O-GlcNAc Research Articles

Ac34deoGlcNAz: A Selective Probe for Identifying O-GlcNAc-Modified Proteins in Prostate Cancer.

O-GlcNAcylation is a prevalent protein modification in eukaryotic cellsandincreasing evidences indicated that over-expressed O-GlcNAcylation are intimately linked to the development and prognosis of prostate cancer.Thus, exploring this modification in the context of prostate cancer is vital for understanding the underlying mechanisms and hopefully used forfuture targeted therapies. In this paper, we use our previously established metabolic probes to comprehensively compare the labeling efficiency ofO-GlcNAc modified proteins in PC3 cells. Our results demonstrated that all the tested probes were non-toxic to PC3 cells and only Ac4GlcNAz, Ac4GalNAzand Ac34deoGlcNAzexhibited robust labeling signals amongst the probes. Further investigations by western blot and flow cytometry analysis revealed that Ac34deoGlcNAzwas a specific and efficient probe for intracelluar protein labeling with negligible S-glyco-modification signal. In addition, proteomic analysis further confirmed that 94% of the proteins identified byAc34deoGlcNAzwere in the form of O-linked GlcNAc, these enriched O-GlcNAcylated proteins were mainly involved in the regulated processes of prostate cancer. Our results here together prove Ac34deoGlcNAzis a safe and reliable probefor metabolic labeling O-GlcNAc modified proteins in prostate cancer, providing a mean to fully exploitthe regulatory mechanism of O-GlcNAcylation in the process of prostate cancer.

NMR Shows Why a Small Chemical Change Almost Abolishes the Antimicrobial Activity of Glycocin F.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)2 structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the Cα proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 μM cf. 1.5 nM). A comparison of the GccFα-methylSer18 NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury.

Regulation of Primary Cilium Length by O-GlcNAc during Neuronal Development in a Human Neuron Model.

The primary cilium plays critical roles in the homeostasis and development of neurons. Recent studies demonstrate that cilium length is regulated by the metabolic state of cells, as dictated by processes such as glucose flux and O-GlcNAcylation (OGN). The study of cilium length regulation during neuron development, however, has been an area left largely unexplored. This project aims to elucidate the roles of O-GlcNAc in neuronal development through its regulation of the primary cilium. Here, we present findings suggesting that OGN levels negatively regulate cilium length on differentiated cortical neurons derived from human-induced pluripotent stem cells. In neurons, cilium length increased significantly during maturation (after day 35), while OGN levels began to drop. Long-term perturbation of OGN via drugs, which inhibit or promote its cycling, during neuron development also have varying effects. Diminishing OGN levels increases cilium length until day 25, when neural stem cells expand and undergo early neurogenesis, before causing cell cycle exit defects and multinucleation. Elevating OGN levels induces greater primary cilia assembly but ultimately results in the development of premature neurons, which have higher insulin sensitivity. These results indicate that OGN levels and primary cilium length are jointly critical in proper neuron development and function. Understanding the interplays between these two nutrient sensors, O-GlcNAc and the primary cilium, during neuron development is important in paving connections between dysfunctional nutrient-sensing and early neurological disorders.

Reduced O-GlcNAcylation diminishes cardiomyocyte Ca2+ dependent facilitation and frequency dependent acceleration of relaxation

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIβ isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

Inhibiting O-GlcNAcylation impacts p38 and Erk1/2 signaling and perturbs cardiomyocyte hypertrophy

The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.

Abstract 12874: A Novel Mechanism in the Pathogenesis of Heart Failure With Preserved Ejection Fraction Through Hexokinase Mitochondrial Dislocation and Substrate Shuttling Into the O-Glcnacylation Machinery

Introduction: Endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF), however, the mechanism is unclear. ECs are highly dependent on glycolysis; however, it is not known how their metabolic profile is altered in the setting of HFpEF. Hexokinase 1 (HK1) carries the first step in glycolysis and is mostly bound to the mitochondria in ECs. Objective: We sought to elucidate the role of EC glucose metabolism, and specifically HK1, in the development of HFpEF. Results: We first showed that among glycolytic enzymes, HK1 is highly specific to ECs, suggesting the significance of HK1 in EC physiology. Although HK1 mRNA and protein levels are not changed, we noted increased dislocation of HK1 from the mitochondria in ECs from HFpEF mice. To study the role of HK1 dislocation in the development of HFpEF, we generated mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag (ΔE1HK1). ΔE1HK1mice develop impaired cardiac relaxation at 20 weeks of age and HFpEF by 40 weeks, and ECs from these mice displayed impaired angiogenic potential. Since HK1 is involved in glucose metabolism, we then performed metabolomic studies and demonstrated that intermediates in hexosamine biosynthetic pathway (HBP) are significantly decreased in ECs from ΔE1HK1 mice. To assess the mechanism for the decrease in HBP, we assessed protein modification, and noted reduced protein N-glycosylation and increased O-GlcNAcylation in ECs from ΔE1HK1. We also demonstrated that inhibition of the key enzyme in O-GlcNAcylation (OGT) reverses the EC dysfunction noted in ΔE1HK1 mice, indicating that O-GlcNAcylation is responsible for the angiogenic defect in ECs from ΔE1HK1. Finally, to provide a mechanism for altered protein glycosylation with HK1 mitochondrial dislocation, we showed that HK1 associates with N-glycosylation machinery when it is attached to mitochondria, while it binds to OGT when it is dislocated from mitochondria. Conclusion: Our studies demonstrate a role for HK1 mitochondrial binding and protein O-GlcNAcylation in the pathogenesis of HFpEF, and that HK1 cellular localization plays a major role in the fate of HBP intermediates into either N-glycosylation or O-GlcNAcylation machinery in ECs.

Integration of O-GlcNAc into Stress Response Pathways.

The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked βN-acetylglucosamine (O-GlcNAc) has emerged as a dynamic and essential post-translational modification of mammalian proteins. O-GlcNAc is cycled on and off over 5000 proteins in response to diverse stimuli impacting protein function and, in turn, epigenetics and transcription, translation and proteostasis, metabolism, cell structure, and signal transduction. Environmental and physiological injury lead to complex changes in O-GlcNAcylation that impact cell and tissue survival in models of heat shock, osmotic stress, oxidative stress, and hypoxia/reoxygenation injury, as well as ischemic reperfusion injury. Numerous mechanisms that appear to underpin O-GlcNAc-mediated survival include changes in chaperone levels, impacts on the unfolded protein response and integrated stress response, improvements in mitochondrial function, and reduced protein aggregation. Here, we discuss the points at which O-GlcNAc is integrated into the cellular stress response, focusing on the roles it plays in the cardiovascular system and in neurodegeneration.

Recombinant norovirus capsid protein VP1 (GII.4) expressed in H5 insect cells exhibits post-translational modifications with potential impact on lectin activity and vaccine design.

Although surface proteins of most enveloped viruses are glycosylated, among non-enveloped viruses only few express glycoproteins in their capsid as infective virions. Noroviruses belong to the latter group and are known to express one major capsid protein (VP1) that lacks genuine glycosylation. In the context of vaccine development based on virus-like particles (VLPs) and in searches for food additives offering potential prophylactic or therapeutic applications an increasing number of reports refers to the use of VLPs that were produced as secretory products in insect cells. We asked the question whether recombinant VLPs (GII.4 Sydney, 2012) produced via the baculovirus vector in H5 insect cells may be glycosylated in the protruding domains that are involved in receptor binding and immune reactivity. Mass spectrometric analysis of tryptic VP1 peptides prior to and after beta-elimination Michael addition in 70% ethylamine revealed Thr238, and Ser519 in the P1 domain, and Thr350, Thr369, Thr371, and Thr381 in the P2 domain as modified. Thr65, Ser67, and Thr350 were revealed by liquid chromatography-mass spectrometry to carry HexNAc or Hex-HexNAc modifications, respectively. Monosaccharide analysis by gas chromatography-mass spectrometry confirmed the presence of GlcNAc on VLP protein, whereas immunoassays with lectins and antibodies demonstrated O-linked GlcNAc on VP1 protein. Post-translational modifications of virus capsid proteins may contribute to a modulation of immunodominant surface epitopes and need to be considered in anti-norovirus vaccine design. Some modifications are located near amino acid side chains involved in the binding of blood group active sugar receptors.

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion–associated protein Zyxin to regulate UV-induced cell death

Zyxin is a zinc-binding phosphoprotein known to regulate cell migration, adhesion, and cell survival. Zyxin also plays a role in signal transduction between focal adhesions and the nuclear compartment. However, the mechanism of Zyxin shuttling to nucleus is still unclear. Here, we identify that the GlcNAc transferase (O-linked GlcNAc [O-GlcNAc] transferase) can O-GlcNAcylate Zyxin and regulate its nuclear localization. We show that O-GlcNAc transferase O-GlcNAcylates Zyxin at two residues, serine 169 (Ser-169) and Ser-246. In addition, O-GlcNAcylation of Ser-169, but not Ser-246, enhances its interaction with 14-3-3γ, which is a phosphoserine/threonine-binding protein and is reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3γ could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export signal sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that the nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death.

The Beginner's Guide to O-GlcNAc: From Nutrient Sensitive Pathway Regulation to Its Impact on the Immune System.

The addition of N-acetyl glucosamine (GlcNAc) on the hydroxy group of serine/threonine residues is known as O-GlcNAcylation (OGN). The dynamic cycling of this monosaccharide on and off substrates occurs via O-linked β-N-acetylglucosamine transferase (OGT) and O-linked β-N-acetylglucosaminase (OGA) respectively. These enzymes are found ubiquitously in eukaryotes and genetic knock outs of the ogt gene has been found to be lethal in embryonic mice. The substrate scope of these enzymes is vast, over 15,000 proteins across 43 species have been identified with O-GlcNAc. OGN has been known to play a key role in several cellular processes such as: transcription, translation, cell signaling, nutrient sensing, immune cell development and various steps of the cell cycle. However, its dysregulation is present in various diseases: cancer, neurodegenerative diseases, diabetes. O-GlcNAc is heavily involved in cross talk with other post-translational modifications (PTM), such as phosphorylation, acetylation, and ubiquitination, by regulating each other’s cycling enzymes or directly competing addition on the same substrate. This crosstalk between PTMs can affect gene expression, protein localization, and protein stability; therefore, regulating a multitude of cell signaling pathways. In this review the roles of OGN will be discussed. The effect O-GlcNAc exerts over protein-protein interactions, the various forms of crosstalk with other PTMs, and its role as a nutrient sensor will be highlighted. A summary of how these O-GlcNAc driven processes effect the immune system will also be included.

Decreased O‐GlcNAcylation and mitochondrial dysfunction are involving in low glucose induced Alzheimer’s disease‐like phenotype in induced pluripotent stem cell‐derived neurons

AbstractBackgroundEvidence supporting that glucose metabolic abnormalities occur prior to Alzheimer’s disease (AD) symptoms. Reduced O‐GlcNAc (OGN) levels likely arise from impaired glucose metabolism or availability and correlates with AD pathogenesis. So far, the mechanism of sAD and the role of OGN in AD pathology remained largely unknown due to a lack of human sAD model.MethodWe generated human cortical neurons from human‐induced pluripotent stem cells and treated neurons with glucose reduction media. FluoroJADE staining and cell viability assays were used to study the effects of low glucose on the degenerative status and cell viability of neurons. Abnormal protein production, phosphorylation, and accumulation were detected by western blotting and immunofluorescence staining using antibody against p‐tau and beta‐amyloid. Neurite and synaptic structure were observed by immunofluorescence staining, while neuronal network activity was detected by multi‐electrode array electrophysiological analyses. Mitochondrial abnormalities, including increased oxidative stress, reduced membrane potential, and mitochondrial dysfunction, were evaluated using CM‐H2DCFDA, JC‐1 and Seahorse, respectively.ResultWe show that lowering glucose level to 2mM leads to dramatic increases of neuron degeneration on day 3 and 5 of treatment and decreased cell viability on day 7. Interestingly, long‐term low glucose treatment induces AD features in neurons, including abnormal hyperphosphorylated tau accumulation and increasing beta‐amyloid production. Besides, glucose deficiency also causes decreased neurite coverage, synapse density, and neuron network activity. Furthermore, we find that O‐GlcNAc levels are significantly reduced soon after low glucose treatment and remain low until the end of experiment. Raising O‐GlcNAc levels by thiamet‐G, inhibitor of O‐GlcNAcase, even in low glucose treated neurons, rescues low glucose‐induced AD phenotypes. Moreover, our data shows that O‐GlcNAc dysregulation causes mitochondrial abnormalities, which occur before any other degenerative phenotype appeared, and may be one of the underlying mechanisms of sAD onset and pathogenesis.ConclusionWe established a human neuron model in which the pathological features are reproduced by glucose deficiency. This platform can serve as a tool for better understanding molecular processes involved in neurodegeneration in sAD. Our results also suggest that dysregulated O‐GlcNAc levels and mitochondrial dysfunction by glucose deficiency are involved in the onset and progression of sAD.

SHCBP1 interacting with EOGT enhances O-GlcNAcylation of NOTCH1 and promotes the development of pancreatic cancer

O-GlcNAcylation is important in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The glycosyltransferase EGF domain-specific O-linked GlcNAc transferase (EOGT) acts as a key participant in glycosylating NOTCH1. High-throughput sequencing of specimens from 30 advanced PDAC patients identified SHCBP1 and EOGT as factors of poor prognosis. We hypothesized that they could mediate PDAC progression by influencing NOTCH1 O-GlcNAcylation. Thus, 186 PDAC tissue specimens were immunostained for EOGT and SHCBP1. Pancreatic cancer cell lines and nude mouse models were used for in vitro and in vivo experiments. Respectively, The protein expression of EOGT and SHCBP1 was significantly elevated and correlated with worse prognosis in PDAC patients. In vitro, SHCBP1 overexpression promoted pancreatic cancer cell proliferation, migration and invasion, while knocking down SHCBP1 and EOGT inhibited these malignant processes. In vivo data showed that SHCBP1 overexpression promoted xenograft growth and lung metastasis and shortened survival in mice, whereas knocking down either EOGT or SHCBP1 expression suppressed xenograft growth and metastasis and prolonged survival. We further clarified the molecular mechanisms by which EOGT and SHCBP1 enhance the O-GlcNAcylation of NOTCH1, Subsequently promoting the nuclear localization of the Notch intracellular domain (NICD) and inhibiting the transcription of E-cadherin and P21 in pancreatic cancer cells.

Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms.

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Potential Roles of O-GlcNAcylation in Primary Cilia- Mediated Energy Metabolism.

The primary cilium, an antenna-like structure on most eukaryotic cells, functions in transducing extracellular signals into intracellular responses via the receptors and ion channels distributed along it membrane. Dysfunction of this organelle causes an array of human diseases, known as ciliopathies, that often feature obesity and diabetes; this indicates the primary cilia’s active role in energy metabolism, which it controls mainly through hypothalamic neurons, preadipocytes, and pancreatic β-cells. The nutrient sensor, O-GlcNAc, is widely involved in the regulation of energy homeostasis. Not only does O-GlcNAc regulate ciliary length, but it also modifies many components of cilia-mediated metabolic signaling pathways. Therefore, it is likely that O-GlcNAcylation (OGN) plays an important role in regulating energy homeostasis in primary cilia. Abnormal OGN, as seen in cases of obesity and diabetes, may play an important role in primary cilia dysfunction mediated by these pathologies.

Disruption of retinoid homeostasis induces RBP4 overproduction in diabetes: O-GlcNAcylation involved

BackgroundRetinol-binding protein 4 (RBP4) is elevated and associated with inflammation in metabolic diseases. Disruption of the retinol cascade and O-GlcNAcylation of the RBP4 receptor (STRA6) are found in diabetic kidneys. ObjectivesWe investigated whether the disruption of the retinol cascade induces RBP4 overproduction and if O-linked GlcNAc modification targets RBPR2 and contributes to the disruption of retinol cascades in diabetic livers. MethodsWestern blot or immunohistochemistry for RBPR2, CRBP1, LRAT, RALDH, RARα, RARγ, RXRα, RBP4, GFAT, OGT, OGA and inflammatory markers, as well as ELISA for RBP4, were performed in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. Immunoprecipitation and dual fluorescence staining were used to explore O-GlcNAc-modified RBPR2 and RBP4 binding activity on RBPR2. Transfection of the CRBP1 gene was done to verify whether a disrupted retinol cascade induces RBP4 overproduction. OGT silencing was done to investigate the association of O-GlcNAcylation with the disruption of retinol cascade. ResultsDisruption of retinol cascade, RBP4 overproduction, O-GlcNAcylation of RBPR2, decreased RBP4 binding activity on RBPR2 and inflammation were found in livers of db/db and ob/ob mice and high glucose-cultured hepatocytes. CRBP1 gene transfection reversed the suppression of the cellular retinol cascade and simultaneously attenuated the RBP4 overproduction and inflammation in high glucose-treated hepatocytes. The silencing of OGT reversed the disruption of the cellular retinol cascade, RBP4 overproduction and inflammation induced by high glucose in hepatocytes. ConclusionsThis study indicates that the disruption of cellular retinol cascade is strongly associated with RBP4 overproduction and inflammation in diabetic livers. RBPR2 is one target for high glucose-mediated O-linked GlcNAc modification, which causes liver retinol dyshomeostasis.

Expression of O-GlcNAc transferase in osteosarcoma and its effect on proliferation and sensitivity to cisplatin

Expression of O-GlcNAc transferase in osteosarcoma and its effect on proliferation and sensitivity to cisplatin

Cereal grass juice in wound healing: hormesis and cell-survival in normal fibroblasts, in contrast to toxic events in cancer cells.

Natural products and traditional medicines are of great importance. Recent studies have demonstrated, that cereal grass juice improves wound healing, however the cellular and molecular mechanisms underlying these processes have not been fully characterized. Also, the full phytochemical characteristics of freshly squeezed juices obtained from cereal grasses is still missing. Thus, in this study a multi-dimensional analysis of juice parameters like refraction value, pH, chlorophyll and flavonoids content as well as antioxidant properties was performed. The results demonstrate that the effect induced by freshly squeezed cereal juices is strictly cell type-dependent. In this study, it is shown for the first time, that in normal fibroblasts (BJ cells) low dose cereal grass juices exhibit strong adaptive response through hormetic mechanism mediated by NF-κB/HO-1 and insulin/IGF-1 anti-oxidant pathways. As consequence, the process of wound healing is significantly upregulated. In cancer cells (ES-2 cells), despite anti-oxidant defense mechanism activation, levels of ROS and RNS are elevated. This leads to enhanced O-GlcNAcylation, DNA damage and cell cycle arrest, and as a result impaired wound healing. This study provides insights into the underlying mechanisms through which cereal grass juices activate hermetic adaptation response in normal fibroblasts, and induce cytotoxic and genotoxic events in cancer cells.

Mitochondrial Protein Poldip2 (Polymerase Delta Interacting Protein 2) Controls Vascular Smooth Muscle Differentiated Phenotype by O-Linked GlcNAc (N-Acetylglucosamine) Transferase-Dependent Inhibition of a Ubiquitin Proteasome System.

The mitochondrial Poldip2 (protein polymerase interacting protein 2) is required for the activity of the tricarboxylic acid cycle. As a consequence, Poldip2 deficiency induces metabolic reprograming with repressed mitochondrial respiration and increased glycolytic activity. Though homozygous deletion of Poldip2 is lethal, heterozygous mice are viable and show protection against aneurysm and injury-induced neointimal hyperplasia, diseases linked to loss of vascular smooth muscle differentiation. Thus, we hypothesize that the metabolic reprograming induced by Poldip2 deficiency controls VSMC differentiation. To determine the role of Poldip2-mediated metabolic reprograming in phenotypic modulation of VSMC. We show that Poldip2 deficiency in vascular smooth muscle in vitro and in vivo induces the expression of the SRF (serum response factor), myocardin, and MRTFA (myocardin-related transcription factor A) and dramatically represses KLF4 (Krüppel-like factor 4). Consequently, Poldip2-deficient VSMC and mouse aorta express high levels of contractile proteins and, more significantly, these cells do not dedifferentiate nor acquire macrophage-like characteristics when exposed to cholesterol or PDGF (platelet-derived growth factor). Regarding the mechanism, we found that Poldip2 deficiency upregulates the hexosamine biosynthetic pathway and OGT (O-linked N-acetylglucosamine transferase)-mediated protein O-GlcNAcylation. Increased protein glycosylation causes the inhibition of a nuclear ubiquitin proteasome system responsible for SRF stabilization and KLF4 repression and is required for the establishment of the differentiated phenotype in Poldip2-deficient cells. Our data show that Poldip2 deficiency induces a highly differentiated phenotype in VSMCs through a mechanism that involves regulation of metabolism and proteostasis. Additionally, our study positions mitochondria-initiated signaling as key element of the VSMC differentiation programs that can be targeted to modulate VSMC phenotype during vascular diseases.