Wang Y, Zhou X, Zhang Y, Yang F, Chen Y, Wu Y, Zhang L, Tan J, Luo W, Zhou J, Yan Y.
OGT-enriched hepatocyte-derived extracellular vesicles promote capillarization of liver sinusoidal endothelial cells in metabolic dysfunction-associated steatotic liver disease.
Cellular and molecular gastroenterology and hepatology202641513003
Abstract: The global incidence of metabolic dysfunction-associated steatotic liver disease (MASLD) has risen dramatically. The capillarization of liver sinusoidal endothelial cells (LSEC) represents a crucial target for intervention in MASLD. However, the regulatory mechanisms underlying LSEC capillarization in MASLD remain unclear. Angiopoietin-2 (Ang-2) serves as a key regulator of vascularization.
Yang F, Chen Y, Zheng G, Gu K, Fan L, Li T, Zhu L, Yan Y.
LIMA1 O-GlcNAcylation Promotes Hepatic Lipid Deposition through Inducing β-catenin-Regulated FASn Expression in Metabolic Dysfunction-Associated Steatotic Liver Disease.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)202539921472
Abstract: Hepatic lipid deposition is a key factor in progressing metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigates the impact of the LIM domain and actin-binding protein 1 (LIMA1) on hepatic steatotic in MASLD and explore the underlying mechanisms. Increased levels of LIMA1 is observed in both serum and serum sEV of metabolic dysfunction-associated steatohepatitis (MASH) patients compared to healthy controls, with AUROC values of 0.76 and 0.86, respectively. Furthermore, increased LIMA1 O-GlcNAcylation is observed in mouse models of MASLD, and steatotic hepatocytes. Mechanistic studies revealed that steatosis upregulated Host cell factor 1 (HCF1) and O-GlcNAc transferase (OGT) expression, leading to catalyzed O-GlcNAcylation at the T662 site of LIMA1 and subsequent inhibition of its ubiquitin-dependent degradation. O-GlcNAcylation of LIMA1 enhances hepatocyte lipid deposition by activating β-catenin/FASn-associated signaling. Additionally, compared with their AAV8-TBG-LIMA1-WT counterparts, AAV8-TBG-LIMA1ΔT662 injection exhibited decreases in systemic insulin resistance, steatosis severity, inflammation and fibrosis in HFD-fed and CDAHFD-fed LIMA1 HKO (hepatocyte-specific knockout) mice. Moreover, LTH-sEV-mediated delivery of LIMA1 promoted MASLD progression by promoting hepatic stellate cell (HSC) activation. The findings suggest that serum sEV LIMA1 may be a potential noninvasive biomarker and therapeutic target for individuals with MASH.
Wang M, Qiao L, Jin L, Chen Y, Wen X, Wang H.
OGT-regulated O-GlcNAcylation promotes the malignancy of colorectal cancer by activating STAT2 to induce macrophage M2: OGT protein macromolecule action.
International journal of biological macromolecules2025
311(Pt 3)
40348242
Abstract: O-GlcNAcylation, as an important glycosylation modification, plays a key role in cancer progression. The purpose of this study was to investigate the role of OGT (O-GlcNAc transferase) in the regulation of O-GlcNAcylation and macrophage M2 polarization, especially in the malignant progression of cancer. The expression of OGT in COAD (colorectal adenocarcinoma) was identified by bioinformatics analysis, and its function was detected by cell culture and stimulation techniques. The OGT expression system was constructed by plasmid construction and cell transfection, and the expression changes of OGT and related genes were analyzed by RNA extraction and quantitative real-time PCR (qRT-PCR). Western blot was used to detect protein expression, transmission electron microscopy (TEM) was used to observe cell ultrastructure, and EdU staining, CCK-8 method, wound healing test, Transwell migration and invasion test were used to evaluate cell proliferation and migration. Immunofluorescence staining was used to detect intracellular markers, and the interaction between OGT and STAT2 was analyzed by co-immunoprecipitation (Co-IP) and ubiquitination experiments. Finally, statistical analysis was performed to evaluate the significance of the experimental results. We found that OGT is highly expressed in COAD and promotes M2-type polarization of macrophages through exosomes derived from colorectal cancer. In M2-type tumor-associated macrophages (TAMs), the synergistic effect of OGT and the deubiquitination enzyme USP18 promoted the deubiquitination of STAT2, thereby enhancing the degree of M2 polarization.
Zhou Y, Xu M, Yu P, Wang W, Cheng L, Jiao X, Zhang Q, Xiong H, Yu P, Chen Y, Xu F, Shen Y.
Empagliflozin Downregulates AMP-Activated Protein Kinaseα O-GlcNAcylation to Ameliorate Hepatic Steatosis.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology2025
39(20)
41122918
Abstract: The efficacy of the SGLT2 inhibitor empagliflozin (EMPA) in mitigating hepatic steatosis in patients with type 2 diabetes mellitus and metabolic dysfunction-associated steatotic liver disease (MASLD) has been previously demonstrated. However, the underlying mechanisms remain unclear. In this study, we investigated the role of EMPA in alleviating hepatic steatosis through the modulation of O-GlcNAcylation. High-glucose (HG)-induced alpha mouse liver 12 (AML12) cells, mouse primary hepatocytes (MPHs), and murine MASLD models (high-fat diet-fed and ob/ob mice) were used to examine the effects of EMPA. Protein O-GlcNAcylation, lipid accumulation, and AMP-activated protein kinase α (AMPKα) regulation were evaluated using Western blotting, immunostaining, and siRNA knockdown. Our findings showed that protein O-GlcNAcylation levels were elevated in both in vitro and in vivo models. EMPA treatment reduced O-GlcNAcylation and ameliorated lipid accumulation in HG-induced AML12 cells, MPHs, and MASLD models. Knockdown of O-GlcNAc transferase (OGT) decreased O-GlcNAcylation levels and lipid accumulation in HG-induced AML12 cells. Additionally, OGT knockdown altered both O-GlcNAcylated and phosphorylated AMPKα levels. In these models, EMPA administration decreased O-GlcNAcylated AMPKα while increasing phosphorylated AMPKα. This study further identified serine 344, threonine 447, and serine 501 as critical O-GlcNAcylation sites on AMPKα2. Mutation of these residues to alanine in AMPKα2 attenuated lipid accumulation in AML12 cells, with no additional improvement observed following EMPA treatment. In summary, EMPA effectively improves hepatic steatosis by modulating the O-GlcNAcylation states of AMPKα. Identification of specific O-GlcNAcylation sites on AMPKα2 highlights their importance in the therapeutic mechanism of EMPA in improving hepatic steatosis.
Li T, Sun W, Zhu S, He C, Chang T, Zhang J, Chen Y.
T-2 Toxin-Mediated β-Arrestin-1 O-GlcNAcylation Exacerbates Glomerular Podocyte Injury via Regulating Histone Acetylation.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)2024
11(7)
38083975
Abstract: T-2 toxin causes renal dysfunction with proteinuria and glomerular podocyte damage. This work explores the role of metabolic disorder/reprogramming-mediated epigenetic modification in the progression of T-2 toxin-stimulated podocyte injury. A metabolomics experiment is performed to assess metabolic responses to T-2 toxin infection in human podocytes. Roles of protein O-linked-N-acetylglucosaminylation (O-GlcNAcylation) in regulating T-2 toxin-stimulated podocyte injury in mouse and podocyte models are assessed. O-GlcNAc target proteins are recognized by mass spectrometry and co-immunoprecipitation experiments. Moreover, histone acetylation and autophagy levels are measured. T-2 toxin infection upregulates glucose transporter type 1 (GLUT1) expression and enhances hexosamine biosynthetic pathway in glomerular podocytes, resulting in a significant increase in β-arrestin-1 O-GlcNAcylation. Decreasing β-arrestin-1 or O-GlcNAc transferase (OGT) effectively prevents T-2 toxin-induced renal dysfunction and podocyte injury. Mechanistically, O-GlcNAcylation of β-arrestin-1 stabilizes β-arrestin-1 to activate the mammalian target of rapamycin (mTOR) pathway as well as to inhibit autophagy during podocyte injury by promoting H4K16 acetylation. To sum up, OGT-mediated β-arrestin-1 O-GlcNAcylation is a vital regulator in the development of T-2 toxin-stimulated podocyte injury via activating the mTOR pathway to suppress autophagy. Targeting β-arrestin-1 or OGT can be a potential therapy for T-2 toxin infection-associated glomerular injury, especially podocyte injury.
Li Y, Yang Z, Chen J, Chen Y, Jiang C, Zhong T, Su Y, Liang Y, Sun H.
OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli.
Molecules (Basel, Switzerland)2023
28(5)
36903375
Abstract: O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.
Yan S, Peng B, Kan S, Shao G, Xiahou Z, Tang X, Chen YX, Dong MQ, Liu X, Xu X, Li J.
Polo-like kinase 1 (PLK1) O-GlcNAcylation is essential for dividing mammalian cells and inhibits uterine carcinoma.
The Journal of biological chemistry2023
299(2)
36626982
Abstract: The O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates intracellular O-GlcNAcylation modification. O-GlcNAcylation occurs on Ser/Thr residues and is important for numerous physiological processes. OGT is essential for dividing mammalian cells and is involved in many human diseases; however, many of its fundamental substrates during cell division remain unknown. Here, we focus on the effect of OGT on polo-like kinase 1 (PLK1), a mitotic master kinase that governs DNA replication, mitotic entry, chromosome segregation, and mitotic exit. We show that PLK1 interacts with OGT and is O-GlcNAcylated. By utilizing stepped collisional energy/higher-energy collisional dissociation mass spectrometry, we found a peptide fragment of PLK1 that is modified by O-GlcNAc. Further mutation analysis of PLK1 shows that the T291A mutant decreases O-GlcNAcylation. Interestingly, T291N is a uterine carcinoma mutant in The Cancer Genome Atlas. Our biochemical assays demonstrate that T291A and T291N both increase PLK1 stability. Using stable H2B-GFP cells, we found that PLK1-T291A and PLK1-T291N mutants display chromosome segregation defects and result in misaligned and lagging chromosomes. In mouse xenograft models, we demonstrate that the O-GlcNAc-deficient PLK1-T291A and PLK1-T291N mutants enhance uterine carcinoma in animals. Hence, we propose that OGT partially exerts its mitotic function through O-GlcNAcylation of PLK1, which might be one mechanism by which elevated levels of O-GlcNAc promote tumorigenesis.
Cai M, Chen Y, Lin Y, Hu Z, Li L, Huang H, Lin J.
SIRT1 Asn346 sugar chain promoting collagen deacetylation protective effect on osteoblasts under stress.
Biochemical and biophysical research communications2023
682
37806254
Abstract: Silencing type information regulator homolog 1 (SIRT1) is a class of nicotinamide adenine dinucleotide (NAD+) dependent deacetylases, which is the convergence point of important physiological processes in vivo, namely, osteoblast aging, energy metabolism, and bone remodeling. To verify whether the O-acetylglucosamine (O-GlcNAc) modification of SIRT1 in the nucleus of osteoblasts enhances its deacetylase activity under stress and protects osteoblasts through the RANK/RANKL signaling pathway by collagen deacetylation. The R language and online data research identified SIRT1 as being involved in bone metabolism. Enrichment analysis showed that SIRT1 is involved in osteoblast transcription, apoptosis, and deacetylation pathways. Interactive Immuno-blotting and immunofluorescence experiments revealed that SIRT1 and O-glycosylation catalytic enzyme (OGT) were localized in the nucleus. Mass Spectrometry analysis showed that O-glycosylation occurred on the asparagine at the 346th position of SIRT1, and N346th was located in the central domain of SIRT1. Furthermore, the protein structure analysis of PyMol also proved that the OGT binding region was in the central domain of SIRT1. Under physiological conditions, both wtSIRT1 and SIRT1N346R can inhibit RANKL-mediated transcriptional activation. The RT-PCR detection results showed that wtSIRT1 reduced RANKL transcription under the conditions of apoptotic agent treatment. The finding that SIRT1 can regulate the physiological process of bone remodeling through the RANK/RANKL signaling pathway in osteoblasts under stress. The O-glycosylation and deacetylation activity of SIRT1 significantly increased, regulating the balance between osteoblast survival and apoptosis by deacetylation of key proteins such as RANKL.
Chen Y, Wan R, Zou Z, Lao L, Shao G, Zheng Y, Tang L, Yuan Y, Ge Y, He C, Lin S.
O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins.
Nature cell biology2023
25(11)
37945829
Abstract: N6-methyladenosine (m6A) is the most abundant internal mRNA nucleotide modification in mammals, regulating critical aspects of cell physiology and differentiation. The YTHDF proteins are the primary readers of m6A modifications and exert physiological functions of m6A in the cytosol. Elucidating the regulatory mechanisms of YTHDF proteins is critical to understanding m6A biology. Here we report a mechanism that protein post-translational modifications control the biological functions of the YTHDF proteins. We find that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications. O-GlcNAcylation attenuates the translation-promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. We further demonstrate that O-GlcNAc modifications on YTHDF1 and YTHDF3 regulate the assembly, stability and disassembly of stress granules to enable better recovery from stress. Therefore, our results discover an important regulatory pathway of YTHDF functions, adding an additional layer of complexity to the post-transcriptional regulation function of mRNA m6A.
Chen Y, Tang F, Qin H, Yue X, Nie Y, Huang W, Ye M.
Endo-M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O-GlcNAcylation Analysis.
Angewandte Chemie (International ed. in English)2022
61(23)
35289036
Abstract: To selectively enrich O-linked β-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.
Chen YF, Zhu JJ, Li J, Ye XS.
O-GlcNAcylation increases PYGL activity by promoting phosphorylation.
Glycobiology2022
32(2)
34939084
Abstract: O-GlcNAcylation is a post-translational modification that links metabolism with signal transduction. High O-GlcNAcylation appears to be a general characteristic of cancer cells. It promotes the invasion, metastasis, proliferation and survival of tumor cells, and alters many metabolic pathways. Glycogen metabolism increases in a wide variety of tumors, suggesting that it is an important aspect of cancer pathophysiology. Herein we focused on the O-GlcNAcylation of liver glycogen phosphorylase (PYGL)-an important catabolism enzyme in the glycogen metabolism pathway. PYGL expressed in both HEK 293T and HCT116 was modified by O-GlcNAc. And both PYGL O-GlcNAcylation and phosphorylation of Ser15 (pSer15) were decreased under glucose and insulin, whereas increased under glucagon and Na2S2O4 (hypoxia) conditions. Then, we identified the major O-GlcNAcylation site to be Ser430, and demonstrated that pSer15 and Ser430 O-GlcNAcylation were mutually reinforced. Lastly, we found that Ser430 O-GlcNAcylation was fundamental for PYGL activity. Thus, O-GlcNAcylation of PYGL positively regulated pSer15 and therefore its enzymatic activity. Our results provided another molecular insight into the intricate post-translational regulation network of PYGL.
Chen Y, Qin H, Yue X, Zhou J, Liu L, Nie Y, Ye M.
Highly Efficient Enrichment of O-GlcNAc Glycopeptides Based on Chemical Oxidation and Reversible Hydrazide Chemistry.
Analytical chemistry2021
93(49)
34846842
Abstract: Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups. The labeled O-GlcNAc glycopeptides could be efficiently enriched based on the equilibrium between the hydrazine and oxime bonds. Good specificity of the glycopeptide enrichment was observed from the mixtures of glycopeptide and non-glycopeptides using the CHO-GlcNAc method. Then, it was applied to analyze O-GlcNAcylation in the nucleus of HeLa cells, and 829 potential O-GlcNAcylation sites on 274 glycoproteins were identified, including the two readers of m6A (YTHDF1 and YTHDF3), which could provide clues for the mechanism of crosstalk between O-GlcNAcylation and other PTMs of proteins and RNA. Thus, this method could be a versatile tool for the proteomic analysis of O-GlcNAcylation.
Hu J, Gao Q, Yang Y, Xia J, Zhang W, Chen Y, Zhou Z, Chang L, Hu Y, Zhou H, Liang L, Li X, Long Q, Wang K, Huang A, Tang N.
Hexosamine biosynthetic pathway promotes the antiviral activity of SAMHD1 by enhancing O-GlcNAc transferase-mediated protein O-GlcNAcylation.
Theranostics2021
11(2)
33391506
Abstract: Rationale: Viruses hijack the host cell machinery to promote viral replication; however, the mechanism by which metabolic reprogramming regulates innate antiviral immunity in the host remains elusive. Herein, we explore how the hexosamine biosynthesis pathway (HBP) and O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulate host antiviral response against hepatitis B virus (HBV) in vitro and in vivo.Methods: We conducted a metabolomics assay to evaluate metabolic responses of host cells to HBV infection. We systematically explored the role of HBP and protein O-GlcNAcylation in regulating HBV infection in cell and mouse models. O-linked N-acetylglucosamine (O-GlcNAc) target proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation assays. Additionally, we also examined uridine diphosphate (UDP)-GlcNAc biosynthesis and O-GlcNAcylation levels in patients with chronic hepatitis B (CHB). Results: HBV infection upregulated GLUT1 expression on the hepatocyte surface and facilitated glucose uptake, which provides substrates to HBP to synthesize UDP-GlcNAc, leading to an increase in protein O-GlcNAcylation. Pharmacological or transcriptional inhibition of HBP and O-GlcNAcylation promoted HBV replication. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) on Ser93 stabilizes SAMHD1 and enhances its antiviral activity. Analysis of clinical samples revealed that UDP-GlcNAc level was increased, and SAMHD1 was O-GlcNAcylated in patients with CHB. Conclusions: HBP-mediated O-GlcNAcylation positively regulates host antiviral response against HBV in vitro and in vivo. The findings reveal a link between HBP, O-GlcNAc modification, and innate antiviral immunity by targeting SAMHD1.
Tan W, Jiang P, Zhang W, Hu Z, Lin S, Chen L, Li Y, Peng C, Li Z, Sun A, Chen Y, Zhu W, Xue Y, Yao Y, Li X, Song Q, He F, Qin W, Pei H.
Posttranscriptional regulation of de novo lipogenesis by glucose-induced O-GlcNAcylation.
Molecular cell2021
81(9)
33657401
Abstract: O-linked β-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.
Wu ZL, Luo A, Grill A, Lao T, Zou Y, Chen Y.
Fluorescent Detection of O-GlcNAc via Tandem Glycan Labeling.
Bioconjugate chemistry2020
31(9)
32830957
Abstract: O-GlcNAcylation is a reversible serine/threonine glycosylation on cytosolic and nuclear proteins that are involved in various regulatory pathways. However, the detection and quantification of O-GlcNAcylation substrates have been challenging. Here, we report a highly efficient method for the identification of O-GlcNAc modification via tandem glycan labeling, in which O-GlcNAc is first galactosylated and then sialylated with a fluorophore-conjugated sialic acid residue, therefore enabling highly sensitive fluorescent detection. The method is validated on various proteins that are known to be modified by O-GlcNAcylation including CK2, NOD2, SREBP1c, AKT1, PKM, and PFKFB3, and on the nuclear extract of HEK293 cells. Using this method, we then report the evidence that hypoxia-inducible factor HIF1α is a potential target for O-GlcNAcylation, suggesting a possibly direct connection between the metabolic O-GlcNAc pathway and the hypoxia pathway.
Wong KKL, Liu TW, Parker JM, Sinclair DAR, Chen YY, Khoo KH, Vocadlo DJ, Verheyen EM.
The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila.
Proceedings of the National Academy of Sciences of the United States of America2020
117(4)
31932432
Abstract: Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.
Yang SZ, Xu F, Yuan K, Sun Y, Zhou T, Zhao X, McDonald JM, Chen Y.
Regulation of pancreatic cancer TRAIL resistance by protein O-GlcNAcylation.
Laboratory investigation; a journal of technical methods and pathology2020
100(5)
31896813
Abstract: TRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling.
Zhu G, Qian M, Lu L, Chen Y, Zhang X, Wu Q, Liu Y, Bian Z, Yang Y, Guo S, Wang J, Pan Q, Sun F.
O-GlcNAcylation of YY1 stimulates tumorigenesis in colorectal cancer cells by targeting SLC22A15 and AANAT.
Carcinogenesis2019
40(9)
30715269
Abstract: Emerging studies have revealed that O-GlcNAcylation plays pivotal roles in the tumorigenesis of colorectal cancers (CRCs). However, the underlying mechanism still remains largely unknown. Here, we demonstrated that Yin Yang 1 (YY1) was O-GlcNAcylated by O-GlcNAc transferase (OGT) and O-GlcNAcylation of YY1 could increase the protein expression by enhancing its stability. O-GlcNAcylation facilitated transformative phenotypes of CRC cell in a YY1-dependent manner. Also, O-GlcNAcylation stimulates YY1-dependent transcriptional activity. Besides, we also identified the oncoproteins, SLC22A15 and AANAT, which were regulated by YY1 directly, are responsible for the YY1 stimulated tumorigenesis. Furthermore, we identified the main putative O-GlcNAc site of YY1 at Thr236, and mutating of this site decreased the pro-tumorigenic capacities of YY1. We concluded that O-GlcNAcylation of YY1 stimulates tumorigenesis in CRC cells by targeting SLC22A15 and AANAT, suggesting that YY1 O-GlcNAcylation might be a potential effective therapeutic target for treating CRC.
Hu CM, Tien SC, Hsieh PK, Jeng YM, Chang MC, Chang YT, Chen YJ, Chen YJ, Lee EYP, Lee WH.
High Glucose Triggers Nucleotide Imbalance through O-GlcNAcylation of Key Enzymes and Induces KRAS Mutation in Pancreatic Cells.
Cell metabolism2019
29(6)
30853214
Abstract: KRAS mutations are the earliest events found in approximately 90% of pancreatic ductal adenocarcinomas (PDACs). However, little is known as to why KRAS mutations preferentially occur in PDACs and what processes/factors generate these mutations. While abnormal carbohydrate metabolism is associated with a high risk of pancreatic cancer, it remains elusive whether a direct relationship between KRAS mutations and sugar metabolism exists. Here, we show that under high-glucose conditions, cellular O-GlcNAcylation is significantly elevated in pancreatic cells that exhibit lower phosphofructokinase (PFK) activity than other cell types. This post-translational modification specifically compromises the ribonucleotide reductase (RNR) activity, leading to deficiency in dNTP pools, genomic DNA alterations with KRAS mutations, and cellular transformation. These results establish a mechanistic link between a perturbed sugar metabolism and genomic instability that induces de novo oncogenic KRAS mutations preferentially in pancreatic cells.
Chen Y, Zhu G, Liu Y, Wu Q, Zhang X, Bian Z, Zhang Y, Pan Q, Sun F.
O-GlcNAcylated c-Jun antagonizes ferroptosis via inhibiting GSH synthesis in liver cancer.
Cellular signalling2019
63
31394193
Abstract: Ferroptosis is a metabolism-related cell death. Stimulating ferroptosis in liver cancer cells is a strategy to treat liver cancer. However, how to eradicate liver cancer cells through ferroptosis and the obstacles to inducing ferroptosis in liver cancer remain unclear. Here, we observed that erastin suppressed the malignant phenotypes of liver cancer cells by inhibiting O-GlcNAcylation of c-Jun and further inhibited protein expression, transcription activity and nuclear accumulation of c-Jun. Overexpression of c-Jun-WT with simultaneous PuGNAc treatment conversely inhibited erastin-induced ferroptosis, whereas overexpression of c-Jun-WT alone or overexpression of c-Jun-S73A (a non-O-GlcNAcylated form of c-Jun) with PuGNAc treatment did not exert a similar effect. GSH downregulation induced by erastin was restored by overexpression of c-Jun-WT with simultaneous PuGNAc treatment. In addition, overexpression of c-Jun-WT, but not its S73A mutant, induced PSAT1 and CBS transcription via directly binding to their promoter regions, suggesting that GSH synthesis is regulated by O-GlcNAcylated c-Jun. A positive correlation between c-Jun O-GlcNAcylation and GSH was observed in clinical samples. Collectively, O-GlcNAcylated c-Jun represents an obstructive factor to ferroptosis, and targeting O-GlcNAcylated c-Jun might be helpful for treating liver cancer.
Yao D, Xu L, Xu O, Li R, Chen M, Shen H, Zhu H, Zhang F, Yao D, Chen YF, Oparil S, Zhang Z, Gong K.
O-Linked β-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.
Arteriosclerosis, thrombosis, and vascular biology2018
38(6)
29622561
Abstract: Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection.
Chen Y, Liu R, Chu Z, Le B, Zeng H, Zhang X, Wu Q, Zhu G, Chen Y, Liu Y, Sun F, Lu Z, Qiao Y, Wang J.
High glucose stimulates proliferative capacity of liver cancer cells possibly via O-GlcNAcylation-dependent transcriptional regulation of GJC1.
Journal of cellular physiology2018
234(1)
30078215
Abstract: Although it is generally accepted that diabetes is one of the most important risk factors for liver cancer, the underlying mechanism is still not well understood. The purpose of the current study is to further investigate how high concentrations of glucose (HG), a major symptom of diabetes, stimulate the development of liver malignancy. Using data mining, gap junction protein gamma 1 (GJC1) was identified as a critical proto-oncoprotein that is essential for the HG stimulation of proliferative capacity in liver cancer cells. Furthermore, enhanced transcriptional expression of GJC1 might occur after stimulation by HG. A transcription factor zinc finger protein 410 (APA1)-binding motif was found to be located at the -82 to -77 nt region within the GJC1 promoter. Without APA1, HG was unable to increase GJC1 expression. Interestingly, APA1, but not GJC1, can be O-GlcNAcylated in liver cancer cells. Moreover, O-GlcNAcylation is essential for HG-induced APA1 binding to the GJC1 promoter. Notably, global O-GlcNAcylation and expression of APA1 and GJC1 were highly elevated in liver cancer patients with diabetes compared to those in patients without diabetes. The HG-stimulated proliferative capacity was abolished upon decreasing O-GlcNAcylation, which could be reversed gradually by the simultaneous overexpression of APA1 and GJC1. Therefore, GJC1 could be a potential target for preventing liver cancer in patients with diabetes.
Liu B, Wang J, Li M, Yuan Q, Xue M, Xu F, Chen Y.
Inhibition of ALDH2 by O-GlcNAcylation contributes to the hyperglycemic exacerbation of myocardial ischemia/reperfusion injury.
Oncotarget2017
8(12)
28038474
Abstract: Although hyperglycemia is causally related to adverse outcomes after myocardial ischemia/reperfusion (I/R), the underlying mechanisms are largely unknown. Here, we investigated whether excessive O-linked-N-acetylglucosamine (O-GlcNAc) modification of acetaldehyde dehydrogenase 2 (ALDH2), an important cardioprotective enzyme, was a mechanism for the hyperglycemic exacerbation of myocardial I/R injury. Both acute hyperglycemia (AHG) and diabetes (DM)-induced chronic hyperglycemia increased cardiac dysfunction, infarct size and apoptosis index compared with normal saline (NS)+I/R rats (P<0.05). ALDH2 O-GlcNAc modification was increased whereas its activity was decreased in AHG+I/R and DM+I/R rats. High glucose (HG, 30mmol/L) markedly increased ALDH2 O-GlcNAc modification compared with Con group (5mmol/L) (P<0.05). ALDH2 O-GlcNAc modification was increased by 62.9% in Con+PUGNAc group whereas it was decreased by 44.1% in Con+DON group compared with Con group (P<0.05). Accordingly, ALDH2 activity was decreased by 18.1% in Con+PUGNAc group whereas it was increased by 17.9% in Con+DON group. Moreover, DON decreased levels of 4-hydroxy-2-nonenal (4-HNE), aldehydes, protein carbonyl accumulation and apoptosis index compared with HG+H/R group (P<0.05). Alda-1, a specific activator of ALDH2, significantly decreased ALDH2 O-GlcNAc modification and improved infarct size, apoptosis index and cardiac dysfunction induced by I/R combined with hyperglycemia. These findings demonstrate that ALDH2 O-GlcNAc modification is a key mechanism for the hyperglycemic exacerbation of myocardial I/R injury and Alda-1 has therapeutic potential for inducing cardioprotection.
Chen Y, Jin L, Xue B, Jin D, Sun F, Wen C.
NRAGE induces β-catenin/Arm O-GlcNAcylation and negatively regulates Wnt signaling.
Biochemical and biophysical research communications2017
487(2)
28427939
Abstract: The Wnt pathway is crucial for animal development, as well as tumor formation. Understanding the regulation of Wnt signaling will help to elucidate the mechanism of the cell cycle, cell differentiation and tumorigenesis. It is generally accepted that in response to Wnt signals, β-catenin accumulates in the cytoplasm and is imported into the nucleus where it recruits LEF/TCF transcription factors to activate the expression of target genes. In this study, we report that human NRAGE, a neurotrophin receptor p75 (p75NTR) binding protein, markedly suppresses the expression of genes activated by the Wnt pathway. Consistent with this finding, loss of function of NRAGE by RNA interference (RNAi) activates the Wnt pathway. Moreover, NRAGE suppresses the induction of axis duplication by microinjected β-catenin in Xenopus embryos. To our surprise, NRAGE induces nuclear localization of β-catenin and increases its DNA binding ability. Further studies reveal that NRAGE leads to the modification of β-catenin/Arm with O-linked beta-N-acetylglucosamine (O-GlcNAc), and failure of the association between β-catenin/Arm and pygopus(pygo) protein, which is required for transcriptional activation of Wnt target genes. Therefore, our findings suggest a novel mechanism for regulating Wnt signaling.
Qin W, Lv P, Fan X, Quan B, Zhu Y, Qin K, Chen Y, Wang C, Chen X.
Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis.
Proceedings of the National Academy of Sciences of the United States of America2017
114(33)
28760965
Abstract: O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2'-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo.