Switch of phosphorylation to O-GlcNAcylation of AhR contributes to vascular oxidative stress induced by benzo[a]pyrene

Benzo[a]pyrene(B[a]P)is a food contaminant toxic for cardiovascular diseases.The nuclear translocation of Arylhydrocarbon receptor(AhR)plays an important role in B[a]P-induced oxidative stress and vascular diseases.We confi rmed that B[a]P promoted ROS production in vascular smooth muscle cells(VSMCs)in vitro and in vivo,associated with the nuclear translocation of AhR.It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR.However,from the posttranslational modifi cation level,the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear.Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation,and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs,suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR.Next,siRNAs of O-linked N-acetylglucosamine transferase(OGT),O-GlcNAcase(OGA)and OGA inhibitor PUGNAc were used.SiOGT blocks but siOGA and PUGNAc promote B[a]P-dependent AhR nuclear translocation and oxidative stress.Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR.Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress.Our fi ndings highlight a new perspective for AhR nuclear translocation regulated by the competitive modifi cation between phosphorylation and O-GlcNAcylation.

Research progress on O-GlcNAcylation in the occurrence,development,and treatment of colorectal cancer

For a long time,colorectal cancer(CRC)has been ranked among the top cancerrelated mortality rates,threatening human health.As a significant posttranslational modification,O-GlcNAcylation plays an essential role in complex life activities.Related studies have found that the occurrence,development,and metastasis of CRC are all related to abnormal O-GlcNAcylation and participate in many critical biological processes,such as gene transcription,signal transduction,cell growth,and differentiation.Recently,nucleotide sugar analogs,tumorspecific carbohydrate vaccine,SIRT1 longevity gene,dendritic cells as targets,and NOTCH gene have become effective methods to induce antitumor therapy.Not long ago,checkpoint kinase 1 and checkpoint kinase 2 were used as therapeutic targets for CRC,but there are still many problems to be solved.With an in-depth study of protein chip,mass spectrometry,chromatography,and other technologies,O-GlcNAcylation research will accelerate rapidly,which may provide new ideas for the research and development of antitumor drugs and the discovery of new CRC diagnostic markers.

O-GlcNAcylation regulates phagocytosis by promoting Ezrin localization at the cell cortex

O-GlcNAcylation is a post-translational modification that serves as a cellular nutrient sensor and participates in multiple physiological and pathological processes.However,it remains uncertain whether O-GlcNAcylation is involved in the regulation of phagocytosis.Here,we demonstrate a rapid increase in protein OGlcNAcylation in response to phagocytotic stimuli.Knockout of the O-GlcNAc transferase or pharmacological inhibition of O-GlcNAcylation dramatically blocks phagocytosis,resulting in the disruption of retinal structure and function.Mechanistic studies reveal that the O-GlcNAc transferase interacts with Ezrin,a membrane-cytoskeleton linker protein,to catalyze its O-GlcNAcylation.Our data further show that Ezrin OGlcNAcylation promotes its localization to the cell cortex,thereby stimulating the membrane-cytoskeleton interaction needed for efficient phagocytosis.These findings identify a previously unrecognized role for protein O-GlcNAcylation in phagocytosis with important implications in both health and diseases.

Protein O-GlcNAcylation homeostasis regulates facultative heterochromatin to fine-tune sog-Dpp signaling during Drosophila early embryogenesis

Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly short gastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.

Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter

O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.

Regulation of the urea cycle by CPS1 O-GlcNAcylation in response to dietary restriction and aging

O-linked N-acetyl-glucosamine glycosylation(O-GlcNAcylation)of intracellular proteins is a dynamic process broadly implicated in age-related disease,yet it remains uncharacterized whether and how O-GlcNAcylation contributes to the natural aging process.O-GlcNAc transferase(OGT)and the opposing enzyme O-GlcNAcase(OGA)control this nutrient-sensing protein modification in cells.Here,we show that global O-GlcNAc levels are increased in multiple tissues of aged mice.In aged liver,carbamoyl phosphate synthetase 1(CPS1)is among the most heavilyO-GlcNAcylated proteins.CPS1O-GlcNAcylation is reversed by calorie restriction and is sensitive to genetic and pharmacological manipulations of theO-GlcNAc pathway.High glucose stimulates CPS1O-GlcNAcylation and inhibits CPS1 activity.Liver-specific deletion of OGT potentiates CPS1 activity and renders CPS1 irresponsive to further stimulation by a prolonged fasting.Our results identify CPS1 O-GlcNAcylation as a key nutrient-sensing regulatory step in the urea cycle during aging and dietary restriction,implying a role for mitochondrial O-GlcNAcylation in nutritional regulation of longevity.

The role of O-GlcNAcylation in innate immunity and inflammation

O-linkedβ-N-acetylglucosaminylation(O-GlcNAcylation)is a highly dynamic and widespread post-translational modification(PTM)that regulates the activity,subcellular localization,and stability of target proteins.O-GlcNAcylation is a reversible PTM controlled by two cycling enzymes:O-linked N-acetylglucosamine transferase and O-GlcNAcase.Emerging evidence indicates that O-GlcNAcylation plays critical roles in innate immunity,inflammatory signaling,and cancer development.O-GlcNAcylation usually occurs on serine/threonine residues,where it interacts with other PTMs,such as phosphorylation.Thus,it likely has a broad regulatory scope.This review discusses the recent research advances regarding the regulatory roles of O-GlcNAcylation in innate immunity and inflammation.A more comprehensive understanding ofO-GlcNAcylation could help to optimize therapeutic strategies regarding inflammatory diseases and cancer.

O-GlcNAcylation in Ventral Tegmental Area Dopaminergic Neurons Regulates Motor Learning and the Response to Natural Reward

Protein O-GlcNAcylation is a post-translational modification that links environmental stimuli with changes in intracellular signal pathways,and its disturbance has been found in neurodegenerative diseases and metabolic disorders.However,its role in the mesolimbic dopamine(DA)system,especially in the ventral tegmental area(VTA),needs to be elucidated.Here,we found that injection of Thiamet G,an O-GlcNAcase(OGA)inhibitor,in the VTA and nucleus accumbens(NAc)of mice,facilitated neuronal O-GlcNAcylation and decreased the operant response to sucrose as well as the latency to fall in rotarod test.Mice with DAergic neuron-specific knockout of O-GlcNAc transferase(OGT)displayed severe metabolic abnormalities and died within 4–8 weeks after birth.Furthermore,mice specifically overexpressing OGT in DAergic neurons in the VTA had learning defects in the operant response to sucrose,and impaired motor learning in the rotarod test.Instead,overexpression of OGT in GABAergic neurons in the VTA had no effect on these behaviors.These results suggest that protein O-GlcNAcylation of DAergic neurons in the VTA plays an important role in regulating the response to natural reward and motor learning in mice.

O-GlcNAcylation Coordinates Glutaminolysis by Regulating the Stability and Membrane Trafficking of ASCT2 in Hepatic Stellate Cells

Background and Aims:Recognition of excessive activa-tion of hepatic stellate cells(HSCs)in liver fibrosis prompt-ed us to investigate the regulatory mechanisms of HSCs.We aimed to examine the role of O-GlcNAcylation modifica-tion of alanine,serine,cysteine transporter 2(ASCT2)in HSCs and liver fibrosis.Methods:The expression of O-Glc-NAcylation modification in fibrotic mice livers and activated HSCs was analyzed by western blotting.Immunoprecipita-tion was used to assess the interaction of ASCT2 and O-Glc-NAc transferase(OGT).In addition,ASCT2 protein stability was assayed after cycloheximide(CHX)treatment.The O-GlcNAcylation site of ASCT2 was predicted and mutated by site-directed mutagenesis.Real-time PCR,immunofluores-cence,kit determinations and Seahorse assays were used to clarify the effect of ASCT2 O-GlcNAcylation on HSC glu-taminolysis and HSC activation.Western blotting,immuno-chemistry,and immunohistofluorescence were used to ana-lyze the effect of ASCT2 O-GlcNAcylation in vivo.Results:We observed significantly increased O-GlcNAcylation modi-fication of ASCT2.ASCT2 was found to interact with OGT to regulate ASCT2 stability.We predicted and confirmed that O-GlcNAcylation of ASCT2 at Thr122 site resulted in HSCs activation.We found Thr122 O-GlcNAcylation of ASCT2 me-diated membrane trafficking of glutamine transport and attenuated HSC glutaminolysis.Finally,we validated the expression and function of ASCT2 O-GlcNAcylation after in-jection of AAV8-ASCT2 shRNA in CCl4-induced liver fibrosis mice in vivo.Conclusions:Thr122 O-GlcNAcylation regu-lation of ASCT2 resulted in stability and membrane traf-ficking-mediated glutaminolysis in HSCs and liver fibrosis.Further studies are required to assess its role as a putative therapeutic target.

O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

利用CRISPR/Cas9系统建立成体心肌细胞特异性Oga基因敲除小鼠模型

目的 利用腺相关病毒9(AAV9)介导的CRISPR/Cas9系统构建成体心肌细胞特异性Oga基因敲除小鼠模型,来研究内源性OGA在成体心脏稳态维持中的功能。方法 首先,筛选靶向Oga编码区的有效sgRNA,构建包装表达有效sgRNA的AAV9病毒。其次,建立心肌细胞特异性SpCas9表达小鼠(α-MHC^(Cas9)),并通过腹腔注射方式将AAV9-sgOga注射到小鼠体内。通过qPCR和Western blot印记杂交实验检测Oga的表达情况,确定Oga敲除是否成功。最后,通过组织学分析心脏结构,以及超声心动图分析心脏功能,来分析Oga对心脏稳态维持的影响。结果 筛选到2条可以有效靶向Oga编码区的sgRNA,通过AAV9递送实现了Oga基因在心脏组织的有效敲除,且导致O-GlcNAcylation表达显著上升;组织学分析和超声心动图分析发现基因敲除小鼠心脏结构及功能在基础水平与对照小鼠无显著变化。结论 利用AAV9介导的CRISPR/Cas9系统成功建立了成体心肌细胞Oga基因敲除的小鼠模型,为研究OGA介导的O-GlcNAcylation清除在心脏稳态维持中的功能和机制提供了有效的小鼠模型。

Ac_(3)6deoGlcNAz could selectively label O-GlcNAc modified proteins with minimal S-glyco-modification

A novel metabolic chemical reporter of Ac_(3)6deo Glc NAz was developed and confirmed as an effective probe for O-Glc NAc modification. Ac_(3)6deo Glc NAz labeling predominantly occurs in intracellular OGlc NAcylated proteins rather than cell-surface glycoproteins. Of note, it could reduce the artificial S-glycomodification compared to Ac_(4)Gal NAz and Ac_(4)Glc NAz. This new reporter allows to be widely used in the field of proteomic identification of O-Glc NAcylation.

O-GlcNAc修饰调节生物节律研究进展

生物体的睡眠/觉醒、进食等行为以及各种生理、生化、代谢过程都遵循着大约24 h的周期性变化,称为昼夜节律(circadian rhythms)。昼夜节律与能量代谢之间存在着紧密的联系。位于下丘脑视交叉上核(suprachiasmatic nuclei,SCN)的中枢生物钟与外周组织细胞中的生物钟共同组成了哺乳动物的昼夜节律系统。以CLOCK/BMAL1异二聚体为核心的转录/翻译负反馈环保障了节律系统的正常运行。各种蛋白质翻译后修饰参与了昼夜节律的调控。综述了氧连β-N-乙酰葡糖胺修饰(O-Glc NAcylation)在调节昼夜节律中发挥的重要作用。O-Glc NAc修饰可以增强一些生物钟蛋白的稳定性及转录活性,也可以影响其他一些生物钟蛋白的磷酸化及细胞定位。抑制生物钟蛋白的O-Glc NAc修饰导致细胞节律衰弱和多种节律基因表达下调。研究表明,O-Glc NAc作为机体能量代谢的感受器参与了多条细胞代谢相关信号转导通路的调节,O-Glc NAc修饰为能量代谢影响昼夜节律提供了一条新的途径。

Xenopus GLP-1-based glycopeptides as dual glucagon-like peptide 1 receptor/glucagon receptor agonists with improved in vivo stability for treating diabetes and obesity

Peptide dual agonists toward both glucagon-like peptide 1 receptor(GLP-1R)and glucagon receptor(GCGR)are emerging as novel therapeutics for the treatment of type 2 diabetes mellitus(T2DM)patients with obesity.Our previous work identified a Xenopus GLP-1-based dual GLP-1R/GCGR agonist termed xGLP/GCG-13,which showed decent hypoglycemic and body weight lowering activity.However,the clinical utility of xGLP/GCG-13 is limited due to its short in vivo half-life.Inspired by the fact that O-GlcNAcylation of intracellular proteins leads to increased stability of secreted proteins,we rationally designed a panel of O-GlcNAcylated xGLP/GCG-13 analogs as potential long-acting GLP-1R/GCGR dual agonists.One of the synthesized glycopeptides 1f was found to be equipotent to xGLP/GCG-13 in cell-based receptor activation assays.As expected,O-GlcNAcylation effectively improved the stability of xGLP/GCG-13 in vivo.Importantly,chronic administration of 1f potently induced body weight loss and hypoglycemic effects,improved glucose tolerance,and normalized lipid metabolism and adiposity in both db/db and diet induced obesity(DIO)mice models.These results supported the hypothesis that glycosylation is a useful strategy for improving the in vivo stability of GLP-1-based peptides and promoted the development of dual GLP-1R/GCGR agonists as antidiabetic/antiobesity drugs.