Neddylation and cardiac protein quality control

项目摘要 蛋白质稳态的破坏导致心肌细胞（CM）功能障碍和死亡，并且越来越多地被认为是心肌细胞损伤的原因。 被认为在人类心肌病和心力衰竭中起因果作用。泛素（Ub）和Ub样蛋白 修饰不同的蛋白质底物并扩大蛋白质组的功能多样性。大量的工作 这表明了靶向这些蛋白质修饰剂对抗心脏病的令人兴奋的潜力。然而，在这方面， NEDD 8是一种新的Ub样蛋白，其在心脏中的功能作用仍然知之甚少。Neddylation 通过NEDD 8特异性E1-E2-E3酶将NEDD 8共价连接至靶蛋白。在此前的融资 在心脏周期中，我们发现neddylation在患者和动物模型的心脏衰竭中失调。 心脏病我们进一步证明了neddylation和deneddylation之间的平衡是必不可少的 心脏稳态，证明了这一事实，扰动的deneddylation沉淀心脏， 心肌病和心力衰竭，而在发育中的心脏中阻断neddylation引起心室 致密化不全和围产期致死。尽管neddylation对器官发育至关重要， 发育，其在完全分化的有丝分裂后组织中的功能重要性尚不清楚。该补助金涉及 一个新的联系neddylation和线粒体完整性在成年CM。的药理学抑制 neddylation促进线粒体融合，破坏线粒体膜电位， 新生儿CM的线粒体呼吸。NAE 1（NEDD 8 E1酶的调节亚基）在 成年CM也导致线粒体延长和线粒体质量受损。机械地说， neddylation修饰各种细胞蛋白以调节线粒体动力学和线粒体自噬。条件 敲除（KO）成年小鼠心脏中的NAE 1导致扩张型心肌病、心力衰竭，并最终导致心脏衰竭。 过早死亡在临床试验中，向癌症患者给予neddylation抑制剂MLN 4924， 严重的心脏毒性这些令人信服的已发表和初步数据构成了我们中心假设的基础 neddylation是通过调节线粒体动力学来实现正常心脏功能所必需的， 线粒体自噬使用新生成的诱导型NAE 1 KO小鼠和MLN 4924，Aim 1将建立 neddylation在成年健康和衰竭心脏中的意义，并确定neddylation对 线粒体功能目的2将剖析neddylation控制线粒体的分子基础 动力学，并将测试线粒体形态的正常化是否有益于NAE 1 KO心脏。目标3将 阐明neddylation调节线粒体自噬的新的不依赖于Parkin的机制，并测试是否 neddylation的恢复减轻了NAE 1 KO小鼠的心功能障碍。这项研究将是第一个建立 neddylation在成年CM中调节线粒体适应性和功能的作用未得到重视。而且 将为目前应用的neddylation抑制剂的潜在心脏毒性提供新的见解 在癌症治疗中，从而推进了心脏肿瘤学领域。

PROJECT SUMMARY Disruption of protein homeostasis causes cardiomyocyte (CM) dysfunction and death, and is increasingly recognized to play a causal role in human cardiomyopathies and heart failure. Ubiquitin (Ub) and Ub-like proteins modify diverse protein substrates and expand the functional diversity of the proteome. An extensive body of work has suggested the exciting potential of targeting these protein modifiers to combat cardiac disease. However, the functional role of NEDD8, a novel Ub-like protein, remains poorly understood in the heart. Neddylation covalently attaches NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Over the previous funding cycle, we discovered that neddylation is dysregulated in the failing hearts of patients and animal models of cardiac diseases. We further demonstrated that a balance between neddylation and deneddylation is essential to cardiac homeostasis, as evidenced by the fact that perturbation of deneddylation precipitated the heart to cardiomyopathy and heart failure, whereas blockade of neddylation in the developing heart caused ventricular noncompaction and perinatal lethality. Although it is becoming apparent that neddylation is essential for organ development, its functional importance in fully differentiated, postmitotic tissues is not clear. This grant addresses a novel linkage between neddylation and mitochondrial integrity in adult CMs. Pharmacological inhibition of neddylation promoted mitochondrial fusion, disrupted mitochondrial membrane potential and impaired mitochondrial respiration in neonatal CMs. Deletion of NAE1 (a regulatory subunit of the NEDD8 E1 enzyme) in adult CMs also resulted in mitochondrial elongation and impaired mitochondrial quality. Mechanistically, neddylation modifies various cellular proteins to regulate mitochondrial dynamics and mitophagy. Conditional knockout (KO) of NAE1 in adult mouse hearts resulted in dilated cardiomyopathy, heart failure, and ultimately premature death. In clinical trials, administration of the neddylation inhibitor MLN4924 to cancer patients elicited severe cardiotoxicity. These compelling published and preliminary data form the basis of our central hypothesis that neddylation is required for normal heart function through regulation of mitochondrial dynamics and mitophagy. Using the newly generated inducible NAE1KO mice and MLN4924, Aim 1 will establish the significance of neddylation in the adult healthy and failing heart and determine the impact of neddylation on mitochondrial function. Aim 2 will dissect the molecular basis by which neddylation controls mitochondrial dynamics and will test if normalization of mitochondrial morphology benefits the NAE1KO heart. Aim 3 will elucidate novel Parkin-independent mechanisms by which neddylation regulates mitophagy and test whether restoration of neddylation attenuates cardiac dysfunction in NAE1KO mice. This study will be the first to establish an unappreciated role for neddylation in regulating mitochondrial fitness and function in adult CMs. Moreover, it will provide novel insight into the potential cardiotoxicity of neddylation inhibitors that are currently being applied in cancer therapy, thus advancing the field of cardio-oncology.