O-linked N-acetylglucosamine Research Articles

O-GlcNAc transferase regulates collagen deposition and fibrosis resolution in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis. We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation. We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition. OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.

Abstract LB293: O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance

Abstract Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs in melanoma. Deploying a model of adaptive cancer drug tolerance H3K4me3 ChIP-seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in vivo. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Gregory Gimenez, Elmar Zuegner, Christoph Magens, Michael Eccles, Mayumi Fujita, Helmut Schaider. O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB293.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

An in-silico analysis of OGT gene association with diabetes mellitus

O-GlcNAcylation is a nutrient-sensing post-translational modification process. This cycling process involves two primary proteins: the O-linked N-acetylglucosamine transferase (OGT) catalysing the addition, and the glycoside hydrolase OGA (O-GlcNAcase) catalysing the removal of the O-GlCNAc moiety on nucleocytoplasmic proteins. This process is necessary for various critical cellular functions. The O-linked N-acetylglucosamine transferase (OGT) gene produces the OGT protein. Several studies have shown the overexpression of this protein to have biological implications in metabolic diseases like cancer and diabetes mellitus (DM). This study retrieved 159 SNPs with clinical significance from the SNPs database. We probed the functional effects, stability profile, and evolutionary conservation of these to determine their fit for this research. We then identified 7 SNPs (G103R, N196K, Y228H, R250C, G341V, L367F, and C845S) with predicted deleterious effects across the four tools used (PhD-SNPs, SNPs&Go, PROVEAN, and PolyPhen2). Proceeding with this, we used ROBETTA, a homology modelling tool, to model the proteins with these point mutations and carried out a structural bioinformatics method– molecular docking– using the Glide model of the Schrodinger Maestro suite. We used a previously reported inhibitor of OGT, OSMI-1, as the ligand for these mutated protein models. As a result, very good binding affinities and interactions were observed between this ligand and the active site residues within 4Å of OGT. We conclude that these mutation points may be used for further downstream analysis as drug targets for treating diabetes mellitus.

Compound SJ-12 attenuates streptozocin-induced diabetic cardiomyopathy by stabilizing SERCA2a

Heart failure (HF) is one of the major causes of death among diabetic patients. Although studies have shown that curcumin analog C66 can remarkably relieve diabetes-associated cardiovascular and kidney complications, the role of SJ-12, SJ-12, a novel curcumin analog, in diabetic cardiomyopathy and its molecular targets are unknown. 7-week-old male C57BL/6 mice were intraperitoneally injected with single streptozotocin (STZ) (160 mg/kg) to develop diabetic cardiomyopathy (DCM). The diabetic mice were then treated with SJ-12 via gavage for two months. Body weight, fast blood glucose, cardiac utrasonography, myocardial injury markers, pathological morphology of the heart, hypertrophic and fibrotic markers were assessed. The potential target of SJ-12 was evaluated via RNA-sequencing analysis. The O-GlcNAcylation levels of SP1 were detected via immunoprecipitation. SJ-12 effectively suppressed myocardial hypertrophy and fibrosis, thereby preventing heart dysfunction in mice with STZ-induced heart failure. RNA-sequencing analysis revealed that SJ-12 exerted its therapeutic effects through the modulation of the calcium signaling pathway. Furthermore, SJ-12 reduced the O-GlcNAcylation levels of SP1 by inhibiting O-linked N-acetylglucosamine transferase (OGT). Also, SJ-12 stabilized Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2a (SERCA2a), a crucial regulator of calcium homeostasis, thus reducing hypertrophy and fibrosis in mouse hearts and cultured cardiomyocytes. However, the anti-fibrotic effects of SJ-12 were not detected in SERCA2a or OGT-silenced cardiomyocytes, indicating that SJ-12 can prevent DCM by targeting OGT-dependent O-GlcNAcylation of SP1.These findings indicate that SJ-12 can exert cardioprotective effects in STZ-induced mice by reducing the O-GlcNAcylation levels of SP1, thus stabilizing SERCA2a and reducing myocardial fibrosis and hypertrophy. Therefore, SJ-12 can be used for the treatment of diabetic cardiomyopathy.

O-GlcNAcylation regulates long-chain fatty acid metabolism by inhibiting ACOX1 ubiquitination-dependent degradation

BackgroundCold as a common environmental stress, causes increased heat production, accelerated metabolism and even affects its production performance. How to improve the adaptability of the animal organism to cold has been an urgent problem. As a key hub of lipid metabolism, the liver can regulate lipid metabolism to maintain energy balance, and O-GlcNAcylation is a kind of important PTMs, which participates in a variety of signaling and mechanism regulation, and at the same time, is very sensitive to changes in stress and nutritional levels, and is the body's “stress receptors” and “nutrient receptors”. Therefore, the aim of this experiment was to investigate the effect of cold-induced O-GlcNAcylation on hepatic lipid metabolism, and to explore the potential connection between O-GlcNAcylation and hepatic lipid metabolism. MethodsTo investigate the loss of O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the precise impacts of additional cold-induced circumstances on liver mass, shape, and metabolic profile, C57 mice were used as an animal model. Using the protein interactions approach, the mechanism of O-GlcNAcylation, as well as the degradation pathway of acyl-Coenzyme A oxidase 1 (ACOX1), were clarified. Additional in vitro analyses of oleic acid (OA) and OGT inhibitor tetraoxan (Alloxan) (Sigma, 2244-11-3) on lipid breakdown in AML-12 cells. ResultsIn C57BL/6 mice, deletion of O-GlcNAcylation disrupted lipid metabolism, caused hepatic edema and fibrosis, and altered mitochondrial apoptosis. This group of modifications was made worse by cold induction. The accumulation of medium- and long-chain fatty acids is a hallmark of lipolysis, which is accelerated by the deletion of O-GlcNAcylation, whereas lipid synthesis is slowed down. The association between ACOX1 and OGT at the K48 gene precludes ubiquitinated degradation.

Protein glycosylation in cardiovascular health and disease.

Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.

O-GlcNAc regulates anti-fibrotic genes in lung fibroblasts through EZH2.

Epigenetic modifications are involved in fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and contribute to the silencing of anti-fibrotic genes. H3K27me3, a key repressive histone mark, is catalysed by the methyltransferase enhancer of Zeste homologue 2 (EZH2), which is regulated by the post-translational modification, O-linked N-Acetylglucosamine (O-GlcNAc). In this study, we explored the effects of O-GlcNAc and EZH2 on the expression of antifibrotic genes, cyclooxygenase-2 (Cox2) and Heme Oxygenase (Homx1). The expression of Cox2 and Hmox1 was examined in primary IPF or non-IPF lung fibroblasts with or without EZH2 inhibitor EZP6438, O-GlcNAc transferase (OGT) inhibitor (OSMI-1) or O-GlcNAcase (OGA) inhibitor (thiamet G). Non-IPF cells were also subjected to TGF-β1 with or without OGT inhibition. The reduced expression of Cox2 and Hmox1 in IPF lung fibroblasts is restored by OGT inhibition. In non-IPF fibroblasts, TGF-β1 treatment reduces Cox2 and Hmox1 expression, which was restored by OGT inhibition. ChIP assays demonstrated that the association of H3K27me3 is reduced at the Cox2 and Hmox1 promoter regions following OGT or EZH2 inhibition. EZH2 levels and stability were decreased by reducing O-GlcNAc. Our study provided a novel mechanism of O-GlcNAc modification in regulating anti-fibrotic genes in lung fibroblasts and in the pathogenesis of IPF.

O-GlcNAcylation stimulates the deubiquitination activity of USP16 and regulates cell cycle progression

Histone 2A monoubiquitination (uH2A) underscores a key epigenetic regulation of gene expression. In this report, we show that the deubiquitinase for uH2A, ubiquitin-specific peptidase 16 (USP16), is modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation involves the installation of the O-GlcNAc moiety to Ser/Thr residues. It crosstalks with Ser/Thr phosphorylation, affects protein-protein interaction, alters enzyme activity or protein folding, and changes protein subcellular localization. In our study, we first confirmed that USP16 is glycosylated on Thr203 and Ser214, as reported in a previous chemoenzymatic screen. We then discovered that mutation of the O-GlcNAcylation site Thr203, which is adjacent to deubiquitination-required Cys204, reduces the deubiquitination activity toward H2AK119ub invitro and in cells, while mutation on Ser214 had the opposite effects. Using USP16 Ser552 phosphorylation-specific antibodies, we demonstrated that O-GlcNAcylation antagonizes cyclin-dependent kinase 1-mediated phosphorylation and promotes USP16 nuclear export. O-GlcNAcylation of USP16 is also required for deubiquitination of Polo-like kinase 1, a mitotic master kinase, and the subsequent chromosome segregation and cytokinesis. In summary, our study revealed that O-GlcNAcylation of USP16 at Thr203 and Ser214 coordinates deubiquitination of uH2A and Polo-like kinase 1, thus ensuring proper cell cycle progression.

Increased O-GlcNAcylation by Upregulation of Mitochondrial O-GlcNAc Transferase (mOGT) Inhibits the Activity of Respiratory Chain Complexes and Controls Cellular Bioenergetics.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. The interplay between O-GlcNAcylation and phosphorylation is critical to control signaling pathways and maintain cellular homeostasis. The addition of O-GlcNAc moieties to target proteins is catalyzed by O-linked N-acetylglucosamine transferase (OGT). Of the three splice variants of OGT described, one is destined for the mitochondria (mOGT). Although the effects of O-GlcNAcylation on the biology of normal and cancer cells are well documented, the role of mOGT remains poorly understood. In this manuscript, the effects of mOGT on mitochondrial protein phosphorylation, electron transport chain (ETC) complex activity, and the expression of VDAC porins were investigated. We performed studies using normal and breast cancer cells with upregulated mOGT or its catalytically inactive mutant. Proteomic approaches included the isolation of O-GlcNAc-modified proteins of the electron transport chain, followed by their analysis using mass spectrometry. We found that mitochondrial OGT regulates the activity of complexes I-V of the respiratory chain and identified a group of 19 ETC components as mOGT substrates in mammary cells. Furthermore, we observed that the upregulation of mOGT inhibited the interaction of VDAC1 with hexokinase II. Our results suggest that the deregulation of mOGT reprograms cellular energy metabolism via interaction with and O-GlcNAcylation of proteins involved in ATP production in mitochondria and its exchange between mitochondria and the cytosol.

Resveratrol alleviates inflammatory bowel disease by inhibiting JAK2/STAT3 pathway activity via the reduction of O-GlcNAcylation of STAT3 in intestinal epithelial cells

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) in the pathogenesis of inflammatory bowel disease (IBD) has been increasingly highlighted in recent studies. It's been reported that signal transducer and activator of transcription 3 (STAT3) O-GlcNAcylation can affect the activity of the Janus kinase2 (JAK2)/STAT3 pathway.Our recent study showed that resveratrol repairsIBDin mice.On this basis,the present study aimed to explore whether the mechanism of IBD repair by resveratrol is associated with STAT3 O-GlcNAcylation. Pretreatment of colitis mice and intestinal epithelial cells with an O-GlcNAcylation promoter (Thiamet G, or Glucosamine) and an O-GlcNAcylation inhibitor (OSMI-1) showed that increased O-GlcNAcylation promoted colitis in mice.The pro-inflammatory cytokines interleukin (IL) -6, IL-1β, and tumor necrosis factor-α (TNF-α) were increased, while the anti-inflammatory cytokine IL-10 was decreased. Moreover, the downstream target proteins of JAK2/STAT3, cyclooxygenase-2 and nitric oxide synthase 2 were up-regulated, Resveratrol treatment mitigated the inflammation by decreasing JAK2/STAT3 activity, as well as STAT3 O-GlcNAcylation. Finally, the correlation between STAT3 glycosylation and phosphorylation in intestinal epithelial cells under the effect of resveratrol was investigated by Immunofluorescence co-localization and immunoprecipitation.The results showed that resveratrol inhibited STAT3 O-GlcNAcylation, thereby inhibiting its phosphorylation, reducing JAK2/STAT3 pathway activity, and alleviating IBD.

Efficient Escorting Strategy for Aggregation-Prone Notch EGF Repeats with Sparcl1.

Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.

HSF1 Increases EOGT-Mediated Glycosylation of Notch1 to Promote IL-1β-Induced Inflammatory Injury of Chondrocytes.

Osteoarthritis (OA) is the most common arthritic disease in humans. Nevertheless, the pathogenic mechanism of OA remains unclear. This study aimed to explore that heat-shock transcription factor 1 (HSF1) facilitated interleukin-1 beta (IL-1β) chondrocyte injury by increasing Notch1 O-linked N-acetylglucosamine (O-GlcNAc) modification level. Human chondrocytes were incubated with 5 ng/ml interleukin-1 beta (IL-1β) for 24 h to establish OA cell model. The messenger RNA (mRNA) or protein expressions were assessed using reverse transcription-quantitative polymerase chain reaction, western blot, or immunofluorescence. Chondrocyte viability was examined by Cell Counting Kit-8 assay. Enzyme-linked immunosorbent assay was employed to detect the secretion levels of interleukin-6 (IL-6) and interleukin-8 (IL-8). Immunoprecipitation was adopted to detect Notch1 O-GlcNAc modification level. The interaction between HSF1 and epidermal growth factor-like (EGF) domain-specific O-GlcNAc transferase (EOGT) promoter was analyzed by dual-luciferase reporter gene and chromatin immunoprecipitation assays. Herein, our results demonstrated that HSF1, EOGT, Notch1, and Notch1 intracellular domain (NICD1) expressions in chondrocytes were markedly increased by IL-1β stimulation. EOGT elevated Notch1 expression in IL-1β-treated chondrocytes by increasing Notch1 O-GlcNAc modification level. EOGT silencing reduced IL-1β-induced chondrocyte inflammatory injury. In addition, HSF1 knockdown relieved IL-1β-induced chondrocyte inflammatory injury. Molecular interaction experiment proved that HSF1 transcriptionally activated EOGT expression in IL-1β-treated chondrocytes. HSF1 promoted IL-1β-induced inflammatory injury in chondrocytes by increasing EOGT-mediated glycosylation of Notch1.

Transcriptome analysis of Chinese mitten crabs (Eriocheir sinensis) gills in response to ammonia stress.

The Chinese mitten crab (Eriocheir sinensis) is an important commercial species in China. E. sinensis is typically farmed in rice-crab symbiosis, as an important ecological farming model. However, E. sinensis is often exposed to a high ammonia environment due to the application of nitrogen fertilizers essential for rice growth. We investigated the molecular mechanisms in the gills of E. sinensis exposed to high ammonia at transcriptional and histological levels. We randomly assigned E. sinensis to two groups (control group, CG; ammonia stress group, AG), and gill samples were excised from the CG and AG groups for histopathological and transcriptome analyses. The histopathological evaluation revealed that ammonia stress damaged the gills of E. sinensis. The transcriptome analysis showed that some essential genes, including Xanthine dehydrogenase (XDH), Ubiquitin C-terminal hydrolase-L3 (UCHL3), O-linked N-acetylglucosamine transferase (OGT), Cathepsin B (CTSB), and Ubiquitin-conjugating enzyme E2 W (UBE2W) changed significantly during ammonia exposure. These genes are related to ammonia detoxification, the immune response, and apoptosis. This study demonstrated the molecular response mechanism of E. sinensis gills to ammonia stress at the transcriptional and histological levels. This study provides insight for further study on the molecular mechanism of ammonia stress in crustaceans and supplies technical support for rice crab symbiosis.

Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress. In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps. In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH. O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.

E. coli-expressed SECRET AGENT O-GlcNAc modifies threonine 829 of GIGANTEA.

The Arabidopsis thaliana glycosyl transferases SPINDLY (SPY) and SECRET AGENT (SEC) modify nuclear and cytosolic proteins with O-linked fucose or O-linked N-acetylglucosamine (O-GlcNAc), respectively. O-fucose and O-GlcNAc modifications can occur at the same sites. SPY interacts physically and genetically with GIGANTEA (GI), suggesting that it could be modified by both enzymes. Previously, we found that, when co-expressed in Escherichia coli, SEC modifies GI; however, the modification site was not determined. By analyzing the overlapping sub-fragments of GI, we identified a region that was modified by SEC in E. coli. Modification was undetectable when threonine 829 (T829) was mutated to alanine, while the T834A and T837A mutations reduced the modification, suggesting that T829 was the primary or the only modification site. Mapping using mass spectrometry detected only the modification of T829. Previous studies have shown that the positions modified by SEC in E. coli are modified in planta, suggesting that T829 is O-GlcNAc modified in planta.

Androgen-repressed lncRNA LINC01126 drives castration-resistant prostate cancer by regulating the switch between O-GlcNAcylation and phosphorylation of androgen receptor.

Prostate cancer (PCa) initially shows satisfactory response to therapies targeting the androgen receptor (AR). However, progression to a castration-resistant stage indicates poor prognosis in PCa patients. AR signalling still plays a central role in most castration-resistant prostate cancers (CRPC). Therefore, unveiling the mechanisms of AR reactivation under androgen-deprived conditions is imperative to discover novel therapeutic targets for CRPC. Using an integrative analysis of the transcriptomics of three independent PCa cohorts and a published landscape of AR-regulated long non-coding RNA (lncRNA), lncRNA LINC01126 was selected as a candidate gene that could drive CRPC progression for further study. Quantitative reverse transcription polymerase chain reaction, in situ hybridisation (ISH) and fluorescent ISH were performed to detect LINC01126 in PCa tissues and cells. The functional role and mechanism of LINC01126 were further investigated using in vitro and in vivo gain and loss of function assays. LINC01126, identified as an AR-repressed lncRNA, was significantly upregulated after AR-targeted therapies. In addition, we found that LINC01126 was upregulated in CRPC and was associated with poor prognosis. We also proved that LINC01126 stabilised AR protein and enhanced AR nuclear translocation and transactivation by promoting the transition from O-GlcNAcylation at threonine 80 to phosphorylation at serine 81 (S81) within the AR protein. Mechanism analysis revealed that LINC01126 facilitates the interaction of CDK9 with AR and impedes the binding of O-linked N-acetylglucosamine (O-GlcNAc) transferase to AR. Consequently, LINC01126 expression was sufficient to activate AR signalling without androgen. LINC01126 overexpression increased, whereas LINC01126 knockdown decreased castration resistance traits in PCa cells in vitro and in vivo. Furthermore, our data showed that LINC01126-targeting antisense oligonucleotides (ASO) substantially inhibited CRPC cells in vitro. Our research expands the functions of AR-regulated lncRNA in sustaining androgen-independent AR activity and promoting CRPC progression and reveals that LINC01126 may be a new therapeutic target for PCa.

Structure-based virtual screening identifies small-molecule inhibitors of O-fucosyltransferase SPINDLY in Arabidopsis.

Protein O-glycosylation is a nutrient signaling mechanism that plays an essential role in maintaining cellular homeostasis across different species. In plants, SPINDLY (SPY) and SECRET AGENT (SEC) posttranslationally modify hundreds of intracellular proteins with O-fucose and O-linked N-acetylglucosamine, respectively. SPY and SEC play overlapping roles in cellular regulation, and loss of both SPY and SEC causes embryo lethality in Arabidopsis (Arabidopsis thaliana). Using structure-based virtual screening of chemical libraries followed by in vitro and in planta assays, we identified a SPY O-fucosyltransferase inhibitor (SOFTI). Computational analyses predicted that SOFTI binds to the GDP-fucose-binding pocket of SPY and competitively inhibits GDP-fucose binding. In vitro assays confirmed that SOFTI interacts with SPY and inhibits its O-fucosyltransferase activity. Docking analysis identified additional SOFTI analogs that showed stronger inhibitory activities. SOFTI treatment of Arabidopsis seedlings decreased protein O-fucosylation and elicited phenotypes similar to the spy mutants, including early seed germination, increased root hair density, and defective sugar-dependent growth. In contrast, SOFTI did not visibly affect the spy mutant. Similarly, SOFTI inhibited the sugar-dependent growth of tomato (Solanum lycopersicum) seedlings. These results demonstrate that SOFTI is a specific SPY O-fucosyltransferase inhibitor that can be used as a chemical tool for functional studies of O-fucosylation and potentially for agricultural management.

Revealing the conformational dynamics of UDP-GlcNAc recognition by O-GlcNAc transferase via Markov state model

The O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a critical post-translational modification and closely linked to various physiological and pathological conditions. The O-GlcNAc transferase (OGT) functions as the only glycosyltransferase of O-GlcNAc glycosylation by transferring GlcNAc from UDP-GlcNAc to serine or threonine residues on protein substrates. The interaction mode of UDP-GlcNAc against OGT has been preliminarily revealed by the crystal structures, yet an atomic-level comprehension for the conformational dynamics of the recognition process remains elusive. Here, we construct the Markov state model based on extensive all-atom molecular dynamics (MD) simulations with an aggregated simulation time of ∼9 μs, and reveal that the UDP-GlcNAc recognition process by OGT encompasses four key metastable states, occurring within an estimated timescale of ∼10 μs. During UDP-GlcNAc recognition process, we find the pyrophosphate moiety (P2O52−) initially anchors to the active pocket via salt bridge and hydrogen bonds, facilitating subsequent binding of the uridine and GlcNAc moieties. Furthermore, the functional roles of K842 involved in the salt bridge with P2O52− were evaluated through extra mutant MD simulations. Overall, our study provides valuable insights into the UDP-GlcNAc recognition mechanism by OGT, which could further aid in mechanistic studies of O-GlcNAc glycosylation and drug development targeting on OGT.

O-GlcNAcylation Inhibition Upregulates Connexin43 Expression in the Endothelium to Protect the Tight Junction Barrier in Diabetic Retinopathy.

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on connexin43 (Cx43) expression and its subsequent effects on tight junction properties in diabetic retinopathy (DR). O-GlcNAcylation levels in primary human retinal vascular endothelial cells (HRVECs) and retinas from rats with diabetes were regulated by treatment with Thiamet G or alloxan. Immunoprecipitation was used to examine the relationship between O-GlcNAcylation and Cx43 expression. Stable overexpression and knockdown of Cx43 in HRVECs were achieved using lentivirus constructs; further, their effects on occludin and zonula occluden-1 (ZO-1) expression and tight junction barrier function were determined. O-GlcNAcylation level increased significantly, whereas Cx43 expression decreased in retinas from rats with diabetes and HRVECs cultured under high-glucose conditions. Immunoprecipitation revealed that Cx43 was modified by O-GlcNAcylation and phosphorylation simultaneously. O-GlcNAcylation inhibition negatively regulated both total Cx43 and phosphorylated Cx43 expression, subsequently disrupting tight junction properties. Conversely, Cx43 overexpression reversed the disruption of tight junction properties and downregulated vascular endothelial growth factor expression. Consistently, Cx43 overexpression increased transendothelial electrical resistance values in HRVEC layers. O-GlcNAcylation negatively regulated Cx43 expression, contributing to the disruption of the blood retinal barrier. However, O-GlcNAcylation inhibition and Cx43 overexpression could reverse the tight junction disruption. Therefore, O-GlcNAcylation inhibition is a potential target for avoiding tight junction disruption through the Cx43 pathway in DR.