Protein O-GlcNAcylation: emerging mechanisms and functions

Abstract

O-GlcNAcylation - the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins - is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels that range from structural and molecular biology to cell signalling and gene regulation to physiology and disease. New mechanisms and functions of O-GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiology can be understood.

Figure 1. Nutrient flux through the hexosamine biosynthetic pathway regulates protein O-GlcNAcylation

a | Glucose (Glc) is taken up from the extracellular milieu by glucose transporter (GLUT) proteins. While a majority of glucose is used for glycolysis and glycogen synthesis, ~2–5% of glucose is channeled into the hexosamine biosynthetic pathway (HBP). Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the rate-limiting step of the HBP, which converts fructose-6-phosphate (Fru-6P) into glucosamine-6-phosphate (GlcN-6P). Subsequent acetylation and uridylation of GlcN-6P yields the donor substrate for protein O-GlcNAcylation, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) catalyze the addition and removal of O-GlcNAc, respectively. Free GlcNAc can be recycled via the GlcNAc salvage pathway, which converts GlcNAc into GlcNAc-6-phosphate (GlcNAc-6P) that can be utilized by the HBP. b | Schematic representation of the OGT and OGA isoforms. The nucleocytoplasmic (ncOGT), mitochondrial (mOGT), and short (sOGT) isoforms of OGT differ in length due to variable numbers of amino-terminal (N-terminal) tetratricopeptide repeats (TPRs) but share common carboxy-terminal (C-terminal) catalytic (CDI and II) and phosphoinositide-binding domains (PPO). mOGT contains a unique N-terminal mitochondrial targeting sequence (MTS). The nucleocytoplasmic (ncOGA) and short (sOGA) isoforms of OGA possess identical N-terminal O-GlcNAc hydrolase domains and central OGT-binding regions; however, sOGA lacks the C-terminal histone acetyltransferase-like (HAT-like) domain present in ncOGA.

Figure 2. Potential mechanisms of O-GlcNAc transferase substrate recognition

a | O-GlcNAc transferase (OGT) may achieve some level of substrate specificity through its amino-terminal tetratricopeptide repeat (TPR) domain. Individually or in combination, TPRs could facilitate substrate recognition by generating unique binding sites that, when occupied, induce a conformational change that permits substrate access to the active site. TPRs 1–6 are required for OGT binding to Sin3a and TPRs 5–6 are required for OGT binding to ten-eleven translocation 2 (TET2), though TPRs 9–12 may also be involved. CDI and II, catalytic domains I and II; PPO, phosphoinositide-binding domain; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine. b | OGT substrate recognition may be additionally mediated by a hierarchy of highly conserved adaptor proteins, each responsible for recognizing and recruiting specific substrates to OGT in a context-dependent manner. For instance, in each of three different nutrient conditions (glucose deprivation, fasting, high glucose), OGT is recruited by a specific adaptor protein (p38, HCF-1, OGA) to O-GlcNAcylate a specific substrate (NF-H, PGC-1α, PKM2), leading to regulation of key downstream cellular pathways (NF-H solubility, gluconeogenic gene expression, aerobic glycolysis). HCF-1, host cell factor C1; OGA, O-GlcNAcase; NF-H, heavy neurofilament polypeptide; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PKM2, pyruvate kinase isoform M2. c | Accumulation of unfolded proteins in the endoplasmic reticulum during cellular stress activates the unfolded protein response, which upregulates hexosamine biosynthetic pathway (HBP) gene expression via the transcription factor spliced X-box binding protein 1 (Xbp1s). Increased production of UDP-GlcNAc by the HBP may lead to non-specific O-GlcNAcylation of unfolded proteins in the cytoplasm. O-GlcNAcylation of unfolded proteins may block their aggregation and proteasomal degradation as well as facilitate their re-folding by chaperones with O-GlcNAc-directed lectin activity (e.g. 70 kDa heat shock protein, Hsp70).

Figure 3. Functions of O-GlcNAcylation in time and space

a | O-GlcNAcylation of transcription factors and RNA polymerase II (Pol II) regulates transcriptional activation and repression. O-GlcNAcylation of nuclear factor kappa B (NF-κB) promotes its nuclear translocation (by blocking its interaction with IκBα) and enhances its DNA binding and transcriptional activity. On the other hand, the glucocorticoid receptor (GR) can directly bind to NF-κB and recruit O-GlcNAc transferase (OGT) to repress NF-κB activity. O-GlcNAcylation of Sp1 increases its nuclear localization and stability but at the same time inhibits its transactivation. O-GlcNAcylation of Pol II on its carboxy-terminal domain (CTD) is important for the assembly of preinitiation complexes at transcription start sites, while removal of O-GlcNAcylation from the Pol II CTD allows for its dynamic phosphorylation during transcription initiation and elongation. Thus, reciprocal O-GlcNAcylation and phosphorylation of the Pol II CTD is essential for maintenance of an unperturbed transcription cycle. Glc, glucose; GLUT, glucose transporter; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine; IκBα, NF-κB inhibitor alpha. b | OGT plays a variety of roles in epigenetic regulation. The OGT/host cell factor C1 (HCF-1) complex binds to ten-eleven translocation (TET) proteins, which catalyze cytosine 5-hydroxymethylation of genomic DNA and thereby promote DNA demethylation. OGT/HCF-1 also binds to BRCA-associated protein 1 (BAP1), which mediates deubiquitylation of histone H2A. OGT also stabilizes enhancer of zeste homolog 2 (EZH2), a component of Polycomb repressive complex 2 (PRC2), to promote histone H3 lysine 27 trimethylation. Furthermore, OGT acts in concert with the Sin3a/histone deacetylase (HDAC) corepressor complex to silence gene expression by promoting histone deacetylation. OGT may also directly modify histones through unknown mechanisms. c | O-GlcNAcylation is involved in the temporal regulation of insulin signalling dynamics. Binding of insulin to the insulin receptor (IR) induces IR autophosphorylation and subsequent tyrosine phosphorylation of insulin receptor substrate (IRS). Phosphorylated IRS binds and activates phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol 3,4,5-trisphosphate (PIP₃). PIP₃ recruits phosphoinositide-dependent kinase 1 (PDK1) and AKT to the plasma membrane, leading to AKT phosphorylation and activation and increased glucose uptake and anabolic cellular metabolism. In response to prolonged insulin stimulation, OGT translocates from the nucleus to the cytoplasm and localizes to the plasma membrane by binding to PIP₃, which leads to tyrosine phosphorylation and activation of OGT by IR. OGT then O-GlcNAcylates and inhibits key insulin signalling mediators such as IRS1, PI3K, PDK1, and AKT, blocking their phosphorylation and/or interactions and thereby facilitating termination of insulin signal transduction.

Figure 4. Nutritional and hormonal regulation of O-GlcNAc signalling

a | Nutrient availability is thought to regulate cellular O-GlcNAcylation levels by controlling flux through the hexosamine biosynthetic pathway (HBP) and thereby determining the abundance of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). We propose that the effect of nutrient availability on O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) expression, OGT-adaptor protein interaction, and high affinity OGT substrate (i.e. unfolded protein) accumulation also drives the nutrient sensitivity of this modification. HCF-1, host cell factor C1. b | In response to systemic changes in metabolic status (feeding, starvation, fasting), hormones (insulin, glucagon, ghrelin) modulate O-GlcNAc signalling in various cell types and tissues (liver, AgRP neurons), leading to regulation of specific cellular pathways (insulin signalling, autophagy, browning of WAT) that generate an appropriate metabolic response (suppression of anabolism, stimulation of gluconeogenesis and ketogenesis, activation of thermogenesis) to the initial stimulus. AgRP, Agouti-related peptide; WAT, white adipose tissue.

Figure 5. Maintenance of O-GlcNAc homeostasis by mutual regulation of O-GlcNAc transferase and O-GlcNAcase

We propose that there exists an “optimal zone” within which global O-GlcNAcylation levels must remain in order to preserve normal cellular function. In response to mild stress stimuli or moderate perturbations in nutrient availability, cellular O-GlcNAcylation levels may be maintained within the “optimal zone” by a “buffering” system consisting of mutual regulation of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) at the transcriptional and post-translational levels. However, severe and chronic insults to the cellular O-GlcNAcylation “buffer” may eventually lead to loss of O-GlcNAc homeostasis, which is an important factor contributing to the pathogenesis of various human diseases.