Functional Analysis of O-GlcNAc Modifications using Synthetic Protein Chemistry O-GlcNAc modification (O-GlcNAcylation) is a dynamic protein-modification that is absolutely required for embryonic development in mammals, and is misregulated in diseases, including diabetes, neurodegeneration and cancer. Although approximately 1000 potential proteins are modified by O-GlcNAc, the effects of the vast majority of these modifications on protein function are completely unknown. This critical lack of knowledge exists in-part because traditional methods are deficient for the study of site-specific O-GlcNAcylation events. The long-term goal of our research program is to understand the consequences of O-GlcNAcylation on proteins that are key to human disease. The objectives of this application are to develop protein engineering strategies that uniquely enable the generation of proteins with site-specific O-GlcNAc modifications and to apply these methods to understand the effects of O-GlcNAcylation on the protein a-synuclein, the aggregation-prone protein in Parkinson's disease. Our preliminary studies demonstrate that homogeneously O-GlcNAcylated proteins can be prepared using synthetic chemistry. Furthermore, we have used synthetic protein chemistry to demonstrate that O-GlcNAcylation blocks a-synuclein aggregation. Guided by these preliminary studies, we will: 1) continue to develop general synthetic-strategies for the preparation of O-GlcNAcylated proteins, 2) investigate the molecular mechanism by which O-GlcNAcylation blocks a-synuclein aggregation and 3) determine the effects of O-GlcNAcylation on the cellular toxicity of a-synuclein. These studies are significant, as the effects of O-GlcNAcylation are almost completely unknown. Additionally, blocking a-synuclein aggregation is a key potential therapeutic strategy in Parkinson's disease. Our approach is also innovative as it enables the effects of O-GlcNAcylation to be directly tested in a site-specific fashion and can be applied to other critical proteins in the future.

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'Functional Analysis of O-GlcNAc Modifications using Synthetic Protein Chemistry' This proposal is aimed at developing and applying strategies to understand the site-specific effects of O-GlcNAc modification on protein function. These studies will enable the study of the consequences of O-GlcNAc modification on proteins that are critical in human diseases including neurodegeneration, cancer and diabetes. This is a key prerequisite to targeting O-GlcNAc as a therapeutic strategy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Bond, Michelle Rueffer
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University of Southern California
Schools of Arts and Sciences
Los Angeles
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Darabedian, Narek; Thompson, John W; Chuh, Kelly N et al. (2018) Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting. Biochemistry 57:5769-5774
De Leon, Cesar A; Lang, Geoffrey; Saavedra, Marcos I et al. (2018) Simple and Efficient Preparation of O- and S-GlcNAcylated Amino Acids through InBr3-Catalyzed Synthesis of ?- N-Acetylglycosides from Commercially Available Reagents. Org Lett 20:5032-5035
Levine, Paul M; De Leon, Cesar A; Galesic, Ana et al. (2017) O-GlcNAc modification inhibits the calpain-mediated cleavage of ?-synuclein. Bioorg Med Chem 25:4977-4982
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De Leon, Cesar A; Levine, Paul M; Craven, Timothy W et al. (2017) The Sulfur-Linked Analogue of O-GlcNAc (S-GlcNAc) Is an Enzymatically Stable and Reasonable Structural Surrogate for O-GlcNAc at the Peptide and Protein Levels. Biochemistry 56:3507-3517
Chuh, Kelly N; Batt, Anna R; Pratt, Matthew R (2016) Chemical Methods for Encoding and Decoding of Posttranslational Modifications. Cell Chem Biol 23:86-107