Jiangsu Provincial Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang No. 24, Nanjing 210009, Jiangsu, China.
State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture, School of Pharmacy, Nanjing University of Chinese Medicine, No. 138 Xianlin Avenue, Nanjing 210023, Jiangsu, China.
J Proteome Res. 2024 Oct 4;23(10):4614-4625. doi: 10.1021/acs.jproteome.4c00525. Epub 2024 Sep 24.
Lysine lactylation has recently been discovered and demonstrated to be an essential player in immunity, cancer and neurodegenerative diseases. Genetic code expansion (GCE) technique is powerful in uncovering lactylation functions, since it allows site-specific incorporation of lactyllysine (Klac) into proteins of interest (POIs) in living cells. However, the inefficient uptake of Klac into cells, due to its high hydrophilicity, results in limited expression of lactylated POIs. To address this challenge, here we designed esterified Klac derivatives, exemplified by ethylated Klac (KlacOEt), to enhance Klac's lipophilicity and improve its cellular uptake. The expression level of site-specifically lactylated POIs was doubled using KlacOEt in both and HEK293T cells. Immunoprecipitation mass spectrometry analysis verified the significantly increased yield of the precisely lactylated fructose-bisphosphate aldolase A using KlacOEt. Furthermore, in conjunction with the Target Responsive Accessibility Profiling approach, we found that lactylation at ALDOA-K147 altered the protein's conformation, which may explain the lactylation-induced reduction in enzyme activity. Together, we demonstrate that, through enhancing the yield of lactylated proteins with Klac esters via GCE, we are able to site-specifically reveal the effects of lactylation on POIs' interactions, conformations and activities using a suite of functional proteomics and biochemical tools.
赖氨酸内酯化最近被发现并被证明是免疫、癌症和神经退行性疾病的重要参与者。遗传密码扩展（GCE）技术在揭示内酯化功能方面非常强大，因为它允许在活细胞中将乳酰赖氨酸（Klac）特异性地掺入感兴趣的蛋白质（POI）中。然而，由于 Klac 的高亲水性，其进入细胞的效率不高，导致乳酰化 POI 的表达有限。为了解决这个挑战，我们在这里设计了酯化 Klac 衍生物，例如 KlacOEt，以提高 Klac 的亲脂性并改善其细胞摄取。使用 KlacOEt，在 和 HEK293T 细胞中，特异性乳酰化 POI 的表达水平提高了一倍。免疫沉淀质谱分析证实，使用 KlacOEt 可显著增加精确乳酰化果糖-1,6-二磷酸醛缩酶 A 的产量。此外，结合靶标响应可及性分析方法，我们发现 ALDOA-K147 上的内酯化改变了蛋白质的构象，这可能解释了内酯化导致酶活性降低的原因。总之，我们通过使用 GCE 用 Klac 酯提高乳酰化蛋白的产量，展示了使用一系列功能蛋白质组学和生化工具，能够特异性地揭示内酯化对 POI 相互作用、构象和活性的影响。
