Pluripotent stem cells (PSCs) can be expanded in vitro in different culture conditions, resulting in a spectrum of cell states with distinct properties. Understanding how PSCs transition from one state to another, ultimately leading to lineage-specific differentiation, is important for developmental biology and regenerative medicine. Although there is significant information regarding gene expression changes controlling these transitions, less is known about post-translational modifications of proteins. Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group. Here, we employed affinity purification of crotonylated peptides and liquid chromatography–tandem mass spectrometry (LC–MS/MS) to systematically profile protein crotonylation in mouse PSCs in different states including ground, metastable, and primed states, as well as metastable PSCs undergoing early pluripotency exit. We successfully identified 3628 high-confidence crotonylated sites in 1426 proteins. These crotonylated proteins are enriched for factors involved in functions/processes related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Moreover, we found that increasing the cellular levels of crotonyl-coenzyme A (crotonyl-CoA) through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation, consistent with previous observations showing that enhanced proteasome activity helps to sustain pluripotency. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.
多能干细胞（pluripotent stem cells，PSCs）可在体外不同培养条件下维持不同的多能性状态。理解PSCs如何在不同多能性状态之间转换并最终向特定谱系分化，是发育生物学和再生医学领域的重要问题。尽管我们对多能性转变过程中的基因表达改变和调控已有相当多的了解，但有关蛋白质翻译后修饰层面的认知还很缺乏。作为一种新发现的蛋白质修饰，蛋白质赖氨酸可以被巴豆酰化修饰。我们利用特异性识别巴豆酰化肽段的抗体，对基态、亚稳态和始发态PSCs以及早期分化细胞中的巴豆酰化修饰蛋白质进行亲和纯化及液相色谱-质谱联用蛋白质组学定量分析。我们在1426个蛋白上鉴定得到3628个高可信度的巴豆酰化位点。这些巴豆酰化蛋白富集与多能性有关的RNA生物合成，中心碳代谢和蛋白酶体等功能。此外，与先前报道的增强蛋白酶体活性可以促进PSCs多能性相一致，我们发现通过添加巴豆酸提高细胞内巴豆酰辅酶A的水平，可以促进亚稳态PSCs的蛋白酶体活性并延缓分化。我们提供的PSCs蛋白质巴豆酰化修饰图谱有助于未来深入研究巴豆酰化修饰对多能性的调控作用，也为研究其他细胞命运转变中的代谢作用提供深入的见解。





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