Neddylation and mitophagy in cardiac aging

心脏衰老中的 Neddylation 和线粒体自噬

基本信息

批准号：
10419019
负责人：
Nuo Sun
金额：
$ 59.76万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10419019
关键词：
Adult Adverse effects Age Aging Algorithm Design Algorithms Animal Model Binding Proteins Biological Assay Cardiac Cardiac Myocytes Cardiovascular Physiology Cardiovascular system Cell Culture Techniques Collection Complex Cullin 2 Protein Data Development Disease Elderly Enzymes Genetic Goals HIF1A gene HRK gene Heart Heart failure Homeostasis Human Image Intervention Light Link Longevity Mediating Mitochondria Molecular Mus Myocardial dysfunction Myocardial tissue Pathologic Pathway interactions Patients Pharmacology Physiological Post-Translational Protein Processing Proteins Quality Control RBX1 gene Reagent Regulation Reporter Research Role Scaffolding Protein Therapeutic Tissue Sample Ubiquitin Ubiquitin Like Proteins Ubiquitination VHL protein age effect age related aged base cardioprotection cardiovascular health combat disorder risk elongin B heart function hypoxia inducible factor 1 improved in vivo monitoring induced pluripotent stem cell inhibitor innovation insight mortality mouse model normal aging novel novel therapeutic intervention preservation prevent receptor spatiotemporal therapeutic target ubiquitin-protein ligase young adult

项目摘要

PROJECT SUMMARY A decline in mitochondrial quality and activity has been associated with normal aging and correlated with the development of a wide range of age-related diseases. Therefore, rejuvenating mitochondrial function or improving mitochondrial quality control might be an effective strategy to combat aging. Mitophagy is an essential mitochondrial quality control mechanism that mediates the lysosomal clearance of damaged mitochondria. Increasing lines of evidence have established the longevity-extending effects of enhanced mitophagy in various model organisms. Interestingly, recent studies suggest that augmented mitophagy may counteract aging- associated cardiac dysfunction. Therefore, identifying more efficient and specific agents that can modulate the clearance of defective mitochondria via mitophagy are likely to have significant therapeutic benefits. We conducted high-content image-based assays for mitophagy modulators using the pH-dependent fluorescent mitophagy reporter, mt-Keima. We identified the selective neddylation inhibitor, MLN4924, to be the most effective mitophagy activator. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Of note, our mechanistic studies suggest MLN4924 effectively blocks neddylation of Cullin 2, a component of the elongins B/C-Cullin 2-Rbx1 (Ring-Box 1)-VHL (Von Hippel-Lindau protein) E3 ubiquitin ligase complex (CRL2VHL). The inhibition leads to an accumulation of the Hypoxia Inducible Factor 1 Subunit Alpha (HIF1α), a CRL2VHL substrate, and subsequent activation of the BCL2-interacting protein 3 (BNIP3), a mitochondrial receptor for mitophagy induction. These results provide a novel connection between neddylation and mitophagy. This project aims to delineate the novel mechanistic link between mitophagy and neddylation, and to determine whether mitophagy represents a novel mechanism and therapeutic target for treating age-related cardiac dysfunction. These studies will be facilitated by our recently described mt-Keima mouse model to monitor in vivo cardiac mitophagy. Additionally, we will utilize a set of innovative reagents to genetically and pharmacologically modulate neddylation. To directly assess the role of neddylation in the heart, we have generated mice with the cardiomyocyte-specific deletion of NAE1, encoding a subunit of the E1 neddylation activating enzyme. In Aim 1 of the proposed studies, our goal is to determine the mechanisms by which inhibiting neddylation regulates mitophagy in cardiomyocytes and the heart. In Aim 2 of the proposed studies, we will genetically and pharmacologically manipulate neddylation in the adult heart using mouse models that lack NAE1 or are treated with MLN4924. We will determine whether restoring mitophagy via inhibiting neddylation ameliorates age-related cardiac dysfunction. Completing the proposed studies will produce critical insights into the role of mitophagy in age-related pathological conditions, and will fundamentally advance our understanding of the mechanisms of mitochondrial quality control in the heart.

项目概要线粒体质量和活性的下降与正常衰老有关，并与因此，恢复线粒体功能或预防多种与年龄相关的疾病的发生。改善线粒体质量控制可能是对抗衰老的有效策略。线粒体质量控制机制，介导受损线粒体的溶酶体清除。越来越多的证据证实增强线粒体自噬在多种疾病中具有延长寿命的作用。模型生物体最近的研究表明，增强的线粒体自噬可能会对抗衰老。因此，寻找可以调节相关心脏功能障碍的更有效和特异性的药物。通过线粒体自噬清除有缺陷的线粒体可能具有显着的治疗效果。使用 pH 依赖性荧光对线粒体自噬调节剂进行高内涵图像分析我们发现选择性 neddylation 抑制剂 MLN4924 是最有效的线粒体自噬报告基因。有效的线粒体自噬激活剂是一种附着泛素样蛋白的翻译后修饰。值得注意的是，我们的机制研究表明，NEDD8 通过 NEDD8 特异性 E1-E2-E3 酶作用于蛋白质靶标。 MLN4924 有效阻断 Cullin 2 的 neddylation，Cullin 2 是 elongins B/C-Cullin 2-Rbx1 (Ring-Box 1)-VHL（Von Hippel-Lindau 蛋白）E3 泛素连接酶复合物 (CRL2VHL) 的抑制导致。缺氧诱导因子 1 亚基 Alpha (HIF1α)（CRL2VHL 底物）的积累，以及随后的激活 BCL2 相互作用蛋白 3 (BNIP3)，这是一种用于诱导线粒体自噬的线粒体受体。结果提供了 neddylation 和线粒体自噬之间的新颖联系。该项目旨在描述这种新颖的联系。线粒体自噬和neddylation之间的机制联系，并确定线粒体自噬是否代表一种新颖的治疗与年龄相关的心脏功能障碍的机制和治疗目标将得到促进。通过我们最近描述的 mt-Keima 小鼠模型来监测体内心脏线粒体自噬。利用一组创新试剂从遗传和药理学上调节 neddylation 来直接评估。为了研究neddylation在心脏中的作用，我们培育出了心肌细胞特异性缺失NAE1的小鼠，编码 E1 neddylation 激活酶的亚基在拟议研究的目标 1 中，我们的目标是确定抑制neddylation调节心肌细胞和心脏线粒体自噬的机制。在拟议研究的目标 2 中，我们将从遗传和药理学角度操纵成人的 neddylation 我们将使用缺乏 NAE1 或接受 MLN4924 治疗的小鼠模型来检测心脏是否恢复。线粒体自噬通过抑制neddylation改善与年龄相关的心脏功能障碍。研究将对线粒体自噬在与年龄相关的病理状况中的作用产生重要的见解，并将从根本上增进我们对心脏线粒体质量控制机制的理解。