O-GlcNAc modification of small heat shock proteins enhances their anti-amyloid chaperone activity

Abstract

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.

Advances in chemical probing of protein O-GlcNAc glycosylation: structural role and molecular mechanisms

“…, HSP27, αA-crystallin and αB-crystallin). 188 Combining protein semisynthesis and biochemical experiments, Pratt and coworkers investigated the functional effects of site-specific α-synuclein O -GlcNAcylation, showing that the modification reduced protein aggregation and toxicity in vitro . 185–187 Most recently, following a similar multidisciplinary approach the same group probed the impact of O -GlcNAc on the semisynthetic sHSPs mentioned above and found that glycosylation improves the chaperone activity of these proteins against amyloid formation.…”

“… 185–187 Most recently, following a similar multidisciplinary approach the same group probed the impact of O -GlcNAc on the semisynthetic sHSPs mentioned above and found that glycosylation improves the chaperone activity of these proteins against amyloid formation. 188 Furthermore, the non-hydrolysable S -GlcNAcylated casein kinase II (CKII) (at native Ser347) 189 and α-synuclein (at native Ser87) 190 have also been prepared by chemical ligation, highlighting S -GlcNAc as an enzymatically stable, suitable structural mimic of O -GlcNAc, as shown by biological studies and two-dimensional nuclear magnetic resonance (NMR) combined with computational modelling. 190 …”

“…A recent MD simulation study of O -GlcNAcylated synuclein proposed that O -GlcNAc modification suppresses oligomer formation by preventing intermolecular hydrogen-bonding interactions between monomers via steric effects, providing some insights into the molecular mechanism of O -GlcNAc-induced inhibition of α-synuclein oligomerisation. 216 In the case of the semi-synthetic, differentially O -GlcNAcylated sHSPs prepared by Pratt and coworkers, 188 the enhanced chaperone activity and aggregation-protective effect caused by the glycan were found to stem from its ability to disrupt the auto-regulatory interaction of the proteins’ IXI motif with their chaperone cleft. Using biophysical and computational modelling techniques, they showed that O -GlcNAcylation decreased the binding of the IXI sequence of HSP27 to its chaperone groove, leading to the formation of larger HSP27 oligomers and a conformational rearrangement in a more active state.…”

“… 216 Moreover, the Pratt group recently showed how sHSP O -GlcNAc glycosylation at the IXI motif perturbs its interaction with the chaperone cleft, a glycan-promoted disruption that prevents the ability of this motif to intramolecularly compete with substrate binding, improving sHSP chaperone activity. 188 …”

O-GlcNAc modification of MYPT1 modulates lysophosphatidic acid–induced cell contraction in fibroblasts

“…Of note, overall O -GlcNAc levels are altered in several human diseases. For example, O -GlcNAc levels are lower in patients with Alzheimer’s disease compared with healthy age-matched individuals ( 12 , 13 , 14 , 15 ). In contrast, the modification levels are elevated in every type of cancer examined compared with healthy tissue ( 16 , 17 , 18 ).…”