Small Molecule-Activated O-GlcNAcase for Spatiotemporal Removal of O-GlcNAc in Live Cells.
Small Molecule-Activated O-GlcNAcase for Spatiotemporal Removal of O-GlcNAc in Live Cells.
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DOI：
10.1021/acschembio.2c00894
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发表时间：
2023-01
影响因子：
4
通讯作者：
Yun Ge;Hailin Lu;Bo Yang;Christina M. Woo
中科院分区：
文献类型：
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作者：
Yun Ge;Hailin Lu;Bo Yang;Christina M. Woo
营养传感器 O-连接 N-乙酰氨基葡萄糖 (O-GlcNAc) 是在数千种核细胞质蛋白上发现的翻译后修饰。细胞中的 O-GlcNAc 水平以时间和空间方式动态响应环境信号，导致信号转导和功能效应改变。 O-GlcNAc 水平的时空调节将加速 O-GlcNAc 的功能询问和细胞行为的操纵以获得期望的结果。在这里，我们报告了一种通过设计与 4-羟基他莫昔芬 (4-HT) 触发的内含肽融合的 O-GlcNAcase (OGA) 来时空减少活细胞中 O-GlcNAc 的策略。经过合理的蛋白质工程和优化后，我们鉴定了一种 OGA-intein 变体，其去糖苷酶活性可以通过 4-HT 以时间和剂量依赖性方式在所需的亚细胞区室中触发。最后，我们证明 OGA-内含肽融合物的 4-HT 激活同样可以通过减少 O-GlcNAc 来增强对乳腺癌细胞的抑制作用。通过化学激活的 OGA-内含肽融合对 O-GlcNAc 进行时空控制将有助于活细胞中 O-GlcNAc 的操作和功能理解。
The nutrient sensor O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification found on thousands of nucleocytoplasmic proteins. O-GlcNAc levels in cells dynamically respond to environmental cues in a temporal and spatial manner, leading to altered signal transduction and functional effects. The spatiotemporal regulation of O-GlcNAc levels would accelerate functional interrogation of O-GlcNAc and manipulation of cell behaviors for desired outcomes. Here, we report a strategy for spatiotemporal reduction of O-GlcNAc in live cells by designing an O-GlcNAcase (OGA) fused to an intein triggered by 4-hydroxytamoxifen (4-HT). After rational protein engineering and optimization, we identified an OGA-intein variant whose deglycosidase activity can be triggered in the desired subcellular compartments by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated that 4-HT activation of the OGA-intein fusion can likewise potentiate inhibitory effects in breast cancer cells by virtue of the reduction of O-GlcNAc. The spatiotemporal control of O-GlcNAc through the chemically activatable OGA-intein fusion will facilitate the manipulation and functional understanding of O-GlcNAc in live cells.