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.

项目摘要 成年心肌细胞（CM）严重依赖于线粒体的正常功能来提供燃料 收缩（ATP）和调节其他重要的细胞过程。线粒体的健康是由 自噬的选择性形式，称为线粒体自噬，其去除泛素（Ub）标记的受损线粒体。 我们目前对线粒体自噬的理解是基于两个关键蛋白的作用，Ub连接酶帕金和 激酶PINK 1，其通过磷酸化的Ub链标记功能障碍的线粒体降解。 令人惊讶的是，帕金消融术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，提示存在尚待确定的、不依赖于Parkin的调控机制 CM中的线粒体更新。考虑到成年心脏中强大的线粒体吞噬活性和 由于各种心脏疾病中的线粒体自噬受损，越来越需要鉴定新的、不依赖于帕金森病的 心脏线粒体自噬的调节器。在这项提议的初步数据中，我们记录了一种新的机制， Cullin-Ring Ub连接酶5（CRL 5）与心脏中的线粒体质量控制。CRL 5是一种多蛋白复合物， 由RING盒蛋白RBX 2、支架蛋白Culllin 5（Cul 5）、衔接蛋白Elongin B/C和 各种底物受体蛋白。CRL 5的激活需要Cul 5的neddylation，这是一个 将小的Ub样蛋白NEDD 8缀合至靶蛋白。通过控制蛋白质的周转 作为底物，CRL 5参与几种生物过程和人类疾病，但在心脏中的作用不是 还不知道。抑制neddylation（控制CRL 5活性的上游信号传导），强烈抑制 线粒体泛素化和线粒体自噬，neddylation缺陷的小鼠由于 受损的线粒体自噬和线粒体功能障碍。蛋白质组学分析确定RBX 2和Cul 5相关 与线粒体在稳态;和他们的招聘线粒体是上调后，线粒体 损害RBX 2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，但被Cul 5概括。此外，在成年小鼠中RBX 2的缺失引起 受损的线粒体积累并导致心力衰竭。这些发现支持了我们的中心假设 RBX 2-CRL 5 Ub连接酶介导线粒体泛素化和线粒体自噬调节心肌细胞 在体内平衡和压力下的功能和生存。提出了三个目标。目标1将确定 使用RBX 2和Cul 5缺陷小鼠在健康正常心脏中RBX 2-CRL 5在线粒体转换中的作用。目的2 将通过鉴定其底物受体来鉴定CRL 5介导的线粒体自噬的分子基础， 底物和支架蛋白在线粒体和研究之间的相互作用CRL 5和PINK 1。 目的3将测试调节RBX 2以改善压力下的线粒体自噬和心脏损伤的可行性。 超负荷的心脏这项研究是重要的，因为它将确定CRL 5作为线粒体Ub连接酶，以维持 线粒体的完整性，并因此提供用于治疗心力衰竭的潜在治疗靶标。