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Technology Project 2
Primary PI - Gerald Hart
O-GlcNAc Tool Development: Crosstalk Between GlcNAcylation and Phosphorylation
Dr. Hart discovered this dynamic protein modification over twenty-five years ago. It is now clear that GlcNAcylation rivals protein phosphorylation in terms of abundance, dynamic cycling, and distribution on nuclear and cytoplasmic proteins (Figure). In fact, recent quantitative glycomic and phospho-proteomic studies have shown that the crosstalk or interplay between phosphorylation GlcNAcyation is extensive. So far, more than fifty kinases are known to be GlcNAcylated. Likewise the O-GlcNAc Transferase (OGT) is activated by Tyr phosphorylation via the insulin receptors, and by Ser (Thr) phosphorylation by other kinases. Thus, virtually dynamic GlcNAcylation influences all signaling pathways and transcription factors.
Understanding GlcNAcylation has been slow, due to the extraordinary difficulty in detecting and quantifying this PTM by standard methods. During the last NHLBI proteomics contract we systematically addressed these technical issues. These studies culminated in the development of a MS method to simultaneously quantify site occupancy for both O-GlcNAc and phosphate on hundreds of proteins.
Overall Research Plan: Our plan is to exploit the new technologies our group has developed to address key questions about HF and cardiac dysfunction associated with diabetes.
Goal 1: Determine how HF affects GlcNAcylation of mitochondrial proteins, and whether increased GlcNAcylation in the mitochondria contribute to mitochondrial dysfunction and ROS production.
Rationale: It is well established that increased stress causes a rapid and dramatic increase in GlcNAcylation of many proteins in the myocyte. Excitingly, recent data has shown that increased GlcNAcylation impairs mitochondrial functions.
Goal 2: Determine whether genetic and pharmacological approaches that specifically raise or lower global GlcNAcylation exacerbate or ameliorate cardiac dysfunction seen in HF.
Rationale: We and others have developed both genetic and pharmacological tools that specifically lower or raise global levels of GlcNAcylation without directly affecting other metabolites. Identification of OGT targeting proteins in the heart, will allow more specific targeting of GlcNAcylation on individual proteins.
Goal 3: Develop additional MRM based tools and site specific GlcNAc Abs to study key proteins.
Rationale: It is clear that MRM approaches can be used to track and quantify certain PTM. We will develop MRM assays for monitoring site specific GlcNAcylation in a higher throughput and quantitative manner.