Pratt, M. R. | Search bioRxiv

bioRxiv2019

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

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-GlcNAcylation 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-GlcNAcylation increases the ability of sHSPs to block the amyloid formation of both -synuclein and A{beta}. Mechanistically, we show that O-GlcNAc near the sHSP IXI-domain prevents its ability to intramolecularly compete with substrate binding. Our results have important implications for neurodegenerative diseases associated with amyloid formation and potentially other areas of sHSP biology.

biochemistry↗

bioRxiv2019

MYPT1 O-GlcNAcylation controls the sensitivity of fibroblasts to sphingosine-1-phosphate mediated cellular contraction

Many intracellular proteins can be modified by N-acetylglucosamine, a posttranslational modification known as O-GlcNAc. Because this modification is found on serine and threonine side-chains, O-GlcNAc has the potential to dynamically regulate cellular signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. First, we find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P). In follow-up mechanistic investigations, we show that this O-GlcNAc dependence lies in the signaling pathway through the S1PR2 receptor and subsequent activation of the Rho and Rho kinase. This pathway typically culminates in the phosphorylation of myosin light chain (MLC), resulting in myosin activation and cellular contraction. We discovered that O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Therefore, MYTP1 O-GlcNAc levels function to regulate the sensitivity of cells to S1P-mediated cellular contraction. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing, where increased levels of the modification may inhibit S1P-mediated healing phenotypes in fibroblasts.

cell biology↗