Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating-protein associated with synucleinopathies, including Parkinson’s disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically-relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory-effect on α-synuclein aggregation, whilst not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

The process of neurodegeneration in Parkinson’s Disease is intimately associated with the aggregation of the protein α-synuclein into toxic oligomers and fibrils. Interestingly, many of these protein aggregates are found to be posttranslationally modified by ubiquitin at several different lysine residues. However, the inability to generate homogeneously ubiquitin modified α-synuclein at each site has prevented the understanding of the specific biochemical consequences. We have used protein semi-synthesis to generate nine site-specifically ubiquitin modified α-synuclein derivatives and have demonstrated that different ubiquitination sites have differential effects on α-synuclein aggregation.

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.

Sweet relief: the Parkinson's disease- associated protein α-synuclein is post-translationally modified by N-acetylglucosamine (O-GlcNAc), but the biochemical consequences are unknown. Here we show that an O-GlcNAc-modified peptide does not participate in α-synuclein aggregation, thus suggesting that O-GlcNAc might directly inhibit aggregation in cells.

No abstract