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Principal Investigator: John A. Hanover
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2024
Award: $1,318,649
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
A dynamic cycle of addition and removal of O-GlcNAc in the nucleus and cytoplasm mediates a final step in the hexosamine signaling pathway. The targets of this modification are nuclear pore complexes, transcription complexes, proteasomes and signaling kinases. Based on the targets modified by O-GlcNAc, we proposed that the enzymes of O-GlcNAc metabolism modulate nuclear transport, transcription, cell growth, and apoptosis in response to nutrient availability. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway (HBP) acting as a cellular sensor of nutrient availability. By integrating these signals, the HBP regulates expression of a number of gene products that include leptin. In skeletal muscle, flux through the HBP correlates with the degree of insulin resistance. The HBP is also linked to pathways regulating cell proliferation and apoptosis; fibroblasts that cannot acetylate UDP-GlcNAc exhibit defects in proliferation, adhesiveness and resistance to apoptotic stimuli. Thus, by generating UDP-GlcNAc, the HBP may be viewed as a nutrient-sensing signaling pathway. We seek to determine how O-GlcNAc participates in this signaling cascade.
We are testing the hypothesis that differentially targeted isoforms of the enzymes of O-GlcNAc metabolism mediate this glycan-dependent signaling pathway. By responding to nutrient levels, this pathway modulates gene expression, cell growth and programmed cell death. We expressed fully functional OGT and O-GlcNAcase isoforms in E. coli. We recently solved the structure of the superhelical TPR (tetratricopeptide repeat) domain of OGT that mediates the recognition of target proteins and showed that exhibits structural similarities to importin alpha. Consistent with a role as a signaling molecule, we showed that OGT modifies glycogen synthase kinase-3 and casein kinase, two enzymes regulating glycogen synthesis. Much of the impact of O-GlcNAc cycling occurs through changes in transcription. Importantly, both histone deacetylases (HDAC) and OGT are recruited to Sin3a transcription-repression complexes. The C-terminus of one isoform of the O-GlcNAcase has been shown to be a histone acetyltransferase (HAT). Therefore, O-GlcNAc appears to be a dynamic participant in histone remodeling complexes. We are currently testing this hypothesis by interfering with O-GlcNAc cycling and examining the subsequent impact on chromatin using CHIP-on-Chip, high throughput sequencing and expression array technologies. Cryoelectron microscopy (CryoEM) is being used to solve the structure of full length, intact OGT and O-GlcNAcase. This technique will be used to explore the numerous protein complexes in which O-GlcNAc cycling occurs.
We have also focused on the catalytic functions of the enzymes of O-GlcNAc cycling. We demonstrated that both isoforms of O-GlcNAcase are active enzymes modulating cellular O-GlcNAc levels. Mutational analysis of OGT and O-GlcNAcase allowed us to define catalytic domains. We showed that OGT isoforms are targeted to both nucleus and mitochondria. The differential localization of mitochondrial and nuclear isoforms of OGT argues that they perform unique intracellular functions in apoptosis, mitochondrial movement and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells; one isoform is nuclear while another accumulates at cellular sites of lipid storage. Small molecule inhibitors and substrates for the enzymes of O-GlcNAc metabolism are under development using both synthetic and natural product approaches. We have proposed that this intracellular glycan modification of Ser/Thr participates in diverse signaling pathways in a manner analogous to protein phosphorylation. In collaborative studies, we have shown that the human OGT gene is subject to X-chromosome imprinting and appears to play a key role in the susceptibility to coronary artery disease. In the immune system, O-GlcNAc cycling is associated with the cytokine storm leading to increased morbidity in viral infections such as COVID-19.
We also have taken Chemical Biology approaches to examine O-GlcNAc cycling. We first developed a chemical method for detecting O-GlcNAc addition. This method is being used for high throughput screening to identify inhibitors of O-GlcNAc cycling. We developed a number of O-GlcNAcase-specific fluorogenic substates and inhibitors that will facilitate dissection of the hexosamine signaling pathway implicated in Type-2 diabetes, obesity and neurodegeneration. These reagents will facilitate the dissection of the nutrient-sensing hexosamine signaling pathway. O-GlcNAc cycling has been linked to Stem Cell self-renewal and DNA damage repair in response to oxidative stress. O-GlcNAc also plays a pivotal role in regulating DNA methylation and de methylation. This nutrient-responsive pathway may be a key component of an environmentally responsive regulation of DNA methylation.
Thus, the enzymes of O-GlcNAc cycling play a key role in the changes in signaling and epigenetic landscape associated with metabolic disease. This pathway may be of great significance in the intrauterine environment to influence the future susceptibility to chronic disease. These ideas are being tested using genetics, biochemistry and imaging in animal models.
Terms: <20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><Abscission><Acetylation><Adhesiveness><Adult-Onset Diabetes Mellitus><Animal Model><Animal Models and Related Studies><Apoptosis><Apoptosis Pathway><Apoptotic><Biochemistry><Biological Chemistry><Biology><COVID-19><CV-19><Catalytic Core><Catalytic Domain><Catalytic Region><Catalytic Site><Catalytic Subunit><Cell Body><Cell Communication and Signaling><Cell Growth in Number><Cell Multiplication><Cell Nucleus><Cell Nucleus Active Transport><Cell Proliferation><Cell Signaling><Cells><Cellular Expansion><Cellular Growth><Cellular Proliferation><Chemicals><Chromatin><Chronic Disease><Chronic Illness><Complex><Coronary Arteriosclerosis><Coronary Artery Disease><Coronary Artery Disorder><Coronary Atherosclerosis><Coronavirus Infectious Disease 2019><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><DNA Damage Repair><DNA Methylation><DNA Methylation Regulation><DNA Repair><Defect><Degenerative Neurologic Disorders><Development><Diabetes Mellitus><Diagnostic><Diathesis><Disease susceptibility><Dissection><E coli><E. coli><Electron Cryomicroscopy><Environment><Enzyme Gene><Enzymes><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Escherichia coli><Excision><Exhibits><Extirpation><Fibroblasts><Future><GSK-3><Gene Chips><Gene Down-Regulation><Gene Expression><Gene Expression Chip><Gene Transcription><GeneChip><Genes><Genetic><Genetic Transcription><Glycans><Glycogen><Glycogen Synthase Kinase 3><HDAC><HDAC Proteins><Hexosamines><High Throughput Assay><High-Throughput Nucleotide Sequencing><High-Throughput Sequencing><Histone Acetylase><Histone Deacetylase><Histones><Human><Image><Immune system><Importin-alpha><Importin-α><Insulin Resistance><Intermediary Metabolism><Intracellular Communication and Signaling><Isoforms><Ketosis-Resistant Diabetes Mellitus><Kinases><Length><Leptin><Link><Lipids><Macropain><Macroxyproteinase><Maturity-Onset Diabetes Mellitus><Mediating><Metabolic Diseases><Metabolic Disorder><Metabolic Processes><Metabolism><Methods><Methylation><Mitochondria><Modern Man><Modification><Molecular><Morbidity><Morbidity - disease rate><Movement><Multicatalytic Proteinase><Mutation Analysis><NIDDM><NLS Receptor><NLS-Binding Protein><NLSBP><NPC><Natural Products><Nerve Degeneration><Nervous System Degenerative Diseases><Neural Degenerative Diseases><Neural degenerative Disorders><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Neuron Degeneration><Non-Insulin Dependent Diabetes><Non-Insulin-Dependent Diabetes Mellitus><Noninsulin Dependent Diabetes><Noninsulin Dependent Diabetes Mellitus><Nuclear><Nuclear Localization Sequence Receptor><Nuclear Localization Signal-Binding Protein><Nuclear Pore Complex><Nuclear Transport><Nucleocytoplasmic Shuttling><Nucleus><Nutrient><Nutrient availability><O-GlcNAcase><Ob Gene Product><Ob Protein><Obese Gene Product><Obese Protein><Obesity><Oxidative Stress><Participant><Pathway interactions><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Play><Polysaccharides><Predisposition><Programmed Cell Death><Proliferating><Prosome><Proteasome><Proteasome Endopeptidase Complex><Protein Isoforms><Protein Phosphorylation><Proteins><Proteosome><RNA Expression><Reagent><Regulation><Removal><Resistance><Role><Signal Pathway><Signal Transduction><Signal Transduction Pathway><Signal Transduction Systems><Signaling><Signaling Molecule><Site><Site-Directed Mutagenesis><Site-Specific Mutagenesis><Skeletal Muscle><Slow-Onset Diabetes Mellitus><Stable Diabetes Mellitus><Stimulus><Structure><Surgical Removal><Susceptibility><T2 DM><T2D><T2DM><Targeted DNA Modification><Targeted Modification><Techniques><Technology><Testing><Therapeutic><Thesaurismosis><Transcription><Transcription Repression><Transcriptional Repression><Transphosphorylases><Type 2 Diabetes Mellitus><Type 2 diabetes><Type II Diabetes Mellitus><Type II diabetes><Unscheduled DNA Synthesis><Viral Diseases><Virus Diseases><Voluntary Muscle><X Chromosome><adiposity><adult onset diabetes><alpha Karyopherins><atherosclerotic coronary disease><biological signal transduction><body movement><casein kinase><cell growth><chronic disorder><coronary arterial disease><coronavirus disease 2019><coronavirus disease-19><coronavirus infectious disease-19><corpulence><cryo-EM><cryoEM><cryogenic electron microscopy><cytokine release syndrome><cytokine storm><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><detection method><detection of nutrient><detection procedure><detection technique><developmental><diabetes><enzyme substrate><epigenetically><expression array><gene expression microarray><gene product><gene repression><gsk-3 Gene Product><high throughput screening><histone acetyltransferase><imaging><imprint><inhibitor><insulin resistant><insulin signaling><insulin tolerance><intra-uterine environment><intrauterine environment><intrauterine milieu><ketosis resistant diabetes><liability to disease><maturity onset diabetes><metabolism disorder><mitochondrial><model of animal><multicatalytic endopeptidase complex><naturally occurring product><neural degeneration><neurodegeneration><neurodegenerative><neurodegenerative illness><neurological degeneration><neuronal degeneration><nucleocytoplasmic transport><nutrient sensing><pathway><peptide O-GlcNAc-beta-N-acetylglucosaminidase><peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase><perception of nutrients><progenitor cell regeneration><progenitor cell self renewal><progenitor regeneration><progenitor self renewal><protein complex><recruit><resection><resistant><response><sensor><small molecular inhibitor><small molecule inhibitor><social role><stem and progenitor cell regeneration><stem and progenitor cell self renewal><stem cell regeneration><stem cell self renewal><sugar nucleotide><type 2 DM><type II DM><type two diabetes><viral infection><virus infection><virus-induced disease>