BACKGROUND Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. METHODS Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. RESULTS The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. CONCLUSIONS Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

中文翻译：

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NAE1 的巴豆酰化通过凝溶胶蛋白 Neddylation 调节心脏肥大。


背景技术心脏肥大及其相关的重构是心力衰竭的主要原因之一。赖氨酸巴豆酰化是最近发现的一种翻译后修饰，其在心脏肥大中的作用仍然很大程度上未知。 NAE1（NEDD8 [神经前体细胞表达发育下调蛋白 8] 激活酶 E1 调节亚基）主要参与蛋白质靶标的 neddylation 修饰。然而，巴豆酰化 NAE1 的功能尚未明确。本研究旨在阐明NAE1巴豆酰化对心脏肥大的影响和机制。方法 通过免疫沉淀和蛋白质印迹测定，检测患有心脏肥大的人类和小鼠受试者的巴豆酰化水平。采用串联质量标签 (TMT) 标记的定量赖氨酸巴豆酰化组分析来鉴定横主动脉缩窄诱导的小鼠心脏肥大模型中的巴豆酰化蛋白。我们生成了携带巴豆酰化缺陷型 K238R（第 238 位点赖氨酸到精氨酸突变）突变 (NAE1 K238R) 的 NAE1 敲入小鼠和表达模拟巴豆酰化 K238Q（第 238 位点赖氨酸到谷氨酰胺突变）突变的 NAE1 敲入小鼠（ NAE1 K238Q）以评估 NAE1 K238 巴豆酰化在病理性心脏肥大中的功能作用。此外，我们结合了免疫共沉淀、质谱和斑点印迹分析，随后采用多种分子生物学方法来鉴定目标 GSN（凝溶胶蛋白）和有助于 NAE1 K238（位点 238 的赖氨酸残基）巴豆酰化功能的相应分子事件。结果小鼠和心脏肥大患者的 NAE1 巴豆酰化水平升高。 定量巴豆酰组学分析表明，K238 是 NAE1 的主要巴豆酰化位点。 NAE1 K238R 敲入小鼠中 K238 巴豆酰化的缺失减轻了心脏肥大并恢复了心脏功能，而 NAE1 K238Q 敲入小鼠中的高巴豆酰化模拟显着增强了横主动脉缩窄诱导的病理性肥厚反应，导致心脏结构和功能受损。携带NAE1 K238R突变体的重组腺病毒载体减弱，而K238Q突变体则加剧Ang II（血管紧张素II）诱导的肥大。从机制上讲，我们将 GSN 确定为 NAE1 的直接目标。 NAE1 的 K238 巴豆酰化促进了 GSN neddylation，从而增强了其蛋白质稳定性和表达。 NAE1 巴豆酰化依赖性 GSN 增加促进了肌动蛋白切断活性，从而导致不利的细胞骨架重塑和病理性肥大的进展。结论我们的研究结果为心脏肥大过程中巴豆酰化对非组蛋白蛋白的先前未被认识的作用提供了新的见解。我们发现NAE1的K238巴​​豆酰化在通过GSN neddylation介导心脏肥大中发挥重要作用，这为病理性肥大和心脏重塑提供了潜在的新治疗靶点。