the molecular underpinnings of CRL5-mediated mitophagy by identifying its substrate receptors, substrates and scaffold proteins in mitochondria and by investigating the interplay between CRL5 and PINK1. Aim 3 will test the feasibility of modulation of RBX2 to improve mitophagy and cardiac injury in pressure- overloaded hearts. This study is significant as it will identify CRL5 as a mitochondrial Ub ligase to maintain mitochondrial integrity and thus provide potential therapeutic targets for treatment of heart failure.