The New Chemical Reporter 6-Alkynyl-6-deoxy-GlcNAc Reveals O-GlcNAc Modification of the Apoptotic Caspases That Can Block the Cleavage/Activation of Caspase-8

Abstract

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-GlcNAc elevation through activation of the hexosamine biosynthetic pathway enhances cancer cell chemoresistance

“…We next investigated the downstream targets of O-GlcNAcylation. A previous study found that the cleavage of apoptotic caspases could be blocked by O-GlcNAcylation 23 . In our study, the major intrinsic apoptotic caspases, namely caspase-3 and 9, were also O-GlcNAcylated in the parental and resistant cells.…”

O-GlcNAc transferase associates with the MCM2–7 complex and its silencing destabilizes MCM–MCM interactions

“…1 a, b). This result is strengthened by two recent works which have identified MCM2, MCM3, MCM4 and MCM5 as O -GlcNAc-modified proteins using metabolic incorporation of chemical GlcNAc analogue probes combined with click chemistry labelling [ 76 , 77 ]. Using a mass-tagging strategy, we show that the O -GlcNAcylated MCM proteins are of low stoichiometry (from less than 3% for MCM2 and MCM4, to 13% for MCM6) (Fig.…”

Metabolic Labeling for the Visualization and Identification of Potentially O‐GlcNAc‐Modified Proteins

“…For example, we have demonstrated that subtle changes in the MCR structure can make them preferential substrates for OGT. This approach has led to the development of multiple more‐selective O‐GlcNAc MCRs including Ac 4 GlcNAlk, Ac 3 6AzGlcNAc, Ac 3 6AlkGlcNAc, and Ac 4 2AzGlc (Chuh et al., 2017; 2014; Zaro et al., 2017; Zaro et al., 2011). Second, treatment of cells with MCRs may cause increased flux into the corresponding donor sugar, resulting in higher than typical amounts of protein modification.…”

“…Several different MCRs have been developed for the investigation of O‐GlcNAcylated proteins (Fig. 1) (Boyce et al., 2011; Chuh et al., 2017; Chuh, Zaro, Piller, Piller, & Pratt, 2014; Hang, Yu, Kato, & Bertozzi, 2003; Hao et al., 2019; Li et al., 2016; Vocadlo, Hang, Kim, Hanover, & Bertozzi, 2003; Zaro, Batt, Chuh, Navarro, & Pratt, 2017; Zaro, Yang, Hang, & Pratt, 2011). Basic Protocol 1 describes a basic procedure for incorporation of metabolic chemical reporters on to live cells, cell harvesting techniques, and CuAAC click chemistry.…”