■心脏肥大及其相关的重塑是心力衰竭的主要原因。赖氨酸巴豆化是最近发现的翻译后修饰,其在心脏肥大中的作用仍然未知。NAE1(NEDD8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的功能尚未确定。本研究旨在阐明NAE1crotonylation对心肌肥厚的影响和机制。
■通过免疫沉淀和蛋白质印迹测定在患有心脏肥大的人和小鼠受试者中检测到巴豆化水平。进行TMT标记的定量赖氨酸巴豆基因组分析以鉴定由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。我们产生了NAE1敲入小鼠,其携带巴豆化缺陷的赖氨酸至精氨酸K238R(在238位点处的赖氨酸至精氨酸突变)突变(NAE1K238R)和表达巴豆化模拟赖氨酸至谷氨酰胺K238Q的NAE1敲入小鼠(在238位点处的赖氨酸至谷氨酰胺突变)突变(NAE1K238Q),以评估NAE1病理性心肌肥大的巴豆化的作用。此外,我们联合免疫共沉淀,质谱,和斑点印迹分析,然后是多种分子生物学方法,以鉴定靶GSN(凝溶胶蛋白)和相应的分子事件,这些分子事件有助于NAE1K238巴豆化的功能。
■在心肌肥厚的小鼠和患者中,NAE1的巴豆化水平升高。定量巴豆酰基组学分析显示,K238是NAE1的主要巴豆酰化位点。NAE1K238R敲入小鼠中K238crotonylation的缺失减轻了心脏肥大并恢复了心脏功能,而NAE1K238Q敲入小鼠中的高巴豆化模拟物显着增强了横主动脉缩窄引起的病理性肥大反应,导致心脏结构和功能受损。携带NAE1K238R突变体的重组腺病毒载体减毒,而K238Q突变体加重了AngII(血管紧张素II)诱导的肥大。机械上,我们确定GSN是NAE1的直接靶标。NAE1的K238crotonylation促进了GSNneddylation,因此,增强其蛋白稳定性和表达。NAE1巴豆化依赖性增加GSN促进肌动蛋白切断活性,这导致不利的细胞骨架重塑和病理性肥大的进展。
■我们的发现提供了新的见解,以了解以前未被认识到的在心肌肥厚过程中的非组蛋白蛋白的作用。我们发现NAE1的K238crotonylation通过GSNneddylation在介导心脏肥大中起重要作用,这为病理性肥大和心脏重塑提供了潜在的新治疗靶点。
UNASSIGNED: 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-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.
UNASSIGNED: Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. 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 lysine to arginine K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking lysine to glutamine 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 crotonylation.
UNASSIGNED: 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.
UNASSIGNED: 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.