Dual-specificity RNA aptamers enable manipulation of target-specific O-GlcNAcylation and unveil functions of O-GlcNAc on ß-catenin.

O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.

Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation.

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1 (Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-ß-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Protein O-GlcNAcylation: emerging mechanisms and functions.

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.

Role of O-Linked N-Acetylglucosamine Protein Modification in Cellular (Patho)Physiology.

In the mid-1980s, the identification of serine and threonine residues on nuclear and cytoplasmic proteins modified by a N-acetylglucosamine moiety (O-GlcNAc) via an O-linkage overturned the widely held assumption that glycosylation only occurred in the endoplasmic reticulum, Golgi apparatus, and secretory pathways. In contrast to traditional glycosylation, the O-GlcNAc modification does not lead to complex, branched glycan structures and is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery, O-GlcNAcylation has been shown to contribute to numerous cellular functions, including signaling, protein localization and stability, transcription, chromatin remodeling, mitochondrial function, and cell survival. Dysregulation in O-GlcNAc cycling has been implicated in the progression of a wide range of diseases, such as diabetes, diabetic complications, cancer, cardiovascular, and neurodegenerative diseases. This review will outline our current understanding of the processes involved in regulating O-GlcNAc turnover, the role of O-GlcNAcylation in regulating cellular physiology, and how dysregulation in O-GlcNAc cycling contributes to pathophysiological processes.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

OGA mutant aberrantly hydrolyzes O-GlcNAc modification from PDLIM7 to modulate p53 and cytoskeleton in promoting cancer cell malignancy.

O-GlcNAcase (OGA) is the only human enzyme that catalyzes the hydrolysis (deglycosylation) of O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation) from numerous protein substrates. OGA has broad implications in many challenging diseases including cancer. However, its role in cell malignancy remains mostly unclear. Here, we report that a cancer-derived point mutation on the OGA's noncatalytic stalk domain aberrantly modulates OGA interactome and substrate deglycosylation toward a specific set of proteins. Interestingly, our quantitative proteomic studies uncovered that the OGA stalk domain mutant preferentially deglycosylated protein substrates with +2 proline in the sequence relative to the O-GlcNAcylation site. One of the most dysregulated substrates is PDZ and LIM domain protein 7 (PDLIM7), which is associated with the tumor suppressor p53. We found that the aberrantly deglycosylated PDLIM7 suppressed p53 gene expression and accelerated p53 protein degradation by promoting the complex formation with E3 ubiquitin ligase MDM2. Moreover, deglycosylated PDLIM7 significantly up-regulated the actin-rich membrane protrusions on the cell surface, augmenting the cancer cell motility and aggressiveness. These findings revealed an important but previously unappreciated role of OGA's stalk domain in protein substrate recognition and functional modulation during malignant cell progression.

Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.

O-GlcNAcylation is the addition of ß-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

Loss of NDST1 N-sulfotransferase activity is associated with autosomal recessive intellectual disability.

Intellectual Disability (ID) is the major cause of handicap, affecting nearly 3% of the general population, and is highly genetically heterogenous with more than a thousand genes involved. Exome sequencing performed in two independent families identified the same missense variant, p.(Gly611Ser), in the NDST1 (N-deacetylase/N-sulfotransferase member 1) gene. This variant had been previously found in ID patients of two other families but has never been functionally characterized. The NDST1 gene encodes a bifunctional enzyme that catalyzes both N-deacetylation and N-sulfation of N-acetyl-glucosamine residues during heparan sulfate (HS) biosynthesis. This step is essential because it influences the downstream enzymatic modifications and thereby determines the overall structure and sulfation degree of the HS polysaccharide chain. To discriminate between a rare polymorphism and a pathogenic variant, we compared the enzymatic properties of wild-type and mutant NDST1 proteins. We found that the p.(Gly611Ser) variant results in a complete loss of N-sulfotransferase activity while the N-deacetylase activity is retained. NDST1 shows the highest and the most homogeneous expression in the human cerebral structures compared to the other members of the NDST gene family. These results indicate that a loss of NDST1 N-sulfation activity is associated with impaired cognitive functions.

N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

O-GlcNAc forces an α-synuclein amyloid strain with notably diminished seeding and pathology.

Amyloid-forming proteins such α-synuclein and tau, which are implicated in Alzheimer's and Parkinson's disease, can form different fibril structures or strains with distinct toxic properties, seeding activities and pathology. Understanding the determinants contributing to the formation of different amyloid features could open new avenues for developing disease-specific diagnostics and therapies. Here we report that O-GlcNAc modification of α-synuclein monomers results in the formation of amyloid fibril with distinct core structure, as revealed by cryogenic electron microscopy, and diminished seeding activity in seeding-based neuronal and rodent models of Parkinson's disease. Although the mechanisms underpinning the seeding neutralization activity of the O-GlcNAc-modified fibrils remain unclear, our in vitro mechanistic studies indicate that heat shock proteins interactions with O-GlcNAc fibril inhibit their seeding activity, suggesting that the O-GlcNAc modification may alter the interactome of the α-synuclein fibrils in ways that lead to reduce seeding activity in vivo. Our results show that posttranslational modifications, such as O-GlcNAc modification, of α-synuclein are key determinants of α-synuclein amyloid strains and pathogenicity.

Glycan-Reactive Innate-like B Cells and Developmental Checkpoints.

Using an Ig H chain conferring specificity for N-acetyl-d-glucosamine (GlcNAc), we developed transgenic (VHHGAC39 TG) mice to study the role of self-antigens in GlcNAc-reactive B-1 B cell development. In VHHGAC39 TG mice, GlcNAc-reactive B-1 B cell development during ontogeny and in adult bone marrow was normal. However, adult TG mice exhibited a block at transitional-2 immature B cell stages, resulting in impaired allelic exclusion and accumulation of a B cell subset coexpressing endogenous Ig gene rearrangements. Similarly, VHHGAC39 B cell fitness was impeded compared with non-self-reactive VHJ558 B TG cells in competitive mixed bone marrow chimeras. Nonetheless, adult VHHGAC39 mice immunized with Streptococcus pyogenes produce anti-GlcNAc Abs. Peritoneal cavity B cells transferred from VHHGAC39 TG mice into RAG-/- mice also exhibited robust expansion and anti-GlcNAc Ab production. However, chronic treatment of young VHHGAC39 mice with GlcNAc-specific mAbs leads to lower GlcNAc-binding B cell frequencies while increasing the proportion of GlcNAc-binding B1-a cells, suggesting that Ag masking or clearance of GlcNAc Ags impedes maturation of newly formed GlcNAc-reactive B cells. Finally, BCR H chain editing promotes expression of endogenous nontransgenic BCR alleles, allowing potentially self-reactive TG B cells to escape anergy or deletion at the transitional stage of precursor B cell development. Collectively, these observations indicate that GlcNAc-reactive B cell development is sensitive to the access of autologous Ags.

SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis.

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

OGT controls mammalian cell viability by regulating the proteasome/mTOR/ mitochondrial axis.

O-GlcNAc transferase (OGT) modifies serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We performed a genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferation induced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cells stems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.

Tools for investigating O-GlcNAc in signaling and other fundamental biological pathways.

Cells continuously fine-tune signaling pathway proteins to match nutrient and stress levels in their local environment by modifying intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) sugars, an essential process for cell survival and growth. The small size of these monosaccharide modifications poses a challenge for functional determination, but the chemistry and biology communities have together created a collection of precision tools to study these dynamic sugars. This review presents the major themes by which O-GlcNAc influences signaling pathway proteins, including G-protein coupled receptors, growth factor signaling, mitogen-activated protein kinase (MAPK) pathways, lipid sensing, and cytokine signaling pathways. Along the way, we describe in detail key chemical biology tools that have been developed and applied to determine specific O-GlcNAc roles in these pathways. These tools include metabolic labeling, O-GlcNAc-enhancing RNA aptamers, fluorescent biosensors, proximity labeling tools, nanobody targeting tools, O-GlcNAc cycling inhibitors, light-activated systems, chemoenzymatic labeling, and nutrient reporter assays. An emergent feature of this signaling pathway meta-analysis is the intricate interplay between O-GlcNAc modifications across different signaling systems, underscoring the importance of O-GlcNAc in regulating cellular processes. We highlight the significance of O-GlcNAc in signaling and the role of chemical and biochemical tools in unraveling distinct glycobiological regulatory mechanisms. Collectively, our field has determined effective strategies to probe O-GlcNAc roles in biology. At the same time, this survey of what we do not yet know presents a clear roadmap for the field to use these powerful chemical tools to explore cross-pathway O-GlcNAc interactions in signaling and other major biological pathways.

O-GlcNAcylation and immune cell signaling: A review of known and a preview of unknown.

The dynamic and reversible modification of nuclear and cytoplasmic proteins by O-GlcNAcylation significantly impacts the function and dysfunction of the immune system. O-GlcNAcylation plays crucial roles under both physiological and pathological conditions in the biochemical regulation of all immune cell functions. Three and a half decades of knowledge acquired in this field is merely sufficient to perceive that what we know is just the prelude. This review attempts to mark out the known regulatory roles of O-GlcNAcylation in key signal transduction pathways and specific protein functions in the immune system and adumbrate ensuing questions toward the unknown functions.

The multifaceted role of intracellular glycosylation in cytoprotection and heart disease.

The modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked ß-N-actylglucosamine (O-GlcNAc) is an essential posttranslational modification that is common in metozoans. O-GlcNAc is cycled on and off proteins in response to environmental and physiological stimuli impacting protein function, which, in turn, tunes pathways that include transcription, translation, proteostasis, signal transduction, and metabolism. One class of stimulus that induces rapid and dynamic changes to O-GlcNAc is cellular injury, resulting from environmental stress (for instance, heat shock), hypoxia/reoxygenation injury, ischemia reperfusion injury (heart attack, stroke, trauma hemorrhage), and sepsis. Acute elevation of O-GlcNAc before or after injury reduces apoptosis and necrosis, suggesting that injury-induced changes in O-GlcNAcylation regulate cell fate decisions. However, prolonged elevation or reduction in O-GlcNAc leads to a maladaptive response and is associated with pathologies such as hypertrophy and heart failure. In this review, we discuss the impact of O-GlcNAc in both acute and prolonged models of injury with a focus on the heart and biological mechanisms that underpin cell survival.

The role of O-GlcNAcylation in RNA polymerase II transcription.

Eukaryotic RNA polymerase II (RNAPII) is responsible for the transcription of the protein-coding genes in the cell. Enormous progress has been made in discovering the protein activities that are required for transcription to occur, but the effects of post-translational modifications (PTMs) on RNAPII transcriptional regulation are much less understood. Most of our understanding relates to the cyclin-dependent kinases (CDKs), which appear to act relatively early in transcription. However, it is becoming apparent that other PTMs play a crucial role in the transcriptional cycle, and it is doubtful that any sort of complete understanding of this regulation is attainable without understanding the spectra of PTMs that occur on the transcriptional machinery. Among these is O-GlcNAcylation. Recent experiments have shown that the O-GlcNAc PTM likely has a prominent role in transcription. This review will cover the role of the O-GlcNAcylation in RNAPII transcription during initiation, pausing, and elongation, which will hopefully be of interest to both O-GlcNAc and RNAPII transcription researchers.

Regulation of protein O-GlcNAcylation by circadian, metabolic, and cellular signals.

O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a dynamic post-translational modification that regulates thousands of proteins and almost all cellular processes. Aberrant O-GlcNAcylation has been associated with numerous diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and type 2 diabetes. O-GlcNAcylation is highly nutrient-sensitive since it is dependent on UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). We previously observed daily rhythmicity of protein O-GlcNAcylation in a Drosophila model that is sensitive to the timing of food consumption. We showed that the circadian clock is pivotal in regulating daily O-GlcNAcylation rhythms given its control of the feeding-fasting cycle and hence nutrient availability. Interestingly, we reported that the circadian clock also modulates daily O-GlcNAcylation rhythm by regulating molecular mechanisms beyond the regulation of food consumption time. A large body of work now indicates that O-GlcNAcylation is likely a generalized cellular status effector as it responds to various cellular signals and conditions, such as ER stress, apoptosis, and infection. In this review, we summarize the metabolic regulation of protein O-GlcNAcylation through nutrient availability, HBP enzymes, and O-GlcNAc processing enzymes. We discuss the emerging roles of circadian clocks in regulating daily O-GlcNAcylation rhythm. Finally, we provide an overview of other cellular signals or conditions that impact O-GlcNAcylation. Many of these cellular pathways are themselves regulated by the clock and/or metabolism. Our review highlights the importance of maintaining optimal O-GlcNAc rhythm by restricting eating activity to the active period under physiological conditions and provides insights into potential therapeutic targets of O-GlcNAc homeostasis under pathological conditions.