Neddylation and mitophagy in cardiac aging

心脏衰老中的 Neddylation 和线粒体自噬

• 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
• 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

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.

项目摘要 线粒体质量和活动的下降与正常衰老有关，并与 各种与年龄有关的疾病的发展。因此，恢复线粒体功能或 改善线粒体质量控制可能是对抗衰老的有效策略。线粒体是必不可少的 线粒体质量控制机制介导了线粒体受损的溶酶体清除率。 越来越多的证据确定了增强的线索延长的影响 模型生物。有趣的是，最近的研究表明，增强线粒体可能会抵消衰老 相关的心脏功能障碍。因此，确定可以调节的更有效和特定的代理 通过线粒体清除线粒体有缺陷的可能性可能具有显着的治疗益处。我们 使用pH依赖性荧光进行了针对线粒体调节剂的高含量基于图像的测定 Mitophagy Reporter，MT-Keima。我们确定了选择性neddylation抑制剂MLN4924，是最多的 有效的线粒体激活剂。 Neddylation是一种连接泛素样蛋白的翻译后修饰 NEDD8通过NEDD8特异性E1-E2-E3酶蛋白靶标。值得注意的是，我们的机械研究表明 MLN4924有效阻止Cullin 2的Neddylation，这是Elongins B/C-Cullin 2-RBX1的成分（环盒） 1）-VHL（von Hippel-Lindau蛋白）E3泛素连接酶复合物（CRL2VHL）。抑制导致 缺氧诱导因子1亚基α（HIF1α），CRL2VHL底物的积累，然后 Bcl2相互作用蛋白3（BNIP3）的激活，一种用于线粒体诱导的线粒体受体。这些 结果提供了尼迪底化和线粒体之间的新联系。该项目旨在描绘小说 线粒体和尼迪底化之间的机械联系，并确定线粒体是否代表一种新颖 治疗与年龄相关的心脏功能障碍的机制和治疗靶点。这些研究将准备 通过我们最近描述的MT-Keima小鼠模型，以监测体内心脏线粒体。此外，我们会的 利用一组创新的试剂对基因和药物进行调节。直接评估 Neddylation在心脏中的作用，我们用Nae1的心肌细胞特异性缺失产生了小鼠 编码E1 NEDDYLATY激活酶的亚基。在拟议的研究的目标1中，我们的目标是 确定抑制Neddylation来调节心肌细胞和心脏线粒体的机制。 在拟议的研究的目标2中，我们将在成年人中普遍和身体操纵Neddylation 心脏使用缺乏NAE1或用MLN4924处理的小鼠模型。我们将确定是否还原 通过抑制和尼尔德化可以缓解与年龄相关的心脏功能障碍。完成建议 研究将对线粒体在与年龄相关的病理状况中的作用产生关键见解，并将 从根本上讲，我们对心脏中线粒体质量控制机制的理解从根本上提高了我们的理解。