BACKGROUND Periodontitis,when exacerbated by diabetes,is characterized by increased M1 macrophage polarization and decreased M2 polarization.O-linkedβ-N-acetylglucosamine(O-GlcNAcylation),catalyzed by O-GlcNAc transf...BACKGROUND Periodontitis,when exacerbated by diabetes,is characterized by increased M1 macrophage polarization and decreased M2 polarization.O-linkedβ-N-acetylglucosamine(O-GlcNAcylation),catalyzed by O-GlcNAc transferase(OGT),promotes inflammatory responses in diabetic periodontitis(DP).Additionally,p38 mitogen-activated protein kinase regulates macrophage polarization.However,the interplay between OGT,macrophage polarization,and p38 signaling in the progression of DP remains unexplored.AIM To investigate the effect of OGT on macrophage polarization in DP and its role in mediating O-GlcNAcylation of p38.METHODS For in vivo experiments,mice were divided into four groups:Control,DP model,model+short hairpin(sh)RNAnegative control,and model+sh-OGT.Diabetes was induced by streptozotocin,followed by ligation and lipopolysaccharide(LPS)administration to induce periodontitis.The impact of OGT was assessed by injecting sh-OGT lentivirus.Maxillary bone destruction was evaluated using micro-computed tomography analysis and tartrateresistant acid phosphatase staining,while macrophage polarization was determined through quantitative real-time polymerase chain reaction(qPCR)and immunohistochemistry.For in vitro experiments,RAW264.7 cells were treated with LPS and high glucose(HG)(25 mmol/L D-glucose)to establish a cell model of DP.OGT was inhibited by OGT inhibitor(OSMI4)treatment and knocked down by sh-OGT transfection.M1/M2 polarization was analyzed using qPCR,immunofluorescence,and flow cytometry.Levels of O-GlcNAcylation were measured using immunoprecipitation and western blotting.RESULTS Our results demonstrated that M1 macrophage polarization led to maxillary bone loss in DP mice,associated with elevated O-GlcNAcylation and OGT levels.Knockdown of OGT promoted the shift from M1 to M2 macrophage polarization in both mouse periodontal tissues and LPS+HG-induced RAW264.7 cells.Furthermore,LPS+HG enhanced the O-GlcNAcylation of p38 in RAW264.7 cells.OGT interacted with p38 to promote its O-GlcNAcylation at residues A28,T241,and T347,as well as its phosphorylation at residue Y221.CONCLUSION Inhibition of OGT-mediated p38 O-GlcNAcylation deactivates the p38 pathway by suppressing its self-phosphorylation,thereby promoting M1 to M2 macrophage polarization and mitigating DP.These findings suggested that modulating macrophage polarization through regulation of O-GlcNAcylation may represent a novel therapeutic strategy for treating DP.展开更多
The aim of this present study was to explore the expression and clinical significance of O-linked N-acetylglucosamine（O-GlcNAc） transferase（OGT） and enzymatic O-linked glycosylation（O-GlcNAcation） through the ad...The aim of this present study was to explore the expression and clinical significance of O-linked N-acetylglucosamine（O-GlcNAc） transferase（OGT） and enzymatic O-linked glycosylation（O-GlcNAcation） through the addition of O-linked-β-N-acetylglucosamine in esophageal squamous cell carcinoma.OGT expression and O-GlcNAcation in 40 samples from patients with esophageal squamous cell carcinoma was detected by immunohistochemical staining with anti-OGT antib ody and O-GlcNAc-specific antibody RL 2,respectively.The relationship between pathological and clinical factors of patients was analyzed.We found that the expression of OGT was higher in esophageal squamous cell carcinoma samples compared to the normal tissues.RL 2 antibody level was positively correlated with OGT expression,and the metastasis of lymph node,which means the level of O-GlcNAcation was high and related to the metastasis of lymph node in esophageal squamous cell carcinoma.In conclusion,OGT activation is the main reason for promoting the level of O-GlcNAcation in esophageal squamous cell carcinoma.O-GlcNAcylation may play an important role in esophageal squamous cell carcinoma.展开更多
The O-linked β-N-acetylglucosamine(O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylat...The O-linked β-N-acetylglucosamine(O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase(OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine(EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.展开更多
Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insu...Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.展开更多
The title compound, spiro[ 1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]- hexane-2,2＇-3＇-（16＇-methoxyacetatyl-4＇-l-menthyloxybutyrolactone）] 1, was obtained via tandem asymmetric double Michael addition/inte...The title compound, spiro[ 1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]- hexane-2,2＇-3＇-（16＇-methoxyacetatyl-4＇-l-menthyloxybutyrolactone）] 1, was obtained via tandem asymmetric double Michael addition/internal nucleophilic substitution of the chiral synthon, 5-l-menthyloxy-3-bromo-2-（5H）-furanone 2 with methoxy α-chloroacetate as a nucleophile under mild conditions, and structurally [letermined by single-crystal X-ray diffraction. Crystal data： C31H47BrO9, Mr = 643.60, orthorhombic, space group P212121. α = 9.6564（7）, b = 14.8994（11）, c = 23.6771（17） A, V= 3406.5（4） A^3, Z = 4, Dc= 1.255 g/cm^3, 2（MoKa） = 0.71073 A,μ= 1.254 mm^-1 and F（000） = 1360. The structure was refined to R =[0.0324 and wR = 0.0737 for 5123 observed reflections （I〉 2σ（I））. The crystallographic results of molecule 1 show that the interesting reaction of 2 with methoxy α-chloroacetate, in the usual manner, gave the spiro-cyclopropane skeleton with O-linked derivative containing multiple stereogenic centers 1 rather than the expected C-linked derivative.展开更多
The title compound, spiro[1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]-hexane-2,2-3-(16-methoxyacetatyl-4-l-menthyloxybutyrolactone)] 1, was obtained via tandem asymmetric double Michael addition/internal nucleop...The title compound, spiro[1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]-hexane-2,2-3-(16-methoxyacetatyl-4-l-menthyloxybutyrolactone)] 1, was obtained via tandem asymmetric double Michael addition/internal nucleophilic substitution of the chiral synthon, 5-l-menthyloxy-3-bromo-2-(5H)-furanone 2 with methoxy α-chloroacetate as a nucleophile under mild conditions, and structurally determined by single-crystal X-ray diffraction. Crystal data: C31H47BrO9, Mr = 643.60, orthorhombic, space group P212121, a = 9.6564(7), b = 14.8994(11), c = 23.6771(17) , V = 3406.5(4) 3, Z = 4, Dc = 1.255 g/cm3, λ(MoKα) = 0.71073 , μ = 1.254 mm-1 and F(000) = 1360. The structure was refined to R = 0.0324 and wR = 0.0737 for 5123 observed reflections (I > 2σ(I)). The crystallographic results of molecule 1 show that the interesting reaction of 2 with methoxy α-chloroacetate, in the usual manner, gave the spiro-cyclopropane skeleton with O-linked derivative containing multiple stereogenic centers 1 rather than the expected C-linked derivative.展开更多
To investigate the modification on the mouse zona pellucida by ethanol activation,the zona pellucida glycoproteins of ethanol-activated eggs was subjectedto SDS-PAGE and HPLC.The SDS-PAGE of zona pellucida samples sho...To investigate the modification on the mouse zona pellucida by ethanol activation,the zona pellucida glycoproteins of ethanol-activated eggs was subjectedto SDS-PAGE and HPLC.The SDS-PAGE of zona pellucida samples showed that the migration profiles of ZP glycoproteins of ethanol-activated eggs and oocytes were similar,except an additional band approximately 20kD was separated from the sample of ethanol-activated eggs under reducing condition.No evident difference between ZP3 of ethanol-activated eggs and unfertilized oo-cytes was observed.HPLC results showed that the O-linked sugars from the sample of etha-nol-activated eggs had three peaks of monosaccharides,which were quite similar to that of the unfertilized oocytes,suggesting both samples had the same three O-linked monosaccharides.However,another two peaks of oligosaccharides were recorded from the sample of ethanol-ac-tivated cgg,with RT values of 12.09 and 8.89.Only another peak with RTs of 9.14 was re-corded from the sample of unfertilized oocytes.Therefore,the ethanol activation had not sig-nificant effect on the ZP3 polypeptide and on the O-linked monosaccharides.The modification by ethanol activation was mainly on the O-linked oligosaccharide chains.展开更多
Investigations from the last four decades have correlated high O-linked N-acetylglucosamine(O-Glc NAc)levels with various cancer types,but it is not known how OGT responds to diverse nutrients to finetune cellular O-G...Investigations from the last four decades have correlated high O-linked N-acetylglucosamine(O-Glc NAc)levels with various cancer types,but it is not known how OGT responds to diverse nutrients to finetune cellular O-Glc NAcylation levels.Herein we identified a critical OGT phosphorylation site by unc-51 like autophagy activating kinase 1(ULK1)under glucose depletion.First,we demonstrated that glucose levels modulate the interaction between OGT and ULK1 and cellular O-Glc NAcylation levels.Low glucose induces high O-Glc NAcylation,which could be reversed by ULK1 inhibition.Then,using mass spectrometry,we showed that ULK1 phosphorylates OGT at Ser576 and stabilizes OGT.Further biochemical experiments revealed that Ser576 phosphorylation inhibits Lys604 ubiquitination by stimulating OGT binding with BAP1,a de-ubiquitinase for OGT.Strikingly,using the OGT S576A knock-in cells,we found that in mouse xenograft models OGTS576A completely abolishes the tumorigenicity of OGT,probably due to low O-Glc NAcylation.In sum,we found that ULK1 phosphorylates OGT at Ser-576 under glucose deprivation,which stabilizes OGT by promoting OGT-BAP1 association and is pivotal for O-Glc NAcylation levels and tumorigenesis.As low glucose is often associated with tumor progression,our work not only unearths a key mechanism of how OGT is regulated by glucose levels,but also offers new therapeutic opportunities targeting OGT.展开更多
The intracellular O-linked N-acetylglucosamine(O-GlcNAc)glycosylation mediates many signal transduction events and regulates tumorigenesis.Previously the RNA N6-methyladenosine(m6 A)reader,YTH(YT521-B homology)domain ...The intracellular O-linked N-acetylglucosamine(O-GlcNAc)glycosylation mediates many signal transduction events and regulates tumorigenesis.Previously the RNA N6-methyladenosine(m6 A)reader,YTH(YT521-B homology)domain 2(YTHDF2),has been shown to be O-GlcNAcylated on Ser-263 during Hepatitis B virus(HBV)infection and promote HBV-related hepatocellular carcinoma.Herein we mapped YTHDF2 O-GlcNAcylation at Thr-49 via electron-transfer dissociation mass spectrometry under unperturbed conditions.We show that YTHDF2 Thr-49 O-GlcNAcylation antagonizes Extracellular-signal regulated kinase(ERK)-dependent phosphorylation at Ser-39 and promotes YTHDF2 degradation.The downstream signaling pathway of YTHDF2 in lung carcinoma is thus upregulated,which leads to the downregulation of c-Myc.We further used mouse xenograft models to show that YTHDF2-T49A mutants increased lung cancer mass and size.Our work reveals a key role of YTHDF2 O-GlcNAcylation in tumorigenesis and suggests that O-GlcNAcylation exerts distinct functions under different biological stress.展开更多
基金Supported by the National Natural Science Foundation of China,No.81973684Natural Science Foundation of Sichuan Province,No.2023NSFSC1760Youth Talent Fund of Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital,No.2021QN09。
文摘BACKGROUND Periodontitis,when exacerbated by diabetes,is characterized by increased M1 macrophage polarization and decreased M2 polarization.O-linkedβ-N-acetylglucosamine(O-GlcNAcylation),catalyzed by O-GlcNAc transferase(OGT),promotes inflammatory responses in diabetic periodontitis(DP).Additionally,p38 mitogen-activated protein kinase regulates macrophage polarization.However,the interplay between OGT,macrophage polarization,and p38 signaling in the progression of DP remains unexplored.AIM To investigate the effect of OGT on macrophage polarization in DP and its role in mediating O-GlcNAcylation of p38.METHODS For in vivo experiments,mice were divided into four groups:Control,DP model,model+short hairpin(sh)RNAnegative control,and model+sh-OGT.Diabetes was induced by streptozotocin,followed by ligation and lipopolysaccharide(LPS)administration to induce periodontitis.The impact of OGT was assessed by injecting sh-OGT lentivirus.Maxillary bone destruction was evaluated using micro-computed tomography analysis and tartrateresistant acid phosphatase staining,while macrophage polarization was determined through quantitative real-time polymerase chain reaction(qPCR)and immunohistochemistry.For in vitro experiments,RAW264.7 cells were treated with LPS and high glucose(HG)(25 mmol/L D-glucose)to establish a cell model of DP.OGT was inhibited by OGT inhibitor(OSMI4)treatment and knocked down by sh-OGT transfection.M1/M2 polarization was analyzed using qPCR,immunofluorescence,and flow cytometry.Levels of O-GlcNAcylation were measured using immunoprecipitation and western blotting.RESULTS Our results demonstrated that M1 macrophage polarization led to maxillary bone loss in DP mice,associated with elevated O-GlcNAcylation and OGT levels.Knockdown of OGT promoted the shift from M1 to M2 macrophage polarization in both mouse periodontal tissues and LPS+HG-induced RAW264.7 cells.Furthermore,LPS+HG enhanced the O-GlcNAcylation of p38 in RAW264.7 cells.OGT interacted with p38 to promote its O-GlcNAcylation at residues A28,T241,and T347,as well as its phosphorylation at residue Y221.CONCLUSION Inhibition of OGT-mediated p38 O-GlcNAcylation deactivates the p38 pathway by suppressing its self-phosphorylation,thereby promoting M1 to M2 macrophage polarization and mitigating DP.These findings suggested that modulating macrophage polarization through regulation of O-GlcNAcylation may represent a novel therapeutic strategy for treating DP.
文摘The aim of this present study was to explore the expression and clinical significance of O-linked N-acetylglucosamine（O-GlcNAc） transferase（OGT） and enzymatic O-linked glycosylation（O-GlcNAcation） through the addition of O-linked-β-N-acetylglucosamine in esophageal squamous cell carcinoma.OGT expression and O-GlcNAcation in 40 samples from patients with esophageal squamous cell carcinoma was detected by immunohistochemical staining with anti-OGT antib ody and O-GlcNAc-specific antibody RL 2,respectively.The relationship between pathological and clinical factors of patients was analyzed.We found that the expression of OGT was higher in esophageal squamous cell carcinoma samples compared to the normal tissues.RL 2 antibody level was positively correlated with OGT expression,and the metastasis of lymph node,which means the level of O-GlcNAcation was high and related to the metastasis of lymph node in esophageal squamous cell carcinoma.In conclusion,OGT activation is the main reason for promoting the level of O-GlcNAcation in esophageal squamous cell carcinoma.O-GlcNAcylation may play an important role in esophageal squamous cell carcinoma.
基金Supported by In part by a grant-in-aid for Challenging Exploratory Research(to Okajima T)from the Japan Society for the Promotion of ScienceScientific Research on Innovative Areas(to Okajima T)from the Ministry of Education,Culture,Sports,Science and Technologya grant from the Takeda Foundation(to Okajima T)
文摘The O-linked β-N-acetylglucosamine(O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase(OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine(EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.
基金financial support from the National Natural Science Foundation of China(Grant No.:31470795)Tianjin Municipal Science and Technology Commission(Grant No.:15JCYBJC24100)the“Fundamental Research Funds for the Central Universities”,Nankai University(Grant No.:63191148)。
文摘Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.
基金Supported by the National Natural Science Foundation of China (No. 29672004)
文摘The title compound, spiro[ 1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]- hexane-2,2＇-3＇-（16＇-methoxyacetatyl-4＇-l-menthyloxybutyrolactone）] 1, was obtained via tandem asymmetric double Michael addition/internal nucleophilic substitution of the chiral synthon, 5-l-menthyloxy-3-bromo-2-（5H）-furanone 2 with methoxy α-chloroacetate as a nucleophile under mild conditions, and structurally [letermined by single-crystal X-ray diffraction. Crystal data： C31H47BrO9, Mr = 643.60, orthorhombic, space group P212121. α = 9.6564（7）, b = 14.8994（11）, c = 23.6771（17） A, V= 3406.5（4） A^3, Z = 4, Dc= 1.255 g/cm^3, 2（MoKa） = 0.71073 A,μ= 1.254 mm^-1 and F（000） = 1360. The structure was refined to R =[0.0324 and wR = 0.0737 for 5123 observed reflections （I〉 2σ（I））. The crystallographic results of molecule 1 show that the interesting reaction of 2 with methoxy α-chloroacetate, in the usual manner, gave the spiro-cyclopropane skeleton with O-linked derivative containing multiple stereogenic centers 1 rather than the expected C-linked derivative.
文摘The title compound, spiro[1-bromo-4-l-menthyloxy-5-oxo-6-oxa-bicyclo[3.1.0]-hexane-2,2-3-(16-methoxyacetatyl-4-l-menthyloxybutyrolactone)] 1, was obtained via tandem asymmetric double Michael addition/internal nucleophilic substitution of the chiral synthon, 5-l-menthyloxy-3-bromo-2-(5H)-furanone 2 with methoxy α-chloroacetate as a nucleophile under mild conditions, and structurally determined by single-crystal X-ray diffraction. Crystal data: C31H47BrO9, Mr = 643.60, orthorhombic, space group P212121, a = 9.6564(7), b = 14.8994(11), c = 23.6771(17) , V = 3406.5(4) 3, Z = 4, Dc = 1.255 g/cm3, λ(MoKα) = 0.71073 , μ = 1.254 mm-1 and F(000) = 1360. The structure was refined to R = 0.0324 and wR = 0.0737 for 5123 observed reflections (I > 2σ(I)). The crystallographic results of molecule 1 show that the interesting reaction of 2 with methoxy α-chloroacetate, in the usual manner, gave the spiro-cyclopropane skeleton with O-linked derivative containing multiple stereogenic centers 1 rather than the expected C-linked derivative.
基金supported in part by a grant from Natural Sciences Foundation of Guangdong Province(No.010398)
文摘To investigate the modification on the mouse zona pellucida by ethanol activation,the zona pellucida glycoproteins of ethanol-activated eggs was subjectedto SDS-PAGE and HPLC.The SDS-PAGE of zona pellucida samples showed that the migration profiles of ZP glycoproteins of ethanol-activated eggs and oocytes were similar,except an additional band approximately 20kD was separated from the sample of ethanol-activated eggs under reducing condition.No evident difference between ZP3 of ethanol-activated eggs and unfertilized oo-cytes was observed.HPLC results showed that the O-linked sugars from the sample of etha-nol-activated eggs had three peaks of monosaccharides,which were quite similar to that of the unfertilized oocytes,suggesting both samples had the same three O-linked monosaccharides.However,another two peaks of oligosaccharides were recorded from the sample of ethanol-ac-tivated cgg,with RT values of 12.09 and 8.89.Only another peak with RTs of 9.14 was re-corded from the sample of unfertilized oocytes.Therefore,the ethanol activation had not sig-nificant effect on the ZP3 polypeptide and on the O-linked monosaccharides.The modification by ethanol activation was mainly on the O-linked oligosaccharide chains.
基金supported by the National Natural Science Foundation of China(NSFC)(92478113,32271285,32071277,82103383)Guangdong Province Basic Research Foundation(2023A1515011842)the Shenzhen Basic Research Foundation(JCYJ20230807095300002)。
文摘Investigations from the last four decades have correlated high O-linked N-acetylglucosamine(O-Glc NAc)levels with various cancer types,but it is not known how OGT responds to diverse nutrients to finetune cellular O-Glc NAcylation levels.Herein we identified a critical OGT phosphorylation site by unc-51 like autophagy activating kinase 1(ULK1)under glucose depletion.First,we demonstrated that glucose levels modulate the interaction between OGT and ULK1 and cellular O-Glc NAcylation levels.Low glucose induces high O-Glc NAcylation,which could be reversed by ULK1 inhibition.Then,using mass spectrometry,we showed that ULK1 phosphorylates OGT at Ser576 and stabilizes OGT.Further biochemical experiments revealed that Ser576 phosphorylation inhibits Lys604 ubiquitination by stimulating OGT binding with BAP1,a de-ubiquitinase for OGT.Strikingly,using the OGT S576A knock-in cells,we found that in mouse xenograft models OGTS576A completely abolishes the tumorigenicity of OGT,probably due to low O-Glc NAcylation.In sum,we found that ULK1 phosphorylates OGT at Ser-576 under glucose deprivation,which stabilizes OGT by promoting OGT-BAP1 association and is pivotal for O-Glc NAcylation levels and tumorigenesis.As low glucose is often associated with tumor progression,our work not only unearths a key mechanism of how OGT is regulated by glucose levels,but also offers new therapeutic opportunities targeting OGT.
基金supported by the National Natural Science Foundation of China(32271285)R&D Program of Beijing Municipal Education Commission(KZ202210028043)+1 种基金supported by Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2021-RC350-002)CAMS Innovation Fund for Medical Sciences(2021I2M-1-026).
文摘The intracellular O-linked N-acetylglucosamine(O-GlcNAc)glycosylation mediates many signal transduction events and regulates tumorigenesis.Previously the RNA N6-methyladenosine(m6 A)reader,YTH(YT521-B homology)domain 2(YTHDF2),has been shown to be O-GlcNAcylated on Ser-263 during Hepatitis B virus(HBV)infection and promote HBV-related hepatocellular carcinoma.Herein we mapped YTHDF2 O-GlcNAcylation at Thr-49 via electron-transfer dissociation mass spectrometry under unperturbed conditions.We show that YTHDF2 Thr-49 O-GlcNAcylation antagonizes Extracellular-signal regulated kinase(ERK)-dependent phosphorylation at Ser-39 and promotes YTHDF2 degradation.The downstream signaling pathway of YTHDF2 in lung carcinoma is thus upregulated,which leads to the downregulation of c-Myc.We further used mouse xenograft models to show that YTHDF2-T49A mutants increased lung cancer mass and size.Our work reveals a key role of YTHDF2 O-GlcNAcylation in tumorigenesis and suggests that O-GlcNAcylation exerts distinct functions under different biological stress.
