Department of Chemistry and §Department of Molecular and Computational Biology, University of Southern California , Los Angeles, California 90089-0744, United States.
J Am Chem Soc. 2017 Jun 14;139(23):7872-7885. doi: 10.1021/jacs.7b02213. Epub 2017 May 31.
O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.
O-连接的 N-乙酰葡萄糖胺修饰（O-GlcNAcylation）是哺乳动物细胞存活所必需的。遗传和生化实验发现，修饰增加会抑制组织和细胞培养中的细胞凋亡，而降低 O-GlcNAcylation 会诱导细胞死亡。然而，O-GlcNAcylation 抑制细胞凋亡的分子机制仍在阐明之中。在这里，我们首先合成了一种新的代谢化学报告物，6-炔基-6-去氧-GlcNAc（6AlkGlcNAc），用于鉴定 O-GlcNAc 修饰的蛋白质。随后对 6AlkGlcNAc 的表征表明，该探针可选择性地掺入 O-GlcNAc 修饰的蛋白质中，而不是细胞表面糖蛋白。利用该探针，我们发现凋亡半胱天冬酶被 O-GlcNAc 修饰，我们使用其他技术对此进行了验证，这提出了修饰可能影响其生物化学的可能性。然后，我们证明了全局 O-GlcNAcylation 水平的变化导致凋亡过程中 caspase-8 激活的动力学发生相反的变化。最后，我们表明 caspase-8 在可以阻止其切割/激活的残基处被修饰。我们的结果首次提供了证据，表明 caspase 可能直接受到 O-GlcNAcylation 的影响，这可能是一种抗细胞凋亡的机制。
