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

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 loweribg 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-6deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modiffed proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylatedProteins-: Over, cell-surface,glycoproteins. Using this prObe, we discover; that the apoptotic "caspases are O-GleN,Acylated which we confirmed using other techniques, raising the possibility that the modification L'affect$ their biochemistry: We then,demon-strate 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-,GlcNACylatiori:s a potential aritiapOptotic mechanism.