O-GlcNAc Transferase Research Articles

Photoactivatable O-GlcNAc transferase library enables covalent chemical capture of solvent-exposed TPR domain interactions.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential, stress-sensing enzyme responsible for adding the O-GlcNAc monosaccharide to thousands of nuclear and cytoplasmic proteins to regulate cellular homeostasis. OGT substrates are found in almost all intracellular processes, and perturbations in protein O-GlcNAc levels have been implicated in proteostatic diseases, such as cancers, metabolic disorders, and neurodegeneration. This broad disease activity makes OGT an attractive therapeutic target; however, the substrate diversity makes pan-inhibition as a therapeutic strategy unfeasible. Rather, a substrate-specific approach to targeting is more advantageous, but how OGT chooses its substrates remains poorly understood. Substrate specificity is controlled by the interactions between OGT's non-catalytic tetratricopeptide repeat (TPR) domain, rather than its glycosyltransferase domain. OGT's TPR domain forms a 100 Å superhelical structure, containing a lumenal surface, known as the substrate-binding surface, and a solvent-exposed surface. To date, there are no tools to site-selectively target regions of the domain and differentiate between the two binding surfaces. Here, we developed a library of recombinant OGT constructs containing site-specifically incorporated photoactivatable unnatural amino acids (UAAs) along the solvent-exposed surface of the TPR domain to covalently capture and map OGT's interactome.

Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT.

The post-translational modification of intracellular proteins through O-linked β-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification has an essential role in signal transduction, gene expression, organelle function and systemic physiology. Here, we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression, which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to localize to specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate the downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool for defining the role of O-GlcNAcylation in cell signaling and physiology.

Rescuable sleep and synaptogenesis phenotypes in a Drosophila model of O-GlcNAc transferase intellectual disability.

O-GlcNAcylation is an essential intracellular protein modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Recently, missense mutations in OGT have been linked to intellectual disability, indicating that this modification is important for the development and functioning of the nervous system. However, the processes that are most sensitive to perturbations in O-GlcNAcylation remain to be identified. Here, we uncover quantifiable phenotypes in the fruit fly Drosophila melanogaster carrying a patient-derived OGT mutation in the catalytic domain. Hypo-O-GlcNAcylation leads to defects in synaptogenesis and reduced sleep stability. Both these phenotypes can be partially rescued by genetically or chemically targeting OGA, suggesting that a balance of OGT/OGA activity is required for normal neuronal development and function.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Opportunities for Therapeutic Modulation of O-GlcNAc.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is an essential, dynamic monosaccharide post-translational modification (PTM) found on serine and threonine residues of thousands of nucleocytoplasmic proteins. The installation and removal of O-GlcNAc is controlled by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery four decades ago, O-GlcNAc has been found on diverse classes of proteins, playing important functional roles in many cellular processes. Dysregulation of O-GlcNAc homeostasis has been implicated in the pathogenesis of disease, including neurodegeneration, X-linked intellectual disability (XLID), cancer, diabetes, and immunological disorders. These foundational studies of O-GlcNAc in disease biology have motivated efforts to target O-GlcNAc therapeutically, with multiple clinical candidates under evaluation. In this review, we describe the characterization and biochemistry of OGT and OGA, cellular O-GlcNAc regulation, development of OGT and OGA inhibitors, O-GlcNAc in pathophysiology, clinical progress of O-GlcNAc modulators, and emerging opportunities for targeting O-GlcNAc. This comprehensive resource should motivate further study into O-GlcNAc function and inspire strategies for therapeutic modulation of O-GlcNAc.

β-Ketoenamine covalent organic framework nanoplatform combined with immune checkpoint blockade via photodynamic immunotherapy inhibit glioblastoma progression

The synergistic approach of combining photodynamic immunotherapy with endogenous clearance of PD-L1 immune checkpoint blockade therapy holds promise for enhancing survival outcomes in glioblastoma (GBM) patients. The observed upregulation of O-GlcNAc glycolysis in tumors may contribute to the stabilization of endogenous PD-L1 protein, facilitating tumor immune evasion. This study presents a pH-adapted excited state intramolecular proton transfer (ESIPT)-isomerized β-ketoamide-based covalent organic framework (COF) nanoplatform (denoted as OT@COF-RVG). Temozolomide (TMZ) and OSMI-4 (O-GlcNAc transferase inhibitor) were integrated into COF cavities, then modified on the surface with polyethylene glycol and the rabies virus peptide RVG-29, showing potential for sensitizing TMZ chemotherapy and initiating photodynamic therapy (PDT). By inhibiting O-GlcNAc and promoting lysosomal degradation of PD-L1, OT@COF-RVG enhanced the effectiveness of immune checkpoint blockade (ICB) therapy. Additionally, treatment with OT@COF-RVG led to a notable elevation in reactive oxygen species (ROS) levels, thereby re-establishing an immunostimulatory state, inducing immunogenic cell death (ICD). In summary, our research unveiled a correlation between O-GlcNAc in GBM and the evasion of immune responses by tumors, while showcasing the potential of OT@COF-RVG in reshaping the immunosuppressive microenvironment of GBM and offering a more effective approach to immunotherapy in clinical settings.

O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication stress

O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus and mitochondria. Recent advent of the mass spectrometry technology has enabled identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, etoposide, diminishes the association. Using higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon etoposide treatment, and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress.

Protein O-GlcNAcylation coupled to Hippo signaling drives vascular dysfunction in diabetic retinopathy

Metabolic disorder significantly contributes to diabetic vascular complications, including diabetic retinopathy, the leading cause of blindness in the working-age population. However, the molecular mechanisms by which disturbed metabolic homeostasis causes vascular dysfunction in diabetic retinopathy remain unclear. O-GlcNAcylation modification acts as a nutrient sensor particularly sensitive to ambient glucose. Here, we observe pronounced O-GlcNAc elevation in retina endothelial cells of diabetic retinopathy patients and mouse models. Endothelial-specific depletion or pharmacological inhibition of O-GlcNAc transferase effectively mitigates vascular dysfunction. Mechanistically, we find that Yes-associated protein (YAP) and Transcriptional co-activator with PDZ-binding motif (TAZ), key effectors of the Hippo pathway, are O-GlcNAcylated in diabetic retinopathy. We identify threonine 383 as an O-GlcNAc site on YAP, which inhibits its phosphorylation at serine 397, leading to its stabilization and activation, thereby promoting vascular dysfunction by inducing a pro-angiogenic and glucose metabolic transcriptional program. This work emphasizes the critical role of the O-GlcNAc-Hippo axis in the pathogenesis of diabetic retinopathy and suggests its potential as a therapeutic target.

ACSS2 regulates ferroptosis in an E2F1-dependent manner in breast cancer brain metastatic cells.

Brain metastasis diagnosis in breast cancer patients is considered an end-stage event. The median survival after diagnosis is measured in months, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to the unique brain environment and are highly dependent on acetate metabolism for growth and survival. However, the signaling pathways that regulate survival in breast cancer brain metastatic (BCBM) tumors are not known. Primary brain tumor cells can convert acetate to acetyl-CoA via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinase-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Here, we show that breast cancer cells selected to metastasize to the brain contain increased levels of O-GlcNAc, OGT and ACSS2-Ser267 phosphorylation compared to parental breast cancer cells. Moreover, OGT and CDK5 are required for breast cancer cell growth in the brain parenchyma in vivo. Importantly, ACSS2 and ACSS2-S267D phospho-mimetic mutant are critical for in vivo breast cancer growth in the brain but not in the mammary fat pad. Mechanistically, we show that ACSS2 regulates BCBM cell survival by suppressing ferroptosis via regulation of E2F1-mediated expression of anti-ferroptotic proteins SLC7A11 and GPX4. Lastly, we show treatment with a novel brain-permeable small molecule ACSS2 inhibitor induced ferroptosis and reduced BCBM growth ex vivo and in vivo . These results suggest a crucial role for ACSS2 in protecting from ferroptosis in breast cancer brain metastatic cells and suggests that breast cancer brain metastatic cells may be susceptible to ferroptotic inducers.

O-GlcNAcylation inhibition redirects the response of colon cancer cells to chemotherapy from senescence to apoptosis.

The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues.

SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing.

O-GlcNAcylation is the reversible post-translational addition of β-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. It plays an important role in several cellular processes through the modification of thousands of protein substrates. O-GlcNAcylation in humans is mediated by a single essential enzyme, O-GlcNAc transferase (OGT). OGT, together with the sole O-GlcNAcase OGA, form an intricate feedback loop to maintain O-GlcNAc homeostasis in response to changes in cellular O-GlcNAc using a dynamic mechanism involving nuclear retention of its fourth intron. However, the molecular mechanism of this dynamic regulation remains unclear. Using an O-GlcNAc responsive GFP reporter cell line, we identify SFSWAP, a poorly characterized splicing factor, as a trans-acting factor regulating OGT intron detention. We show that SFSWAP is a global regulator of retained intron splicing and exon skipping that primarily acts as a negative regulator of splicing. In contrast, knockdown of SFSWAP leads to reduced inclusion of a 'decoy exon' present in the OGT retained intron which may mediate its role in OGT intron detention. Global analysis of decoy exon inclusion in SFSWAP and UPF1 double knockdown cells indicate altered patterns of decoy exon usage. Together, these data indicate a role for SFSWAP as a global negative regulator of pre-mRNA splicing and positive regulator of intron retention.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway.

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation levels in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent mice by repressing the cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt-/-) caused a marked reduction in tumor growth in both syngeneic mice tumor models and a genetic mice colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of the type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas or Sting from Ogt-/- cancer cells restored tumor growth, and this correlated with impaired CD8+ T-cell-mediated antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production in tumors. In summary, our results identify OGT-mediated genomic stability and activate cGAS-STING pathway as an important tumor-cell-intrinsic mechanism to repress antitumor immunity.

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

Inhibition of O-GlcNAc transferase activates type I interferon-dependent antitumor immunity by bridging cGAS-STING pathway

Compromised CDK12 activity causes dependency on the high activity of O-GlcNAc transferase.

O-GlcNAc transferase (OGT) coordinates with regulators of transcription, including cyclin-dependent kinase 12 (CDK12), the major transcription elongation kinase. Here, we use inhibitor- and knockdown-based strategies to show that co-targeting of OGT and CDK12 is toxic to prostate cancer cells. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation and due to its essentiality in higher eukaryotes, it is not an ideal drug target. Our glycoproteomics-data revealed that short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in different models of prostate cancer. By integrating our glycoproteomics-, gene essentiality- and clinical-data from CDK12 mutant prostate cancer patients, we identify the non-essential serine-arginine protein kinase 1 (SRPK1) as a synthetic lethal partner with CDK12-inactivation. Both normal and cancer cells become highly sensitive against inhibitors of OGT and SRPK1 if they have lowered activity of CDK12. Inactivating mutations in CDK12 are enriched in aggressive prostate cancer, and we propose that these patients would benefit from therapy targeting the spliceosome.

Multi-omics after O-GlcNAc alteration identified cellular processes promoting aneuploidy after loss of O-GlcNAc transferase

Pharmacologic or genetic manipulation of O-GlcNAcylation, an intracellular, single sugar post-translational modification, are difficult to interpret due to the pleotropic nature of O-GlcNAc and the vast signaling pathways it regulates. To address this issue, we employed either OGT (O-GlcNAc transferase), OGA (O-GlcNAcase) liver knockouts, or pharmacological inhibition of OGA coupled with multi-Omics analysis and bioinformatics. We identified numerous genes, proteins, phospho-proteins, or metabolites that were either inversely or equivalently changed between conditions. Moreover, we identified pathways in OGT knockout samples associated with increased aneuploidy. To test and validate these pathways, we induced liver growth in OGT knockouts by partial hepatectomy. OGT knockout livers showed a robust aneuploidy phenotype with disruptions in mitosis, nutrient sensing, protein metabolism/amino acid metabolism, stress response, and HIPPO signaling demonstrating how OGT is essential in controlling aneuploidy pathways. Moreover, these data show how a multi-Omics platform can discern how OGT can fine-tune multiple cellular pathways involved in in aneuploidy.

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency.

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1's O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.

The study on the role of O-GlcNAcylation of SIRT3 in regulating mitochondrial oxidative stress during simulate myocardial ischemia-reperfusion.

Myocardial ischemia-reperfusion injury (MIRI) is a significant complication following reperfusion therapy after myocardial infarction. Mitochondrial oxidative stress is a critical factor in MIRI, and Sirtuin 3 (SIRT3), as a major mitochondrial deacetylase, plays a key protective role, with its activity potentially regulated by O-GlcNAcylation. This study used the H9C2 cell line to establish a simulated ischemia/reperfusion (SI/R) model, we utilized co-immunoprecipitated to validate the relationship between O-GlcNAc transferase (OGT) and SIRT3, demonstrated SIRT3 O-GlcNAcylation sites through LC-MS/MS, and performed site mutations using CRISPR/Cas9 technology. The results were validated using immunoblotting. SIRT3 and superoxide dismutase 2 (SOD2) activities were detected using a fluorometric assay, while mitochondrial reactive oxygen species (MROS) levels and cellular apoptosis were assessed using immunofluorescence. We have identified an interaction between SIRT3 and OGT, where SIRT3 undergoes dynamic O-GlcNAcylation at the S190 site, facilitating SIRT3 deacetylase activity. During SI/R, elevated levels of O-GlcNAcylation activate SOD2 by promoting SIRT3 enzyme activity, thereby inhibiting excessive MROS production. This significantly mitigates the occurrence of malignant autophagy in myocardial cells during reperfusion, promoting their survival. Conversely, blocking SIRT3 O-GlcNAcylation at the S190 site exacerbates SI/R injury. We demonstrate that O-GlcNAcylation is a crucial post-translational modification (PTM) of SIRT3 during SI/R, shedding light on a promising mechanism for future therapeutic approaches.

Sodium aescinate alleviates neuropathic pain through suppressing OGT-mediated O-GlcNAc modification of TLR3 to inactivate MAPK signaling pathway

Neuropathic pain results from damage to nerves or the brain, and is characterized by symptoms such as allodynia, spontaneous pain, and hyperalgesia. The causes of this type of pain are intricate, which can make it difficult to treat. Sodium aescinate (SA), a natural extract from horse chestnut tree seeds, has been shown to act as a neuroprotector by inhibiting microglia activation. This study aims to explore the therapeutic potential of SA for neuropathic pain and the molecular mechanisms regulated by SA treatment. Through in vivo animal models and experiments, we found that SA treatment significantly reduced mechanical allodynia and heat hyperalgesia in neuropathic pain models. Additionally, SA inhibited O-GlcNAc-transferase (OGT)-induced O-GlcNAcylation (O-GlcNAc) modification in neuropathic pain mice. OGT overexpression could impede the therapeutic effects of SA on neuropathic pain. Further investigation revealed that Toll-like receptor 3 (TLR3), stabilized by OGT-induced O-GlcNAc modification, could activate the Mitogen activated protein kinase (MAPK) signaling pathway. Further in vivo experiments demonstrated that TLR3-mediated p38 mitogen-activated protein kinase (p38MAPK) activation is involved in SA-mediated relief of neuropathic pain. In conclusion, this study uncovers a novel molecular pathway deactivated by SA treatment in neuropathic pain.

Structure and Inhibition of Insect UDP-N-acetylglucosamine Pyrophosphorylase: A Key Enzyme in the Hexosamine Biosynthesis Pathway.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.