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.
The process of amyloid fibril formation remains one of the primary targets for developing diagnostics and treatments for several neurodegenerative diseases (NDDs). Amyloid-forming proteins such α-Synuclein and Tau, which are implicated in the pathogenesis of Alzheimer's and Parkinson's disease, can form different types of fibril structure, or strains, that exhibit distinct structures, toxic properties, seeding activities, and pathology spreading patterns in the brain. Therefore, understanding the molecular and structural determinants contributing to the formation of different amyloid strains or their distinct features could open new avenues for developing disease-specific diagnostics and therapies. In this work, we report that O-GlcNAc modification of α-Synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by Cryo-EM, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-Synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that post-translational modifications, such as O-GlcNAc modification, of α-Synuclein are key determinants of α-Synuclein amyloid strains and pathogenicity. These findings have significant implications for how we investigate and target amyloids in the brain and could possibly explain the lack of correlation between amyloid burden and neurodegeneration or cognitive decline in some subtypes of NDDs.
dynamical properties of polymer in the lysate environment, we measure the translational diffusion of the test polymer polyethylene glycol (PEG) using diffusion NMR. While PEG is not a biomolecule, it is a useful analog for a disordered protein. Our rheological tests show that while Ficoll is nearly Newtonian even at high concentrations, bacterial cell lysate is markedly viscoelastic. Results will provide a better understanding of the effects of complex biological crowders on polymer diffusion.
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