The cycling of N-acetylglucosamine on Ser(Thr) residues (O-GlcNAcylation) of nuclear, cytoplasmic and mitochondrial proteins serves as a nutrient sensor to regulate signaling, transcription, and cellular physiology. Abnormal O-GlcNAcylation underlies the etiology of diabetes, cancer and Alzheimer's disease. O-GlcNAcylation regulates nearly every aspect of transcription, including RNA polymerase II, histones, DNA methyltransferases, and nearly all transcription factors. Yet we know very little about the mechanisms involved. TATA-binding protein (TBP) is amongst the most important components of the transcription machinery. Our recent studies indicate that TBP regulated by O-GlcNAcylation in response to nutrients. O-GlcNAcylation has extensive crosstalk with protein phosphorylation and other modifications. However, unlike phosphorylation, which is catalyzed by hundreds of kinases, there is only one highly conserved gene encoding O-GlcNAc transferase (OGT). Nonetheless, OGT site-specifically modifies thousands of proteins. Our data indicate that OGT's substrate specificity is not only determined by its specificity for peptide sequence, but also by its transient associations with other proteins, which dynamically target it to specific substrates. OGT targeting interactions are largely mediated by its tetratricopeptide repeats (TPRs). OGT missing TPRs remains active against small peptides, but has poor activity against full-length proteins. Herein we will investigate two major questions related to O-GlcNAcylation:
Specific Aim 1 : Continue to Study Glucose Regulation of TATA-binding protein (TBP) via its O-GlcNAcylation. Hypothesis: At certain promoters, glucose regulates TBP DNA-binding and its ability to bend DNA by altering its O- GlcNAcylation. We will systematically elucidate mechanisms and functions of this nutrient regulation of TBP. Three goals: A. Roles of O-GlcNAcylation in the molecular and cellular properties of TBP? B. Roles of O- GlcNAc in TBP's Interactions in the Transcription Cycle. C. O-GlcNAc's In Vivo Roles in TBP-mediated Gene Expression.
Specific Aim 2 : Continue to Elucidate How OGT is Specifically Targeted to Thousands of Different Protein Substrates? Hypothesis: OGT achieves a high degree of specificity not only by its recognition of peptide sequence, but also by it being targeted to substrates by accessory proteins.
This Aim will systematically evaluate both the general and specific roles of OGT binding partners in OGT's activities toward its many substrates. Given O-GlcNAc's importance to mechanisms of chronic disease, such as glucose toxicity in diabetes, cancer and neurodegeneration, elucidation of these mechanisms will not only be key to understanding transcription and signaling, also to uncovering new avenues for therapeutics.

Public Health Relevance

Decades of work have shown that a cycling sugar modification of proteins in the nucleus and cytoplasm of cells is a major mechanism by which what we eat regulates our cellular physiology. In fact, dysregulation of this sugar cycling contributes to major diseases of aging, such as diabetes, cancer and Alzheimer's disease. This project will investigate how this sugar modification regulates gene expression, and how it is enzymatically added to so many different proteins in cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM116891-01A1
Application #
9172016
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Marino, Pamela
Project Start
2016-08-10
Project End
2020-07-31
Budget Start
2016-08-10
Budget End
2017-07-31
Support Year
1
Fiscal Year
2016
Total Cost
$313,856
Indirect Cost
$121,356
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
Liu, Wei; Han, Guanghui; Yin, Yalin et al. (2018) AANL (Agrocybe aegerita lectin 2) is a new facile tool to probe for O-GlcNAcylation. Glycobiology 28:363-373
Ma, Junfeng; Hart, Gerald W (2017) Analysis of Protein O-GlcNAcylation by Mass Spectrometry. Curr Protoc Protein Sci 87:24.10.1-24.10.16
Lagerlöf, Olof; Slocomb, Julia E; Hong, Ingie et al. (2016) The nutrient sensor OGT in PVN neurons regulates feeding. Science 351:1293-6
Hardivillé, Stéphan; Hart, Gerald W (2016) Nutrient regulation of gene expression by O-GlcNAcylation of chromatin. Curr Opin Chem Biol 33:88-94
Ramirez-Correa, Genaro A; Ma, Junfeng; Slawson, Chad et al. (2015) Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle. Diabetes 64:3573-87
Ma, Junfeng; Liu, Ting; Wei, An-Chi et al. (2015) O-GlcNAcomic Profiling Identifies Widespread O-Linked ?-N-Acetylglucosamine Modification (O-GlcNAcylation) in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function. J Biol Chem 290:29141-53
Banerjee, Partha S; Ma, Junfeng; Hart, Gerald W (2015) Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria. Proc Natl Acad Sci U S A 112:6050-5