The nutrient sensor O-linked N-acetylglucosamine (O-GlcNAc) is installed as a post-translational modification on nucleocytoplasmic proteins. O-GlcNAc’s addition and removal are respectively orchestrated by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), which together maintain homeostasis. Tools that offer spatial and temporal control of O-GlcNAcylation can help provide insight into how O-GlcNAc level dynamics influence biological functions. Ge et al. developed an approach for controlling OGA activation in response to 4-hydroxytamoxifen (4-HT) exposure using a self-splicing intein.
Intein-bearing OGA variants were screened in HEK293T human embryonic kidney cells for decreased O-GlcNAc levels on a substrate protein after exposure to 4-HT, with C181 being the best intein insertion point. Interestingly, 4-HT-mediated OGA–intein activation resulted in the rapid upregulation of both protein and mRNA levels of OGT and downregulation of OGA mRNA levels. Localization of OGA–intein to the cytoplasm or nucleus induced substrate protein deglycosylation in these respective subcellular locations after exposure to 4-HT. 4-HT is the active metabolite of tamoxifen, a breast cancer drug approved by the US Food and Drug Administration, whose activity against MCF7 human breast cancer cells is downregulated when O-GlcNAcylation levels are elevated. The researchers evaluated whether their 4-HT-dependent OGA–intein system could decrease O-GlcNAcylation levels to sensitize MCF7 cells to 4-HT treatment and found that low concentrations of 4-HT suppressed the viability of MCF7 cells stably expressing an active OGA–intein. Given its demonstrated utility, this 4-HT-activatable OGA offers the opportunity to remove O-GlcNAcylation with improved spatial and temporal resolution.
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