Ong Qunxiang, Lim Ler Ting Rachel, Goh Cameron, Liao Yilie, Chan Sher En, Lim Crystal Jing Yi, Kam Valerie, Yap Jerome, Tseng Tiffany, Desrouleaux Reina, Wang Loo Chien, Ler Siok Ghee, Lim Siew Lan, Kim Sun-Yee, Sobota Radoslaw M, Bennett Anton M, Han Weiping, Yang Xiaoyong
Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Department of Comparative Medicine, Yale University School of Medicine, New Haven, CT, USA.
Nat Chem Biol. 2025 Feb;21(2):300-308. doi: 10.1038/s41589-024-01770-7. Epub 2024 Nov 14.
The post-translational modification of intracellular proteins through O-linked β-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O-GlcNAcylation in cell signaling and physiology.
通过O-连接的β-N-乙酰葡糖胺（O-GlcNAc）对细胞内蛋白质进行的翻译后修饰是多细胞生物中一种保守的调节机制。这种动态修饰由O-GlcNAc转移酶（OGT）催化，在信号转导、基因表达、细胞器功能和全身生理中起着至关重要的作用。在这里，我们展示了光控OGT（Opto-OGT），一种光遗传学探针，可通过光刺激对OGT活性进行精确的时空控制。通过将光敏隐花色素蛋白与OGT融合，Opto-OGT可以被蓝光以高时间分辨率强烈且可逆地激活，并且在无光照时表现出最小的背景活性。Opto-OGT的瞬时激活导致mTORC激活和AMPK抑制，这重现了营养感应信号传导。此外，Opto-OGT可以定制以定位于特定的亚细胞位点。通过将OGT靶向质膜，我们证明了响应胰岛素刺激时特定位点的AKT磷酸化和信号输出的下调。因此，Opto-OGT是确定O-GlcNAc糖基化在细胞信号传导和生理学中的作用的有力工具。
