Fellowship/Crawford: Beta-N-Acetylglucosamine Modified Proteins Regulation Platelet Function

The post-translational modification of proteins by the addition of a single beta-N-acetylglucosamine (GlcNAc) has been proposed to serve a protective effect masking residues from phosphorylation, playing a role in cell signaling. More recently this modification has been proposed to serve as a nutrient sensor where by increased protein modifications occur in response to high levels of extracellular glucose resulting in insulin resistance and development of type" diabetes. Initial data have shown that some platelet proteins are modified by O-GlcNAc and levels of this modification change upon platelet activation. Changes in O-GlcNAc modification upon platelet activation indicate a role for this modification in signaling cascades that deserves further investigation. Understanding the role this modification plays in platelet function will lead to a more complete characterization of platelet signaling. Our hypothesis is that dynamic O-GlcNAc regulation is critical for platelet activation and incorrect regulation of this modification results in altered platelet functions. We will address our hypothesis through two specific aims: 1) Elucidate the functional significance of O-GlcNAc modification during platelet activation and 2) Biochemical analysis of O-GlcNAc modifications in human platelets. To address these, we will disrupt the cycling of O-GlcNAc using specific inhibitors and antibodies in a permeablized assay to elucidate its role in platelet aggregation and secretion. We will use newly developed mass spectroscopic techniques along with affinity chromatography to identify modified proteins in platelets and map these modifications to individual residues. Lastly, we intend to correlate phosphorylation and O-GlcNAcylation to platelet signaling cascades using in-gel dyes, western blotting and mass spectroscopy.