Crotonylation of GAPDH regulates human embryonic stem cell endodermal lineage differentiation and metabolic switch

Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs). Here we report the investigation of induced crotonylation in hESCs, which resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. We showed that increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Through large-scale profiling of non-histone protein crotonylation, we showed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs, where crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis. Our study demonstrates that crotonylation of glycolytic enzymes may be crucial to metabolic switching and cell fate determination in hESCs. Compare transcriptome profiling (RNA-seq) of crotonate treated primed human embryonic stem cell H9 and EPS cells with untreated primed H9 cells, EPS cells and endodermal cells.

蛋白质的翻译后修饰对其活性与功能的调控至关重要。作为新发现的酰化修饰类型，非组蛋白巴豆酰化（crotonylation）的研究仍较为有限，尤其在人类胚胎干细胞（human embryonic stem cells, hESCs）中。本研究针对人类胚胎干细胞中的诱导型巴豆酰化展开探究，发现其可使不同多能性状态的人类胚胎干细胞向内胚层谱系分化。研究证实，人类胚胎干细胞中蛋白质巴豆酰化水平升高会伴随转录组改变与糖酵解活性降低。通过大规模非组蛋白巴豆酰化修饰谱分析，我们发现代谢酶是人类胚胎干细胞中诱导型巴豆酰化的主要靶标。本研究进一步发现，甘油醛-3-磷酸脱氢酶（GAPDH）作为关键糖酵解酶，在人类胚胎干细胞向内胚层分化的过程中受巴豆酰化调控：GAPDH的巴豆酰化会降低其酶活性，进而导致糖酵解水平下降。本研究证明，糖酵解相关酶的巴豆酰化或许对人类胚胎干细胞的代谢转换与细胞命运决定具有关键作用。本研究将巴豆酸盐处理的始发态人类胚胎干细胞H9、EPS细胞，与未处理的始发态H9细胞、EPS细胞及内胚层细胞开展转录组测序（RNA-seq）对比分析。