Principal Investigator
Natasha Elizabeth
Zachara
Awardee Organization
Johns Hopkins University
United States
Fiscal Year
2020
Activity Code
U01
Project End Date
NIH RePORTER
For more information, see NIH RePORTER Project 5U01CA230978-03
Tuning and Tagging the O-GlcNAc Modification
Thousands of intracellular proteins are dynamically modified by monosaccharides of O-linked Nacetylglucosamine (O-GlcNAc). The cycling of O-GlcNAc is regulated by the concerted actions of enzymes encoded by just two genes: the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) that add and remove O-GlcNAc, respectively (1-4). Dysregulation of O-GlcNAc cycling, or levels of the nucleotide sugar used by OGT (UDP-GlcNAc), exacerbates the pathophysiology of a host of diseases including type II diabetes, cancer, neurodegeneration, heart failure, hypertension and aging (5-7). While technological and methodological innovations have improved our ability to detect, modulate, and site-map O-GlcNAc (8-13), many of these techniques have not been adopted by the broader scientific community thus inhibiting a mechanistic understanding of the roles that O-GlcNAc plays in potentiating disease. Often, the aforementioned approaches require specialized equipment and reagents, lack the specificity required to study a modification that is cycled by just two enzymes, or lead to significant off-target effects (10, 14-20). Thus, the goal of the studies proposed herein is to generate facile, inexpensive, chemical genetic tools that probe the roles of O-GlcNAc in vitro and in vivo. Specifically, we will address the following aims: Specific Aim #1: Enabling tunable, reversible chemical-genetic regulation of O-GlcNAcylation in an organelle-specific manner. These tools will combine destabilization domain (21-23) and nanotrap (24) technology to enable researchers to modulate the expression of OGT and OGA, as well as key enzymes within the hexosamine biosynthetic pathway, in a spatial, temporal, and dose-dependent manner. These tools will be applied to understanding the role of O-GlcNAc in prostate cancer. Specific Aim 2: Enabling tissue- and organelle-specific tagging of O-GlcNAc-modified proteins. This tool will harness the power of proximity Biotin ligation (25, 26) to enable tagging and enrichment of OGlcNAcylated proteins in a cell-specific or organelle-specific manner. Together, the tools described in this proposal will overcome current experimental limitations associated with studying O-GlcNAc and facilitate studies focused on determining the role of O-GlcNAc at a mechanistic level in a broad range of models.
Publications
- Fahie KMM, Papanicolaou KN, Zachara NE. Integration of O-GlcNAc into Stress Response Pathways. Cells. 2022 Nov 5;11. (21). PMID: 36359905
- Fahie K, Narayanan B, Zahra F, Reeves R, Fernandes SM, Hart GW, Zachara NE. Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine. Current protocols. 2021 May;1(5):e129. PMID: 34004049
- Levine ZG, Potter SC, Joiner CM, Fei GQ, Nabet B, Sonnett M, Zachara NE, Gray NS, Paulo JA, Walker S. Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase. Proceedings of the National Academy of Sciences of the United States of America. 2021 Jan 26;118. (4). PMID: 33419956
- Kaur D, Dorion S, Jmii S, Cappadocia L, Bede JC, Rivoal J. Pseudophosphorylation of Arabidopsis jasmonate biosynthesis enzyme lipoxygenase 2 via mutation of Ser600 inhibits enzyme activity. The Journal of biological chemistry. 2023 Mar;299(3):102898. Epub 2023 Jan 10. PMID: 36639029
- Papanicolaou KN, Jung J, Ashok D, Zhang W, Modaressanavi A, Avila E, Foster DB, Zachara NE, O'Rourke B. Inhibiting O-GlcNAcylation impacts p38 and Erk1/2 signaling and perturbs cardiomyocyte hypertrophy. The Journal of biological chemistry. 2023 Mar;299(3):102907. Epub 2023 Jan 13. PMID: 36642184
- Narayanan B, Zahra F, Reeves RA, Aggarwal A, O'Meally RN, Henry RK, Craven M, Jacobson A, Cole RN, Kohr MJ, Umapathi P, Zachara NE. Differential Detection of O-GlcNAcylated proteins in the heart using antibodies. Analytical biochemistry. 2023 Oct 1;678:115262. Epub 2023 Jul 27. PMID: 37507081
- Zachara NE. Critical observations that shaped our understanding of the function(s) of intracellular glycosylation (O-GlcNAc). FEBS letters. 2018 Dec;592(23):3950-3975. Epub 2018 Nov 24. PMID: 30414174
- Narayanan B, Sinha P, Henry R, Reeves RA, Paolocci N, Kohr MJ, Zachara NE. Cardioprotective O-GlcNAc signaling is elevated in murine female hearts via enhanced O-GlcNAc transferase activity. The Journal of biological chemistry. 2023 Dec;299(12):105447. Epub 2023 Nov 8. PMID: 37949223