Xu Senhan, Yin Kejun, Xu Xing, Fu Longping, Wu Ronghu
School of Chemistry and Biochemistry and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA.
The Scripps Research Institute, La Jolla, CA, USA.
Nat Commun. 2025 Apr 30;16(1):4068. doi: 10.1038/s41467-025-59371-4.
O-GlcNAcylation plays critical roles in the regulation of protein functions and cellular activities, including protein interactions with other macromolecules. While the formation of biomolecular condensates (or biocondensates) regulated by O-GlcNAcylation in a few individual proteins has been reported, systematic investigation of O-GlcNAcylation on the regulation of biocondensate formation remains to be explored. Here we systematically study the roles of O-GlcNAcylation in regulating protein solubility and its impacts on RNA-protein condensates using mass spectrometry-based chemoproteomics. Unexpectedly, we observe a system-wide decrease in the solubility of proteins modified by O-GlcNAcylation, with glycoproteins involved in focal adhesion and actin binding exhibiting the most significant decrease. Furthermore, O-GlcNAcylation sites located in disordered regions and with fewer acidic and aromatic residues nearby are related to a greater drop in protein solubility. Additionally, we discover that a specific group of O-GlcNAcylation events promotes the dissociation of RNA-protein condensates under heat stress, while some enhance the formation of RNA-protein condensates during the recovery phase. Using site mutagenesis, inhibition of O-GlcNAc transferase, and fluorescence microscopy, we validate that O-GlcNAcylation regulates the formation of biocondensates for YTHDF3 and NUFIP2. This work advances our understanding of the functions of protein O-GlcNAcylation and its roles in the formation of biomolecular condensates.
O-连接的N-乙酰葡糖胺化（O-GlcNAcylation）在蛋白质功能和细胞活动的调节中起着关键作用，包括蛋白质与其他大分子的相互作用。虽然已有报道在少数个别蛋白质中，O-GlcNAcylation调节生物分子凝聚物（或生物凝聚物）的形成，但对O-GlcNAcylation在生物凝聚物形成调节方面的系统研究仍有待探索。在此，我们使用基于质谱的化学蛋白质组学系统地研究了O-GlcNAcylation在调节蛋白质溶解度及其对RNA-蛋白质凝聚物影响中的作用。出乎意料的是，我们观察到经O-GlcNAcylation修饰的蛋白质在全系统范围内溶解度下降，其中参与粘着斑和肌动蛋白结合的糖蛋白溶解度下降最为显著。此外，位于无序区域且附近酸性和芳香族残基较少的O-GlcNAcylation位点与蛋白质溶解度的更大下降有关。此外，我们发现特定组的O-GlcNAcylation事件在热应激下促进RNA-蛋白质凝聚物的解离，而在恢复阶段一些事件则增强RNA-蛋白质凝聚物的形成。通过位点诱变、抑制O-GlcNAc转移酶和荧光显微镜，我们验证了O-GlcNAcylation调节YTHDF3和NUFIP2生物凝聚物的形成。这项工作增进了我们对蛋白质O-GlcNAcylation功能及其在生物分子凝聚物形成中作用的理解。
