A major role for the intracellular posttranslational modification O-GlcNAc appears to be the inhibition of protein aggregation. Most of the previous studies in this area have focused on O-GlcNAc modification of the amyloid-forming proteins themselves. Here, we use synthetic protein chemistry to discover that O-GlcNAc also activates the anti-amyloid activity of certain small heat shock proteins (sHSPs), a potentially more important modification event that can act broadly and substoichiometrically. More specifically, we find that O-GlcNAc increases the ability of sHSPs to block the amyloid formation of both α-synuclein and Aβ(1-42). Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Finally, we find that although O-GlcNAc levels are globally reduced in Alzheimer’s disease brains, the modification of relevant sHSPs is either maintained or increased, suggesting a mechanism to maintain these potentially protective O-GlcNAc modifications. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

O-GlcNAc modification of the microtubule associated protein tau and α-synuclein can directly inhibit the formation of the associated amyloid fibers associated with major classes of neurodegenerative diseases. However, the mechanism(s) by which this posttranslational modification (PTM) inhibit amyloid aggregation are still murky. One hypothesis is that O-GlcNAc simply acts as a polyhydroxylated steric impediment to the formation of amyloid oligomers and fibers. Here, we begin to test this hypothesis by comparing the effects of O-GlcNAc to other similar monosaccharidesglucose, N-acetyl-galactosamine (GalNAc), or mannoseon α-synuclein amyloid formation. Interestingly, we find that this quite reasonable hypothesis is not entirely correct. More specifically, we used four types of biochemical and biophysical assays to discover that the different sugars display different effects on the inhibition of amyloid formation, despite only small differences between the structures of the monosaccharides. These results further support a more detailed investigation into the mechanism of amyloid inhibition by O-GlcNAc and has potential implications for the evolution of N-acetyl-glucosamine as the monosaccharide of choice for widespread intracellular glycosylation.

Toxic amyloid aggregates are a feature of many neurodegenerative diseases. A number of biochemical and structural studies have demonstrated that not all amyloids of a given protein are equivalent but rather that an aggregating protein can form different amyloid structures or polymorphisms. Different polymorphisms can also induce different amounts of pathology and toxicity in cells and in mice, suggesting that the structural differences may play important roles in disease. However, the features that cause the formation of polymorphisms in vivo are still being uncovered. Posttranslational modifications on several amyloid forming proteins, including the Parkinson’s disease causing protein α-synuclein, may be one such cause. Here, we explore whether ubiquitination can induce structural changes in α-synuclein aggregates in vitro. We used protein chemistry to first synthesize ubiquitinated analogues at three different positions using disulfide linkages. After aggregation, these linkages can be reversed, allowing us to make relative comparisons between the structures using a proteinase K assay. We find that, while ubiquitination at residue 6, 23, or 96 inhibits α-synuclein aggregation, only modification at residue 96 causes an alteration in the aggregate structure, providing further evidence that posttranslational modifications may be an important feature in amyloid polymorphism formation.

O-GlcNAcylation is a dynamic post-translational modification which affects myriad proteins, cellular functions, and disease states. Its presence or absence modulates protein function via differential protein- and site-specific mechanisms, necessitating innovative techniques to probe the modification in highly selective manners. To this end, a variety of biological and chemical methods have been developed to study specific O-GlcNAc modification events both in vitro and in vivo , each with their own respective strengths and shortcomings. Together, they comprise a potent chemical biology toolbox for the analysis of O-GlcNAcylation (and, in theory, other post-translational modifications) while highlighting the need and space for more facile, generalizable, and biologically authentic techniques.

Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein’s endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein–DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.