糖基化在人类胆管癌 (CCA) 的复杂景观中发挥着关键作用，积极参与驱动肿瘤进展的关键病理生理过程。在各种糖基化修饰中，O-连接的β-N-乙酰基-葡萄糖胺修饰（O-GlcNAcylation）作为一种动态调节剂而出现，影响多种肿瘤相关的生物活性。在这项研究中，我们采用最先进的化学蛋白质组学方法来分析完整的糖肽，揭示了 O-GlcNAcylation 在协调角蛋白 18 (K18) 及其与三羧酸 (TCA) 循环酶的相互作用中的关键作用，特别是异柠檬酸脱氢酶 (IDH)，促进 CCA 进展。我们的研究结果揭示了 O-GlcNAcylation 的复杂机制，揭示了 Ser 30 上 K18 的位点特异性修饰可作为稳定因子，放大细胞周期检查点的表达。这种分子事件错综复杂地促进细胞周期进展并增强 CCA 中的细胞生长。值得注意的是，O-GlcNAcylated K18 和 IDH 之间的相互作用通过下调柠檬酸盐和异柠檬酸盐水平同时升高 α-酮戊二酸 (α-KG) 来协调代谢重编程。这些代谢变化进一步增加了 CCA 的整体致瘤潜力。因此，我们的研究扩展了目前对蛋白质 O-GlcNAcylation 的理解，并为代谢和肿瘤发生的翻译后控制引入了新的复杂性。© 2024 作者。由美国化学会出版。

Glycosylation plays a pivotal role in the intricate landscape of human cholangiocarcinoma (CCA), actively participating in key pathophysiological processes driving tumor progression. Among the various glycosylation modifications, O-linked β-N-acetyl-glucosamine modification (O-GlcNAcylation) emerges as a dynamic regulator influencing diverse tumor-associated biological activities. In this study, we employed a state-of-the-art chemical proteomic approach to analyze intact glycopeptides, unveiling the critical role of O-GlcNAcylation in orchestrating Keratin 18 (K18) and its interplay with tricarboxylic acid (TCA) cycle enzymes, specifically isocitrate dehydrogenases (IDHs), to propel CCA progression. Our findings shed light on the mechanistic intricacies of O-GlcNAcylation, revealing that site-specific modification of K18 on Ser 30 serves as a stabilizing factor, amplifying the expression of cell cycle checkpoints. This molecular event intricately fosters cell cycle progression and augments cellular growth in CCA. Notably, the interaction between O-GlcNAcylated K18 and IDHs orchestrates metabolic reprogramming by down-regulating citrate and isocitrate levels while elevating α-ketoglutarate (α-KG). These metabolic shifts further contribute to the overall tumorigenic potential of CCA. Our study thus expands the current understanding of protein O-GlcNAcylation and introduces a new layer of complexity to post-translational control over metabolism and tumorigenesis.© 2024 The Authors. Published by American Chemical Society.