Department of Cardiovascular Surgery, Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, China (J.J., Kai Wang, C.-Y.L., S.-C.W., L.-Y.Z., X.-M.L., Y.-Q.W., X.-Z.C., R.-F.L., S.-M.Y., Kun Wang).
Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Shandong Provincial Maternal and Child Health Care Hospital affiliated to Qingdao University, Jinan, China (J.J., M.-H.Z., Kun Wang).
Circ Res. 2024 Sep 27;135(8):806-821. doi: 10.1161/CIRCRESAHA.124.324733. Epub 2024 Sep 4.
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.
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.
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.
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.
心肌肥厚及其相关重构是心力衰竭的主要原因之一。赖氨酸巴豆酰化是一种新发现的翻译后修饰，其在心肌肥厚中的作用尚不清楚。NAE1（NEDD8 [神经前体细胞表达的发育下调蛋白 8]-激活酶 E1 调节亚基）主要参与蛋白靶标的 neddylation 修饰。然而，巴豆酰化 NAE1 的功能尚未确定。本研究旨在阐明 NAE1 巴豆酰化对心肌肥厚的影响和机制。
通过免疫沉淀和 Western blot 检测心肌肥厚患者和小鼠的巴豆酰化水平。通过串联质量标签（TMT）标记定量赖氨酸巴豆酰化组分析鉴定横主动脉缩窄诱导的小鼠心肌肥厚模型中的巴豆酰化蛋白。我们生成了携带巴豆酰化缺陷 K238R（位于 238 位的赖氨酸突变为精氨酸）突变的 NAE1 敲入小鼠（NAE1 K238R）和表达巴豆酰化模拟 K238Q（位于 238 位的赖氨酸突变为谷氨酰胺）突变的 NAE1 敲入小鼠（NAE1 K238Q），以评估 NAE1 在 K238 处的巴豆酰化在病理性心肌肥厚中的功能作用。此外，我们结合了免疫共沉淀、质谱和斑点印迹分析，随后进行了多种分子生物学方法，以鉴定靶标 GSN（凝胶蛋白）和对应分子事件，这些事件有助于 NAE1 K238（位于 238 位的赖氨酸残基）巴豆酰化的功能。
在小鼠和心肌肥厚患者中，NAE1 的巴豆酰化水平增加。定量巴豆酰化组学分析显示，K238 是 NAE1 的主要巴豆酰化位点。NAE1 K238R 敲入小鼠中 K238 巴豆酰化的丧失减弱了心肌肥厚并恢复了心脏功能，而 NAE1 K238Q 敲入小鼠中 K238 的超巴豆酰化模拟显著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 突变的重组腺病毒载体减弱了 Ang II（血管紧张素 II）诱导的肥大，而 K238Q 突变则加重了肥大。在机制上，我们鉴定了 GSN 为 NAE1 的直接靶标。NAE1 的 K238 巴豆酰化促进了 GSN 的 neddylation，从而增强了其蛋白稳定性和表达。NAE1 巴豆酰化依赖性 GSN 增加促进了肌动蛋白的切割活性，导致不良的细胞骨架重塑和病理性肥厚的进展。
我们的研究结果提供了关于心肌肥厚过程中非组蛋白蛋白巴豆酰化的新认识。我们发现，NAE1 的 K238 巴豆酰化在通过 GSN neddylation 介导心肌肥厚中起着重要作用，这为病理性肥厚和心脏重构提供了潜在的新治疗靶点。
