Mitochondrial quality control in the heart

心脏线粒体质量控制

• 批准号: 10503851
• 负责人: Huabo Su
• 金额: $ 59.68万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10503851
• 关键词: Ablation; Adaptor Signaling Protein; Address; Adult; Aging; Autophagocytosis; Biochemical; Biological; Biological Assay; Biological Process; Biotinylation; Cardiac; Cardiac Myocytes; Cell Death; Cell physiology; Cullin 5 Protein; Cullin Proteins; Data; Disease; Enzymes; Health; Heart; Heart Diseases; Heart Injuries; Heart failure; Homeostasis; Impairment; In Vitro; Label; Ligase; Link; LoxP-flanked allele; Mediating; Membrane Potentials; Mitochondria; Molecular; Monitor; Multiprotein Complexes; Mus; PINK1 gene; Parkin; Pathologic; Phosphorylation; Phosphotransferases; Physiological; Process; Proteins; Proteomics; Quality Control; Regulation; Reporter; Research; Respiration; Role; Scaffolding Protein; Signal Transduction; Stress; System; Tamoxifen; Testing; Ubiquitin; Ubiquitin Like Proteins; Ubiquitination; base; cardiogenesis; elongin B; experimental study; feasibility testing; heart function; human disease; improved; in vivo; insight; mitochondrial dysfunction; mitochondrial membrane; molecular dynamics; mouse model; novel; overexpression; premature; pressure; protein complex; receptor; recruit; therapeutic target; ubiquitin ligase

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.

项目概要 成体心肌细胞 (CM) 严重依赖线粒体的正常功能来提供燃料 用于收缩（ATP）并调节其他重要的细胞过程。线粒体的健康由以下因素维持 选择性形式的自噬，称为线粒体自噬，可去除泛素 (Ub) 标记的受损线粒体。 我们目前对线粒体自噬的理解基于两种关键蛋白的作用，即 Ub 连接酶 Parkin 和 激酶 PINK1，标记功能失调的线粒体，通过磷酸化的 Ub 链进行降解。 令人惊讶的是，帕金的消融未能影响年轻人和老年人的稳态线粒体自噬或心脏功能。 衰老小鼠，表明存在尚未确定的 Parkin 独立机制控制 CM 中的线粒体周转。鉴于成人心脏中强大的线粒体自噬活性以及 各种心脏病中线粒体自噬受损，越来越需要识别新的、不依赖 Parkin 的线粒体自噬 心脏线粒体自噬的调节因子。在该提案的初步数据中，我们记录了一种新的机制，将 Cullin-Ring Ub 连接酶 5 (CRL5) 在心脏中具有线粒体质量控制。 CRL5是一种多蛋白复合物 由 RING 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 neddylation，该过程 将小型 Ub 样蛋白 NEDD8 与靶蛋白结合。通过控制蛋白质的周转 CRL5 参与多种生物过程和人类疾病，但在心脏中没有作用 尚未知晓。抑制neddylation（控制CRL5活性的上游信号），被强力抑制 线粒体泛素化​​和线粒体自噬，缺乏 neddylation 的小鼠会因以下原因发生心力衰竭： 线粒体自噬受损和线粒体功能障碍。蛋白质组学分析确定 RBX2 和 Cul5 相关 线粒体处于稳态；并且它们对线粒体的募集随着线粒体的增加而上调 损害。 RBX2 是线粒体泛素化​​和周转所必需的，其在线粒体中的作用是 独立于 Parkin，但由 Cul5 重述。此外，成年小鼠中 RBX2 的缺失会引发 受损线粒体的积累并导致心力衰竭。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​​和线粒体自噬来调节心肌细胞 体内平衡和压力下的功能和生存。提出了三个目标。目标 1 将定义以下角色： 使用 RBX2 和 Cul5 缺陷小鼠观察 RBX2-CRL5 在健康正常心脏线粒体周转中的作用。目标2 将通过识别其底物受体来识别 CRL5 介导的线粒体自噬的分子基础， 线粒体中的底物和支架蛋白，并研究 CRL5 和 PINK1 之间的相互作用。 目标 3 将测试调节 RBX2 以改善压力下线粒体自噬和心脏损伤的可行性 超负荷的心。这项研究意义重大，因为它将鉴定 CRL5 作为线粒体 Ub 连接酶以维持 线粒体完整性，从而为心力衰竭的治疗提供潜在的治疗靶点。