The dynamic modification of nucleocytoplasmic proteins by the enzymatic attachment of O-linked N-acetylglucosamine to serine or threonine residues (O-GlcNAc) is now known to be a nutrient & stress sensor, regulating cellular signaling, transcription, proteasomal activity, and cytoskeleton. O-GlcNAc is as abundant as protein phosphorylation in all multi-cellular organisms, and has a dynamic interplay with phosphorylation. Hyperglycemia- and hyperinsulinemia-induced elevation of O-GlcNAc blocks insulin signaling and contributes to glucose toxicity. O-GlcNAcylation of proteins is sensitive to both oxidative stress and to glucose concentrations. O-GlcNAc cycles at different rates on different proteins. These properties will be exploited to develop a simple immunoassay to quantify site-specific O-GlcNAcylation in order to allow easy assessment of the extent and duration of glucose dysregulation in patients. Plan: In the R21 phase, two complementary proteomic approaches (2D DIGE/MS-MS & BEMAD/MS-MS) will be used to identify and quantify O-GlcNAc sites in samples covering a range levels of glucose dysregulation.
Aim 1 of this phase uses the STZ-rat model to relate the amplitude and time course of hyperglycmia to changes in O-GlcNAc at specific sites on blood proteins.
In Aim 2, we apply these proteomic methods to well characterized, masked, human samples from NIDDK and our own 'normal', pre-diabetic and diabetic subjects. The major milestone of the R21 is the identification of O-GlcNAc sites on blood proteins that display a consistent range of responsiveness to the diabetic state. In the R33 phase Aim 3, site specific monoclonal antibodies will be prepared to both the unmodified and the modified sites identified in Aim 2.
In Aim 4, these antibodies will be used to develop simple and rapid quantitative immunoassays. These studies will exploit a ubiquitous, highly-dynamic protein modification that is central to the pathophysiologic abnormalities of diabetes, to develop an assessment tool that will have significant advantages over existing methods. The project also represents a new inter-disciplinary, inter-departmental basic science-clinical research collaboration between Biological Chemistry and The Department of Medicine.
