Figure 2. O-GlcNAcylated HSP27 is a better chaperone against α-synuclein amyloid aggregation.
a) Unmodified and differentially O-GlcNAcylated versions of HSP27 were retrosynthetically deconstructed into a recombinant protein thioester and peptides prepared by solid phase peptide synthesis. b) α-Synuclein alone (50 μM) or in the presence of HSP27 or the indicated O-GlcNAcylated HSP27 proteins (1 μM) was subjected to aggregation conditions (agitation at 37 °C). After different lengths of time, aliquots were removed and analyzed by ThT fluorescence (λex = 450 nm, λem = 482 nm). The y-axis shows fold change in fluorescence compared with α-synuclein alone at t = 0 h. Results are mean ±SEM of n=3 independent experiments. Statistical significance was determined using a one-way ANOVA test followed by Dunnet’s test (α-synuclein alone or plus HSP27 versus O-GlcNAcylated proteins). c) The same reactions were analyzed by TEM after 168 h. d) The same reactions were subjected to the indicated concentrations of proteinase-K (PK) for 30 min before separation by SDS-PAGE and visualization by Coomassie staining. The persistence of bands correlates with the amount of amyloid formation.