Protein O-GlcNAc Transferase - an overview (2023)

In addition, OGT protein levels and total O-GlcNAcylation level were elevated in both human NSCLC cells and mouse lung tumor tissues overexpressing EMT-TFs. The HBP pathway was required for KrasG12D-induced lung tumorigenesis, as OGT deficiency produced using OGT knockout mice was characterized by a significant delay in lung tumor development.

Van:Advances in Cancer Research, 2019

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Cell glycobiology and development; Health and disease in glycomedicine

GW Hart, K. Sakabe, iComprehensive glycoscience, 2007 Diabetes type 2

It is estimated that 170 million people worldwide suffer from diabetes mellitus, of which 90% are type 2, non-insulin dependent diabetes mellitus (NIDDM).100Diabetics exhibit elevated levels of circulating glucose, insulin, and free fatty acids, all of which contribute to the complications associated with this disease, cardiomyopathy, atherosclerosis, retinopathy, and nephropathy.101In fact, tissues normally responsible for clearing plasma glucose become insulin resistant and the liver becomes gluconeogenic. Pancreatic β-cells normally secrete insulin to signal the uptake of glucose into peripheral tissues; however, the function of these cells is compromised in diabetes.102With the increase in obesity and type 2 diabetes in developed countries and the associated increase in cardiovascular disease, the strain on the healthcare system is enormous.103

Since cellular UDP-GlcNAc levels are extremely sensitive to the amount of circulating glucose, and since OGT itself is sensitive to donor sugar nucleotide levels, adjustments in sugar levels also cause rapid fluctuations inO-GlcNAc modification, leading to the assumption thatO-GlcNAc may play a direct molecular role in the pathology of type 2 diabetes. Hyperglycemia-induced increases in UDP-GlcNAc levels would cause aberrant protein O-GlcNAcylation, leading to the myriad complications associated with type 2 diabetes. In fact, several studies support this hypothesis. BothO-diazoacetyl-I-serine (azaserine) and DON, glutamine analogs that irreversibly inhibit GFAT, resulting in lower UDP-GlcNAc levels, inhibited insulin desensitization in adipocytes. Addition of glucosamine to the culture medium, which enters the HBP downstream of GFAT and thereby bypasses it, was 40 times more potent in inducing insulin resistance than glucose alone.40Infusion of rats with glucosamine increased muscle and liver UDP-GlcNAc levels four- to fivefold and induced insulin resistance in skeletal muscle by reducing the ability to take up glucose and synthesize glycogen.104Rats infused with glucosamine also had reduced glucose-induced insulin secretion, mimicking β-cell dysfunction associated with type 2 diabetes.105Ofob/obmice defective in leptin production show symptoms similar to patients with NIDDM and have elevated UDP-GlcNAc in skeletal muscle.106Two-dimensional gel analysis ofOGlcNAc-modified nuclear proteins from rat aortic smooth muscle cells incubated in physiological glucose (5.5 mM) and under hyperglycemic conditions (20 mM) for 5 days showed altered levels of modification in at least 60 proteins.107These studies link hyperglycemia, increased flux through the HBP and thus elevation of the OGT substrate UDP-GlcNAc.

BothOGlcNAc processing enzymes have been implicated in type 2 diabetes. A number of studies have established the importance of OGT in pancreatic β-cell function. Indeed, OGT is most highly enriched in the β-cells, and increasing UDP-GlcNAc levels associated with hyperglycemia are thought to cause altered O-GlcNAcylation that ultimately leads to β-cell dysfunction.108-110A common model for inducing diabetes in rats and mice is by injection with a GlcNAc analog, 2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose (streptozotocin, STZ), which destroys pancreatic β-cells,111mimics β-cell dysfunction. In STZ-induced diabetic rats, not only is O-GlcNAcylation dramatically increased, but OGT protein levels and OGT activity are also increased.110In addition, glucose and glucosamine potentiated STZ toxicity of pancreatic β-cells in mice. Treatment with 50mgkg−1of STZ was insufficient to induce β-cell death alone; however, infusion with glucose or glucosamine induced apoptosis in the β cells.109Targeted overexpression of OGT in the muscles or adipocytes of transgenic mice results in overt symptoms of type 2 diabetes.112Finally,ogt-1 C. elegansshowed altered macronutrient storage by having increased levels of trehalose and glycogen compared to wild-type nematodes.31In addition, mutants had fewer lipid stores.C. eleganswith inactivating mutations in the insulin signaling pathway have extended lifespan by forming dauers.1 augwere able to suppress dauer formation, suggesting that OGT functions by inhibiting insulin signaling.

The enzyme responsible for the removal ofO-GlcNAc,O-GlcNAcase, has been linked to type 2 diabetesO-GlcNAcase has been mapped to the chromosomal location 10q24.1-q24.3, a region associated with age of onset and susceptibility to NIDDM.113While ratting a mouseO-GlcNAcase maps to a locus that also contains the insulin degrading enzyme (IDE) gene,114.115a key enzyme in controlling the action of insulin through its breakdown.116In addition, a single nucleotide polymorphism (SNP) has been found inO-GlcNAcase was associated with type 2 diabetes in Mexican Americans.117This SNP was found in intron 10, caused an alternative splice variant and resulted in a C-terminal deletion and therefore a catalytically inactive mutant ofO-GlcNAcase. This study is consistent with the hypothesis that increased protein O-GlcNAcylation leads to the diabetic phenotype.

The molecular mechanism of insulin action involves the binding of insulin to the insulin receptor. Binding activates the receptor's intrinsic kinase activity, autophosphorylating itself and creating binding sites for adapter molecules such as insulin receptor substrates (IRSs) 1 and 2. Recruitment of phosphatidylinositol-3-kinase (PI3K) initiates a cascade of signal transduction leading to protein kinase. activation B/Akt, transcription of genes involved in proliferation, translocation of the glucose transporter GLUT4 and stimulation of glucose metabolism.118Several recent studies have suggested a possible molecular mechanism for thisO-GlcNAc in insulin resistance. Chemically risingO-GlcNAc levels by incubating adipocytes inO-GlcNAcase inhibitor PUGNAc caused insulin resistance as determined by reduced insulin-stimulated glucose uptake and reduced GLUT4 translocation to the plasma membrane.119,120In addition, decreases in active Akt were observed as its activating phosphorylation was blunted, as was phosphorylation of its substrate glycogen synthase kinase β (GSK3β). Rises inOGlcNAc-modified Akt, IRS-1, and GLUT4 were observed, suggesting that the increase in O-GlcNAcylation of these proteins induces the insulin resistance phenotype by impairing their function. Glycogen synthase (GS), an enzyme that plays a crucial role in glucose metabolism by synthesizing glycogen, is a highly regulated enzyme. In the starved state, it is inhibited by GSK3β to synthesize the rapidly released storage form of glucose. Upon insulin stimulation, GSK3β is inactivated by Akt, relieving the repression of GS. GS is also allosterically regulated by glucose-6-phosphate (G6P). Glycogen synthase has also been shown to be regulated by O-GlcNAcylation.121.122Increased O-GlcNAcylation of GS was observed in adipocytes cultured in high glucose or glucosamine, and this increase inO-GlcNAc modification correlated with decreased activity of the enzyme.O-GlcNAc modification of GS was also increased in the fat pads of streptozotocin-treated diabetic mice with a concomitant decrease in GS activity. These studies indicate that in the diabetic state, key regulatory enzymes within the insulin signaling pathway are abnormally O-GlcNAcylated, leading to insulin resistance.

In addition to proteins directly involved in the insulin signaling pathway, flux through HBP increased and therefore increasedO-GlcNAc leads to the pathophysiology of NIDDM by affecting O-GlcNAcylation of other proteins. It is becoming clear that an important mechanism of "glucose toxicity" is the abnormal increase in glucose levelsO-GlcNAc on key signaling/regulatory proteins. Atherosclerosis, symptomatic of patients with diabetes, is closely related to inflammation, and certainly markers associated with inflammation are found in type 2 diabetics. Nuclear factor κB (NF-κB) is a transcription factor normally sequestered by IκB in the nucleus.123When cells are stimulated, IκB targets proteolytic degradation and NF-κB can translocate to the nucleus and activate transcription of genes involved in stress response, inflammation and apoptosis.124High glucose has been shown to maintain NF-κB activation, and subsequent expression of proinflammatory cytokines is thought to be mediated by hyperglycemic induction of NF-κB.125-127These observations provided the basis for a study showing that NF-κB was O-GlcNAcylated, and increasing flux through HBP, either by incubation in high glucose or in glucosamine, increased O-GlcNAcylation at this transcription factor.128Simultaneously with increaseO-GlcNAc, an increase in NF-κB reporter activity was reported as assessed by luciferase reporter assays and by electrophoretic mobility shift assays (EMSAs). In addition, genes responsive to NF-κB activity and known to be upregulated in the diabetic state are vascular cell adhesion molecule 1 (VCAM-1), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL). - 6 ), was expressed.

Diabetic cardiomyopathy is characterized by altered calcium homeostasis, oxidative stress and altered energy metabolism.129It has been established that hyperglycemia can impair cardiomyocyte contractility130and elevation of intracellular calcium levels.131Men clarket al. provided a link between hyperglycemia and dysfunctional cardiomyocyte function to O-GlcNAcylation.132Increasing UDP-GlcNAc levels by incubating neonatal rat cardiomyocytes in high glucose, glucosamine, or PUGNAc increased intracellular calcium levels. In addition, adenoviral infection withO-GlcNAcase in the presence of high glucose only attenuated the effect of high glucose, suggesting that increase ofO-GlcNAc levels disrupt calcium cycling in cardiomyocytes. SERCA2, the sarcoendoplasmic reticulum (SR) calcium ATPase responsible for calcium reuptake and for diastolic relaxation, was downregulated in cells with increased O-GlcNAcylation as assessed by Northern blotting, Western blotting and luciferase reporter assays. Perhaps the most compelling data pointing to a role forO-GlcNAc in diabetic cardiomyopathy is the overexpressedO-GlcNAcase in STZ-treated diabetic mice improved cardiac contractile function.133Moreover, increasingO- GlcNAc levels with incubation in glucosamine or in PUGNAc attenuated baseline calcium levels in rat neonatal ventricular myocytes, suggesting thatO-GlcNAc can affect calcium homeostasis in cardiomyocytes.134In addition, diabetes-associated O-GlcNAcylation of endothelial nitric oxide synthase blocks its activation by Akt kinase135and directly leads to diabetes-induced erectile dysfunction.136

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The role of post-translational protein modifications on cardiovascular metabolism

Florence Mailleux, ... Luc Bertrand, iBiochimica et Biophysica Acta (BBA) - Molecular basis of disease, 2016

(Video) The split personality of human O-GlcNAc transferase

6 Molecular mechanisms by which cardiac hypertrophy promotes O-GlcNAcylation

As previously mentioned, pro-hypertrophic stimuli induce marked increases in O-GlcNAc levels both in isolated cardiomyocytes and in animal and human models of cardiac hypertrophy. One wonders about the mechanisms involved in this increase in O-GlcNAc. As mentioned earlier, the O-GlcNAcylation process is mainly mediated by three enzymes, GFAT, OGT and OGA, the first being the rate-limiting step in the HBP pathway and the latter two being involved in the direct insertion and removal of the O-GlcNAc group. respectively (figure 1). Lunde and colleagues have largely described the regulation of these enzymes[56]. Regarding pressure overload-induced cardiac hypertrophy, aortic banding in rats increases GFAT2 protein level. Similarly, both OGT mRNA and protein levels are elevated in such a hypertrophic model. These results are supported by the study by Facundo and colleagues, who showed that both treatment of cardiomyocytes for 48 hours with phenylephrine and transverse aortic constriction in mice promote increases in OGT protein levels.[59]. Another model of hypertrophy evaluated by Lunde and colleagues is the SHR rat. SHR rats show higher GFAT2 protein level, increased OGT mRNA and protein levels, while OGA protein level does not change[56]. These authors also showed that patients suffering from aortic stenosis are characterized by a higher OGT mRNA level, with no differences observed for OGA. In contrast, aortic ligation was also found to increase OGA protein levels[56].

These studies shed some light on the molecular mechanism that regulates O-GlcNAc during the development of cardiac hypertrophy. However, several questions remain unanswered. As mentioned above, transverse aortic stenosis is clearly characterized by an increase in O-GlcNAc level. Not only GFAT and OGT, but also OGA, which work in the opposite way, all act similarly upregulated by pressure overload. An explanation can be found in the fact that these data are mainly obtained at the endpoint when cardiac hypertrophy is largely established. As the proposed hypothesis assumes that the O-GlcNAc process is part of the early events involved in the onset of hypertrophy, it will be necessary to examine GFAT, OGT and OGA expression during early time points. Because the molecular mechanisms involved in cardiac hypertrophy are complex (including contractile machinery, mitochondrial modifications, membrane and nuclear events), the subcellular function of these three enzymes would be worthy of further investigation. Their post-translational regulation is another challenging point to investigate. In fact, in the previous chapter we investigated how O-GlcNAcylation regulates the signaling pathways involved in the development of hypertrophy. Conversely, it would be interesting to see if and how these signaling pathways interact with HBP and O-GlcNAc enzymes. In the same context, it is interesting to point out that both GFAT and OGT interact with the AMP-activated protein kinase (AMPK).[107.108]. AMPK is a major sensor of energetic imbalance that exhibits several cardioprotective effects[109.110]. AMPK is activated during the development of cardiac hypertrophy and has a well-described antihypertrophic activity. It can be considered a negative feedback loop that occurs during this pathology, and its pharmacological activation has been shown to be largely protective during the development of hypertrophy[109.110]. AMPK has been shown to phosphorylate GFAT in non-myocytic cell models, modulating its activity[107]. Bullen and colleagues described a strong interplay between AMPK and OGT, both of which regulate each other[108]. In the future, it would be useful to evaluate the putative role of AMPK/O-GlcNAc interaction in hypertrophic hearts. In conclusion, GFAT, OGA and OGT play an important role during the development of cardiac hypertrophy. Pharmacological modulation of these enzymes involved in HBP could represent a future therapeutic approach for the treatment of this heart disease.

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Mechanistic Biology • Chemical Biophysics

Dustin T. King, ... David J. Vocadlo, iCurrent Opinion in Chemical Biology, 2019

Transcriptional activation and repression

New findings are emerging to support that the expression of OGA and OGT is coordinately regulated by the control of transcriptional programs that act to maintain balanced O-GlcNAc levels. For example, increases in OGT lead to concomitant increases in OGA expression, and vice versa, in what appears to be an attempt to buffer the cell against rapid changes in O-GlcNAcylation. In addition, treatment of cells with the selective OGA inhibitor ThiametG increases O-GlcNAc levels and induces a compensatory increase in OGA gene expression and a correlated decrease in OGT abundance [51], and the reverse is also true for OGT inhibitors [52].

We are just beginning to discover the transcriptional programs that control these coordinated shifts in expressionOGTIOGA. For example, OGA acts as a co-activator in combination with the HAT p300 to positively regulate the transcription factor CCAAT/enhancer-binding protein beta, one of the most abundant activators involved inOGTdistrict attorney (Figure 5) [53]. This transcriptional regulatory mechanism allows OGT expression to be tuned to OGA levels to balance O-GlcNAcylation. that tooOGApromoter is sensitive to OGT expression, as illustrated by a recent analysis of O-GlcNAcylation in patients with XLID. An XLID variant L254F-OGT results in reduced L254F-OGT protein levels in isolated lymphoblastoid cells from patients [3]. However, the global abundance of O-GlcNAc appeared unchanged, presumably due to a compensatory decrease in OGA mRNA and downstream OGA levels. OGT appears to be involved in a balance mechanism that regulates global O-GlcNAc levels in XLID through formation of an OGT-mSin3A-HDAC1 repressive complex atOGAproximal promotorregio (Figure 5) [3]. Curiously, E2F transcription factor 1 (E2F1) simultaneously represses transcription in bothaugIogapromoters in a retinoblastoma (pRb)-dependent manner in mice to maintain a balanced mRNA pool (Figure 5) [54].OGTtranscription also appears to be regulated by hepatocyte nuclear factor 1 homolog A, which is itself O-GlcNAc modified. Loss of O-GlcNAc appeared to increase the transcription ofOGT, suggesting a mechanism for autoregulation of O-GlcNAc homeostasis [55]. Overall, a higher-order network of multiple transcriptional mechanisms likely interacts to maintain O-GlcNAc homeostasis, but deciphering this requires both more detailed biochemical and systems-wide studies.

Protein O-GlcNAc Transferase - an overview (1)

Figure 5.Transcriptional and post-transcriptional regulation ofOGAIOGTexpression. OGT is regulated at the transcriptional level by activators (C/EBPβ, Nrf2), which are themselves modulated by secondary regulatory mechanisms. Both OGA and OGT promoters are negatively regulated by E2F1 in an Rb1-pRb-dependent manner. In addition, OGA is thought to be transcriptionally repressed by the OGT-mSin3A-HDAC1 complex. The OGT mRNA transcript contains a retained fourth intron with an O-GlcNAc-sensitive internal splicing silencer (ISS). Under conditions of high O-GlcNAc, ISS prevents cleavage of the retained intron, leading to nuclear degradation of the transcript, while low O-GlcNAc results in cleavage of the intron and subsequent nuclear export. The cytoplasmically located mature OGT and OGA mRNA transcripts are either translated into protein product or can be targeted for degradation via hybridization to miRNAs and subsequent RNA-induced silencing. OGT and EZH2 participate in a negative feedback mechanism to repress the miRNA-101 promoter. C/EBPβ, CCAAT/enhancer-binding protein beta; miRNA, microRNA; Nrf2, ENF-E2-related factor 2; OGA, O-GlcNAc hydrolase; OGT, O-GlcNAc transferase; O-GlcNAc, O-linkedN-acetylglucosamin.

Transcription of O-GlcNAc cyclic enzymes is also dysregulated in response to other specific stimuli. For example, OGT levels are markedly elevated in human papillomavirus (HPV)-induced cervical neoplasms, and transfection of HPV oncogenes into mouse embryonic fibroblasts causes a marked increase inOGTmRNA and protein levels [56]. HPV infection alters the expression of several transcription factors with predicted binding sites withinOGTpromoter. Enhanced overexpression of transcription factors AP-1, SP-1, NF-κB, p65 and c-MYCOGTpromoter activity on a reporter construct [56]. But furtherI liveanalysis of the relative contribution of these transcription factors to endogenous amplificationOGTexpression is required. As a further example, upon lipopolysaccharide stimulation, NF-E2-related factor 2 (Nrf2) bindsaugpromoter and enhances its transcription in bone marrow-derived mouse macrophages, resulting in increased O-GlcNAcylation and inhibition of the pro-inflammatory transcription factor STAT3. Interestingly, Nrf2 is not required for baselineOGTexpression, but rather only increases its abundance in response to stimuli. In addition, the E3 ubiquitin ligase negatively regulates CUL3OGTexpression through its targeted degradation of Nrf2 (Figure 5) [57]. Future efforts should focus on discovering morecis- Indtrans-acting factors that regulate the transcription ofOGTIOGAand elucidate how they are integrated to modify transcriptional programs in response to various stimuli.

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Special section on post-translational modification

Victor M. Darley-Usmar, ... John C. Chatham, iJournal of Molecular and Cellular Cardiology, 2012

(Video) O Linked glycosylation

3 Pharmacological and molecular modulation of O-GlcNAc turnover

Experimentally, O-GlcNAc levels can be increased by activating synthesis, which is usually achieved by increasing extracellular glucose concentrations, adding extracellular glucosamine, or preventing its removal by inhibiting O-GlcNAcase. Hyperglycemia (~25 mM glucose) increases total O-GlcNAc levels in many cell lines, including neonatal and adult cardiomyocytes[34,45,46]. Although some effects of hyperglycemia in cardiomyocytes can be reversed by inhibition of GFAT with either 6-diazo-5-oxo-I-norleucine (DON) of O-diazoacetyl-I-cool (azaserin)[34,46], hyperglycemia clearly has many effects on cellular function apart from increasing metabolism via HBP. While azaserine and DON are commonly used as GFAT inhibitors, they are better characterized as amidotransferase inhibitors.[88]; therefore, conclusions based on its use should be drawn with caution. Glucosamine is readily transported by the glucose transport systems with a KMof 2-4 mM for GLUT1 and GLUT4[89], the primary glucose transporters in cardiomyocytes and then phosphorylated by hexokinase to glucosamine 6-phosphate. Glucosamine 6-phosphate enters HBP directly, bypassing the key regulatory enzyme GFAT (figure 1), resulting in an increase in UDP-GlcNAc and O-GlcNAc levels. In the perfused heart, 5 mM glucosamine leads to a 2- to 3-fold increase in O-GlcNAc levels within 15 min[32]and as little as 100 μM glucosamine has been shown to significantly increase cardiac O-GlcNAc levels[90]. Such studies highlight the dynamic nature of HBP and O-GlcNAc synthesis in the heart; however, increasing UDP-GlcNAc levels may also affect the synthesis of other glycoconjugates[91,92]and increased levels of other intermediates in HBP have the potential to alter other metabolic pathways, such as glycogen synthesis[93]. Nevertheless, increasing OGT protein levels at the cellular level appears to have the same effects on cardiomyocytes as glucosamine supplementation.[33,94], suggesting that the dominant effect of glucosamine, at least in the short term, is to increase cellular O-GlcNAc levels. Several studies have reported that glucosamine supplementation results in depletion of ATP levels[95]; however, we observed no influence of glucosamine treatment on ATP levels in cardiomyocytes or the intact perfused heart[34,36,90]. In addition, up to 10 mM glucosamine also had no negative effect on cardiac function[90].

PUGNAc [O-(2-acetamido-2-desoxy-D-glucopyranosylidene) amino-N-phenylcarbamate] is a commonly used competitive inhibitor of O-GlcNAcase[96,97]and by reducing the removal of O-GlcNAc, this leads to significant increases in cellular O-GlcNAc levels in isolated cardiomyocytes, the isolated perfused heart and in vivo[31,34,38,98]. While PUGNAc is a general tool for increasing O-GlcNAc levels, it is also equally effective as an inhibitor of lysosomal hexosaminidase[99], raising concerns about its specificity. Recently, two new classes of O-GlcNAcase inhibitors have been described; GlcNAcastin[100]and NAG-thiazolin[99.101], both of which show several orders of magnitude greater specificity for O-GlcNAcase relative to other hexosaminidases and with KIin vitro in the pico to nanomolar range. We have successfully used the NAG thiazoline derivatives 1,2 dideoxy-2'-propyl-α-D-glycopyranoso-[2,1-d]-Δ2'-thiazolin (NAG-Bt) og 1,2 dideoxy-2'-ethylamino-α-D-glucopyranoso-[2,1-d]-Δ2'thiazoline (NAG-Ae) to increase O-GlcNAc levels in the perfused heart and cardiomyocytes[37]. In the perfused heart, NAG-Bt had an EC50of ∼30μM to increase total O-GlcNAc levels[37]; in recent studies, we found in cardiomyocytes that NAG-Ae, also known as thiamet-G, had an EC50of ∼100nM to increase O-GlcNAc levels[102]. In addition to effectively increasing O-GlcNAc levels in isolated cardiomyocytes and hearts, preliminary in vivo studies show that i.p. administration of thiamet-G at a dose of 50 mg/kg resulted in a 2–3-fold increase in cardiac O-GlcNAc levels within 2 h of administration (Chatham data not shown). It is also worth noting that Yuzwa et al.[103]reported that thiamet-G added to drinking water was effective in increasing tissue O-GlcNAc levels; however, they did not report O-GlcNAc levels in the heart. We observed no adverse effects of O-GlcNAcase inhibitors on cardiac function in the perfused heart; thus, there appear to be no acute adverse effects of increasing cardiac O-GlcNAc levels by inhibiting O-GlcNAcase.

Although it is possible to pharmacologically increase O-GlcNAc levels by increasing synthesis or inhibiting degradation, there are limited options for reducing O-GlcNAc levels. The most common approach has been to attenuate OGT protein levels at the cellular level, either by using siRNA methods[33]increase O-GlcNAcase expression[40]or to completely remove OGT with Cre-lox technology[30]. In cell culture, OGT-null mouse embryonic fibroblasts (MEFs) grow normally for up to 48 hours, after which growth slows and cell death occurs around 4–5 days[30]. Watson et al.,[104]reported the development of a cardiac-specific, inducible OGT KO mouse and found that a reduction in OGT levels had no adverse effects under basal conditions but accelerated the development of contractile dysfunction in response to pressure overload. The lack of any effects of decreased OGT levels in the absence of additional stress was somewhat surprising since OGT gene deletion usually leads to cell death[30]. The reason for this lack of phenotype is unclear and may be related to transduction mosaicism or incomplete penetrance that may occur in such inducible models, or that although OGT levels are significantly lower, there has been an adaptive response leading to reduced O-GlcNAcase -protein, so O-GlcNAc turnover rates may not be significantly altered. Certainly in other cell systems, deletion of OGT leads to a reduction of the O-GlcNAcase protein in what appears to be an attempt to keep O-GlcNAc levels constant.[30]. However, it is also possible that since cardiomyocytes are essentially terminally differentiated, they can survive on very low levels of OGT until subjected to further stress.

The lack of specific inhibitors of high-affinity OGT was a major limitation in the study of the role of O-GlcNAcs on cardiomyocyte function. Walker and colleagues identified potential OGT inhibitors using in vitro high-throughput screening methods[105]. These compounds subsequently became commercially available as OGT inhibitors (TimTec,LLC); although there have been some reports in cell-based systems showing the effectiveness of these compounds in lowering O-GlcNAc levels[39.106], they have not yet been fully validated as OGT inhibitors and should therefore be used with caution. Indeed, our experience with these compounds to lower O-GlcNAc levels in cardiomyocytes has been unsuccessful, and we found that in an isolated perfused heart model, as little as 5 μM of “TT04” resulted in a significant decrease in cardiac function (Chatham, data not shown). ). Recently, Gloster et al.[107]described a new sulfonated UDP-GlcNAc analog that inhibited OGT in vitro with KIof 8 μM and significantly reduced O-GlcNAc levels in COS-7 cells. Clearly, further studies are needed to better characterize such inhibitors, but if their specificity can be fully validated and any effects on other glycosylation reactions can be characterized, the availability of these or similar compounds could be a useful addition to pharmacological modulation. -GlcNAc turnover of cardiomyocytes. .

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N-acetylglucosamine modification in the lumen of the endoplasmic reticulum

Mitsutaka Ogawa, ... Tetsuya Okajima, iBiochemistry and Biophysics Acta (BBA) - General subjects, 2015

6.2EGOmutation i Adams-Olivers syndrom

Although the biological roles of extracellular O-GlcNAc in mammals have not been elucidated,EGOmutations have been reported in patients with Adams-Oliver syndrome (AOS)[29,51]. AOS is a rare congenital disorder characterized by aplasia cutis congenita (ACC) at the vertex of the scalp and terminal transverse limb defects (TTLDs) (Afb.3ONE). The underlying pathophysiological mechanism in AOS remains unknown, but these defects are thought to be due to a vasculopathy, as cardiovascular defects are occasionally observed in AOS patients. In fact, cutis marmorata telangiectatica congenita (CMTC), atrial septal defect (ASD) and ventricular septal defect (VSD), caused byEGOmutations have been reported in some AOS patients[29,51].

Protein O-GlcNAc Transferase - an overview (2)

Afb.3. Candidate genes for Adams-Oliver syndrome (AOS).

(A) Representative clinical images of individuals from families of AOS caused by EOGT mutation (Reprinted with permission from Macmillan Publishers Ltd: European Journal of Human Genetics, Ref.[49], copyright 2014). AOS is a rare congenital disorder characterized by terminal transverse limb defects (shown in Fig. 3A[a]) and scalp defects (shown in Fig. 3A[b]). (B) Summary of the genetic mutations identified so far in AOS patients. ARHGAP31 and DOCK6 regulate the activity of a key molecule in the actin cytoskeleton (shown in Figblue).RBPJIHAK1is associated with Notch signaling (shown inpink). The biological function ofEGOhas not been clarified (shown ingul).

Three homozygous mutations forEGOhave been reported to date. All of these mutations reduce the ability to O-GlcNAcylate EGF domains. In addition, an EOGTR377Qmutant lost enzyme activity without affecting the ability to bind to EGF domains. These results suggested that reduced glycosyltransferase activity in mutant EOGT proteins and consequent defective O-GlcNAcylation in the ER is the molecular basis of AOS.[28].

In addition toEGO, homozygous mutations ofDOCK6IARHGAP31and heterozygous mutations ofRBPJIHAK1have been reported in AOS patients (Afb.3B)[52-56]. Cardiovascular defects have also been noted inrights 1- IndDOCK6related AOS. Both ARHGAP31 and DOCK6 regulate the activity of important regulatory proteins of the actin cytoskeleton, such as RAC1 and CDC42. Accordingly, fibroblasts were isolated from patients withARHGAP31-related orDOCK6-related AOS showed disorganized cytoskeleton and morphology[53,54]. In contrast to,EGOmutant fibroblasts showed a typical spindle appearance similar to control fibroblasts[51]. Therefore, EOGT does not appear to affect the actin cytoskeleton, although EOGT can affect actin dynamics in restricted cell types other than fibroblasts.

Of the AOS-causing proteins, Notch1, one of four members of the Notch receptor family, is the only protein modified by EOGT. After binding to Notch ligands, Notch receptors undergo proteolytic cleavage, releasing the Notch intracellular domain (NICD). In the absence of NICD, the DNA-binding protein, RBPJ, acts as a transcriptional repressor. In the nucleus, NICD binds RBPJ and Mastermind-like (MAML), forming the RBPJ/NICD/MAML transcriptional activation complex to induce the transcription of Notch target genes[57,58]. The pathogenRBPJmutations have been identified that lead to reduced binding to the Notch target promoter,HES1 [52]. In addition, five different heterozygous Notch1 mutations have been reported: an 85 kb deletion, including part of the promoter and the non-coding first exon; a splice site mutation; a cysteine ​​substitution in the EGF-like 11 domain; a cysteine ​​substitution in the LNR domain; and an Asp1989Asn mutation in ankyrin repeats. However, the effects of these mutations on Notch signaling have not been investigated. It has been reported that both decreased and increased Notch signaling can lead to vascular abnormalities[59]. Thus, the mechanism by whichHAK1mutations and EOGT-catalyzed O-GlcNAcylation that affect the structure and function of Notch receptors may provide insight into the pathophysiology of AOS.

Notch1 EGF domains are modified simultaneously with other EGF domain-specific glycosylations (using O-fucose and O-glucose)[60]. O-fucosylation and O-glucosylation are catalyzed by POFUT1/Ofut1 and POGLUT1/Rumi, respectively[61,62]. Both types of O-glycosylation play key roles in Notch signaling such as trafficking, processing and ligand binding[63-67]. Interestingly, heterozygous mutations (presumed haploinsufficiency) for POFUT1 or POGLUT1 cause Dowling-Dego disease, an autosomal dominant genodermatosis characterized by reticular pigmented anomaly[68-70]. Therefore, O-fucose and O-glucose play an important role in the development of Notch-dependent melanocyte lineages. In contrast, similar anomalies have not been reported in AOS patients. The different contributions of O-fucose/O-glucose and O-GlcNAc in human pathology imply that different O-glycans on Notch receptors have different functions in the regulation of Notch receptors.

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Pathophysiological significance of glycans and glycosylation

Yi Zhu, Gerald W. Hart, iMolecular aspects of medicine, 2021

2.1 O-GlcNAc-transferase (OGT)

O-GlcNAc transferase (OGT) is the only enzyme that O-GlcNAcylates intracellular proteins. It is highly conserved across metazoans, with 97% similarity to humans (A wise man, NP_858058.1) and zebrafish (Danio rerio, NP_001018116.1), and 86% similarity between human and fruit fly (Drosophila melanogaster, NP_724407.1). In humans, this enzyme is encoded by the OGT gene on the X chromosome (Xq13.1) and is associated with the genetic locus for Parkinson's disease and dystonia (Graeber et al., 1992;Shafi et al., 2000). Expression forOGTthe gene is ubiquitous, its mRNA level was found to be highest in the β-cell of the pancreas, brain and thymus, and lowest in the lung and liver (Hannover et al., 1999;Kreppel et al., 1997). Knock it outOGTthe gene is embryonically lethal in most model organisms with the exception ofC. elegans(Forsythe et al., 2006;Shafi et al., 2000).

The crystal structure of OGT has been solved. According to its structure, the enzyme OGT has three domains: a tetratricopeptide repeats (TPR) domain at the N-terminus, which is responsible for target recognition by the enzyme; a catalytic domain at the C-terminus; and a flexible "hinge" area between the two (Lazarus et al., 2011). There are three splice isoforms of OGT that differ in the number of TPRs: a longest isoform containing 13.5 TPRs and located in the nucleus and cytoplasm (nucleocytoplasmic OGT; ncOGT); an isoform possessing 9.5 TPRs and localized to the inner membrane of mitochondria (mitochondrial OGT; mOGT); and a shortest isoform that has only 2.5 TPRs (short form OGT; sOGT) (Hannover et al., 2003;Liu et al., 2019;Love et al., 2003). A nuclear localization signal (NLS) sequence consisting of three amino acid residues (DFP, residues 451–453) is located in the linker region between the TPR and the catalytic domain (SEO et al., 2016). In dividing cells, the OGT protein is more abundant in the nucleus than in the cytoplasm, but it is excluded from the nucleus (Zeidan et al., 2010).

The sugar donor of OGT is uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine biosynthetic pathway (HBP) (Haltiwanger et al., 1992;Wang et al., 1998). To produce UDP-GlcNAc, HBP integrates metabolites of the metabolic pathways of glucose, amino acids, fatty acids and nucleic acids (figure 1). Thus, the level of UDP-GlcNAc is sensitive to all these nutrients, and OGT is highly sensitive to UDP-GlcNAc, both in terms of its activity and substrate selectivity. Thus, UDP-GlcNAc is an important metabolism hub, and O-GlcNAc acts as a "nutrient sensor" in cells (Hart et al., 2011).

Protein O-GlcNAc Transferase - an overview (3)

figure 1.The hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation. HBP integrates nutrient metabolic pathways and provides sugar donor for protein O-GlcNAcylation. HK, hexokinase; GPI, glucose-6-phosphate isomerase; GFAT, glutamine:fructose-6-phosphate amidotransferase; GNPNAT, glucosamine-6-phosphate-N-acetyltransferase; PGM, phosphoglucomutase; UAP, UDP-N-Acetyl Glucosamine Pyrophosphorylase.

To differentially modify thousands of its substrates, the activity and specificity of OGT must be regulated. To date, three ways to tune the function of OGT in a cell have been discovered. First, the activity of OGT is affected by the concentration of UDP-GlcNAc. From 50 nM to 4.8 mM UDP-GlcNAc, OGT showed three clearKMvalues ​​(6µM, 35µM and 217µM). The enzyme is more active at higher UDP-GlcNAc levels (Kreppel and Hart, 1999). Thus, the activity of OGT is changed by the activity of enzymes in HBP and the availability of other metabolites in a cell. Second, like many other proteins, the function of OGT is regulated by post-translational modifications. The small negatively charged phosphate group usually generates a subtle change in the local structure of proteins (Cohen, 2002) and also plays a role in the regulation of OGT. For example, upon insulin stimulation, the insulin receptor (IR) phosphorylates OGT on tyrosine residues and activates the enzyme (Whelan et al., 2008). Phosphorylation at Thr444 by AMP-activated protein kinase (AMPK) alters OGT nuclear localization and substrate selectivity (Bullen et al., 2014). Glycogen synthase kinase 3β (GSK3β) activates OGT by phosphorylating the Ser 3 and Ser 4 residues (Kaasik et al., 2013). In glioblastoma cells, OGT is phosphorylated and activated by calcium/calmodulin-dependent kinase IV (CaMKIV), following KCl-induced depolarization (Song et al., 2008). CaMKII phosphorylates OGT at Ser 20 and in turn increases its activity at Ulk1 (Ruan et al., 2017). OGT is autoglycosylated (Fan et al., 2018;Griffin et al., 2016;Kreppel et al., 1997;Tai et al., 2004). O-GlcNAc at Ser 389 of ncOGT affects nuclear localization in HeLa cells (SEO et al., 2016). On sOGT, O-GlcNAcylation at Thr12 and Ser 56 does not alter activity, but these modifications regulate the substrate selectivity of the enzyme (Liu et al., 2019). Finally, the substrate selectivity of OGT is regulated by some of its binding partners. For example, PGC-1α targets OGT to the transcription factor FoxO1, increases O-GlcNAcylation on FoxO1, and increases its transcriptional activity (Housley et al., 2008,2009).

Surprisingly, OGT also catalyzes a proteolysis reaction. A peptide representing the threonine-rich region of host cell factor-1 (HCF-1) can be cleaved by OGT at a glutamic acid residue. The cleavage requires UDP-GlcNAc and is inhibited by UDP and UDP-5S-GlcNAc. When the glutamic acid at the cleavage site is mutated to a serine, the O-GlcNAcylation reaction occurs instead of proteolysis (Capotosti et al., 2011;Lazarus et al., 2013). These observations suggest that OGT used the glycosylation mechanism to catalyze the hydrolysis of certain peptide sequences. However, the HCF-1 peptide is the only known substrate cleaved by OGT to date.

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An update on the unfolded protein response in brain ischemia: experimental evidence and therapeutic options

Xuan Li, Wei Yang, opNeurochemie International, 2021

3 IRE1 UPR-tak

The central function of the IRE1 branch is mediated via unconventional cleavage ofXbp1mRNA. Under ER stress, IRE1 becomes a functioning endonucleaseXbp1mRNA, removing its 26-nt fragment to generate splicedXbp1mRNA (Xbp1s).Xbp1sis then translated into a 54 kDa protein, XBP1s. XBP1s is a transcription factor that regulates the expression of many genes related to protein homeostasis (Hetz et al., 2020). In particular, XBP1s can modulate the hexosamine biosynthetic pathway (HBP) by upregulating the expression of key enzymes. The HBP produces UDP-GlcNAc, the substrate for the post-translational modification of O-GlcNAcylation. Thus, the XBP1s/HBP/O-GlcNAc axis was proposed, which has been shown to function in the brain and heart (Jiang et al., 2017;Wang et al., 2014). Note that O-GlcNAcylation involves 2 enzymes that add and remove O-GlcNAc from proteins: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively.

It is well establishedXbp1smRNA levels increase rapidly in the brain after ischemic stroke and cardiac arrest (Shen et al., 2018;Yu et al., 2017), indicating activation of the IRE1/XBP1s branch after cerebral ischemia. Early evidence suggesting the role of this branch in cerebral ischemia was largely obtained fromin vitrosurveys. It was proven by dataXbp1splicing is induced in hippocampal neurons exposed to oxygen/glucose deprivation (OGD).in vitroischemia-like model and overexpression ofXbp1ssuppresses OGD-induced neuronal cell death (Ibuki et al., 2012). However,Directevidence for this protective role was long lacking. Recently, the use of both loss-of-function and gain-of-function mouse models has been significantDirectdata on this branch has been generated in relation to stroke and cardiac arrest. Such studies have shown that neuron-specific deletion ofXbp1leads to a worse outcome after transient or permanent ischemic stroke, whereas neuron-specific expression ofXbp1simproves short-term neurological function and reduces infarct volume (Jiang et al., 2017;Wang et al., 2021). Similar results were observed in a mouse model of cardiac arrest (Li et al., 2021).

These XBP1-specific mice were also used to study the XBP1s/HBP/O-GlcNAc axis in the brain. As expected insideXbp1stransgenic mice, key HBP enzymes are upregulated and global O-GlcNAcylation is increased in the brain (Jiang et al., 2017). Interestingly, using a newly developed analytical method, our group further demonstrated that brain UDP-GlcNAc levels are closely related to the activity of the XBP1 pathway, providing direct evidence for the link between XBP1s and HBP (Wang et al., 2021). Pharmacological stimulation of O-GlcNAcylation with thiamet-G, a potent OGA inhibitor, partially reverses the worse stroke outcome inXbp1knockout mus (Wang et al., 2021). Taken together, the IRE1/XBP1 UPR is a pro-survival pathway in brain ischemia, and a critical mechanism underlying its neuroprotective effects is the XBP1/HBP/O-GlcNAc axis.

(Video) Cardiac Protein O-GlcNAcylation Induces Cardiac Hypertrophy and Increases Risk of Heart Failure

Currently, targeting the IRE1 branch in brain ischemia has focused on the XBP1/HBP/O-GlcNAc axis. To stimulate this axis, approaches can be designed to intervene in the O-GlcNAcylation cycle by targeting 2 enzymes (ie, OGA and OGT), or to increase the substrate UDP-GlcNAc by targeting HBP. Both approaches have been used in experimental brain ischemia. We and others have shown that thiamet-G treatment to increase O-GlcNAcylation is beneficial in both stroke and cardiac arrest (Gu et al., 2017;Hij et al., 2017;Jiang et al., 2017;Shen et al., 2018;Wang et al., 2021). For example, mice treated with thiamet-G show smaller infarct volumes and better short-term functional outcome after transient and permanent stroke. Recently, we provided evidence that thiamet-G treatment also improves long-term functional outcome in young mice and old rats after ischemic stroke (Wang et al., 2021). To increase UDP-GlcNAc, glucosamine has been evaluated. Glucosamine can be easily converted to glucosamine-6-phosphate and then enter the HBP flux. Indeed, glucosamine dosing increases O-GlcNAcylation in the brain and exerts beneficial effects in ischemic stroke (both transient and permanent stroke) and cardiac arrest (Gu et al., 2017;Hwang et al., 2010;Li et al., 2021;Wang et al., 2021). For example, glucosamine treatment significantly reduces infarct volume in rats after transient stroke and improves short-term functional recovery in both young and old mice after permanent stroke. In addition, this treatment may also benefit the long-term recovery of neurological function after transient middle cerebral artery occlusion (MCAO; a widespread stroke model) in young mice (Gu et al., 2017). Overall, there is compelling evidence to support the idea that increased O-GlcNAcylation is cytoprotective in brain ischemia.

Due to the lack of resources, pharmacological targeting of the IRE1/XBP1 pathway (not only downstream O-GlcNAcylation) has not been performed in brain ischemia. However, this approach could be very interesting based on the literature, as many studies have shown remarkable positive effects of the IRE1/XBP1 pathway in animal models of various diseases (Casas-Tinto et al., 2011;Cisse et al., 2017). Therefore, the search for small molecules that specifically activate this pathway has attracted much attention. It is important to note that in addition to splicingXbp1mRNA, activated IRE1 can degrade mRNAs through a mechanism called regulated IRE1-dependent decay (RIDD), a process that contributes to apoptosis. Recently, the Wiseman group identified some IRE1/XBP1 activators that selectively activate IRE1-dependent pro-survival XBP1s signaling without promoting the deleterious RIDD process (Grandjean et al., 2020). One of these activators in particular, IXA4, has been shown to ameliorate amyloid precursor protein (APP)-mediated toxicity (Grandjean et al., 2020). Thus, it would be intriguing to assess the therapeutic potential of using IXA4 to target the IRE1/XBP1 pathway in brain ischemia.

Current brain ischemia studies for this branch have mainly focused on neurons. However, when targeting the branch pharmacologically for therapeutic purposes, we must consider other cell types. For example, the IRE1/XBP1 branch also plays a crucial role in glial cells. A recent study has shown that activation of the IRE1/XBP1s pathway in astrocytes promotes their pathogenic activities in experimental autoimmune encephalomyelitis (EAE) by driving pro-inflammatory responses, for example upregulation of inflammatory genes and monocyte recruitment (Wheeler et al., 2019). This critical aspect needs to be clarified in the context of brain ischemia, as neuroinflammation is a crucial contributor to ischemia-induced brain damage.

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O-GlcNAcylation regulation of cellular signaling in cancer

Lorela Ciraku, ... Mauricio J. Reginato, iCellular signaling, 2022

3 Metabolic pathways

3.1 Metabolic sensor

The role of O-GlcNAcylation as a metabolic sensor has been extensively studied[77-79]. Recent findings support regulation of cellular bioenergetics through O-GlcNAc modifications. Although not widely studied, the mitochondrial isoform of OGT may also be O-GlcNAcylate proteins in the mitochondria. It has recently been shown that expression of mitochondrial OGT (mOGT) and O-GlcNAc levels in breast cancer cells is dependent on glucose and that overexpression of mOGT levels leads to increased mitochondrial membrane potential and respiration, leading to an increase in reactive oxygen species (ROS). ) and decrease in cellular ATP. O-GlcNAcylated proteins in cells overexpressing mOGT were found in the mitochondrial matrix and inner membrane, and these identified proteins play a role in mitochondrial respiration, fatty acid metabolism, transport, translation, and apoptosis, suggesting the possible role of O-GlcNAcylation in cellular bioenergetics. and mitochondrial homeostasis and function[80]. Elevated O-GlcNAc is also seen in patients with diabetes mellitus 2 (DM2), mainly characterized by a hyperglycemic state that promotes an increased risk of developing breast cancer[81,82]with poor survival outcomes[83]. In a syngeneic diabetes-induced breast cancer mouse model, tumors from diabetic mice had elevated levels of O-GlcNAc compared with tumors from non-diabetic mice and showed higher malignant tumor characteristics such as vascularization, proliferation, adjusted collagen, and an M2 tumor-associated macrophage (TAM) profile , which promotes a protumor environment. These phenotypes, including O-GlcNAc levels, were reduced after treatment with metformin, a blood glucose-lowering drug[84]. The higher O-GlcNAc levels in tumors from diabetic mice reflect the role of O-GlcNAc as a metabolic sensor of the organism's nutritional status that may contribute to a more aggressive tumor phenotype induced by hyperglycemia.

Despite the fact that elevated O-GlcNAc levels are maintained in many cancer cells, a homeostatic response to O-GlcNAc fluctuations is also active. Homeostasis of cellular O-GlcNAc levels is maintained through feedback regulation of OGT and OGA, whose expression is modulated in response to drastic changes in O-GlcNAc levels, as has been shown in neuroblastoma, leukemia, cervical and colon cancer cell lines[85,86]. A recent study suggests that OGA expression upon induced increased or decreased levels of O-GlcNAc is regulated at the transcriptional level and in an epigenetically dependent mechanism regulated by histone acetylation.[87]. Conversely, increased O-GlcNAc 4E-BP1 induces O-GlcNAcylation at Ser5 and Ser6, which acts as a sensor of O-GlcNAc levels to upregulate OGT protein levels in a feedback loop in lung cancer cells[87].

3.2 Modulation of signal metabolism pathways

Increased glucose uptake diverted to glycolysis is a hallmark of cancer cells originally observed by Otto Warburg[88]. It was recently reported that the reliance of cancer cells on glycolysis rather than oxidative phosphorylation involves increased O-GlcNAcylation. In particular, phosphoglycerate kinase 1 (PGK1), the first ATP-producing enzyme in glycolysis, is O-GlcNAcylated at Thr255, which promotes mitochondrial translocation, where it inhibits pyruvate dehydrogenase and thus reduces oxidative phosphorylation in colon cancer cells.[89]. Another example of the glycolytic-HBP connection regulating tumor cell proliferation includes the elevated O-GlcNAc levels in pancreatic cancer that mediate O-GlcNAcylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) in hypoxic172 conditions. . This competes for phosphorylation at this site by ERK and promotes nuclear translocation of O-GlcNAcylated PFKFB3, preventing p27 hypoxia-induced accumulation and leading to cell cycle progression[90].

Another study suggested control of O-GlcNAc levels through glucose utilization that occurs through regulation of HBP enzymes. Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) was reported to induce an increase in O-GlcNAc levels, which may contribute to epithelial-mesenchymal transition (EMT) in serous ovarian cancer (SOC).[91].

In addition to the clear role of OGT and O-GlcNAcylation in the regulation of tumorigenesis, the role of its counterpart, OGA in cancer, has been investigated to a lesser extent. Singh et al. show that overexpression of OGA induced increased tumor progression in mice through its non-intrinsic acetyltransferase activity. In particular, OGA promotes acetyltransferase activity on pyruvate kinase muscle isoform 2 (PKM2), the enzyme that regulates the conversion of phosphoenolpyruvate (PEP) to pyruvate, by inducing its acetylation, which in turn increases O-GlcNAcylation, which correlates with increased tumorigenesis. OGA overexpression. This regulation is dependent on glucose and inhibits the catalytic activity of PKM2 by blocking its tetramerization, promoting aerobic glycolysis and tumor growth[92]. Taken together, these findings reinforce the dependence of O-GlcNAcylation on nutrient availability and its potential to influence the metabolic rewiring that supports cancer progression.

O-GlcNAcylation has also been linked to regulation of lipogenesis in adipocytes and lipogenic cancers. Studies have shown that in adipocytes, O-GlcNAc levels, regulated by glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), the first rate-limiting enzyme in HBP, increase during adipogenesis and the reduction of UDP-GlcNAc that reduces O-GlcNAcylthio levels results in a reduction of fatty acid synthase (FASN) and other lipid accumulation proteins as well as a blockade in adipogenesis[93]. This regulation is thought to be driven by O-GlcNAcylation of (CCAAT/enhancer-binding protein β) C/EBPβ and (peroxisome proliferator-activated receptor γ) PPARγ[93]. In addition, increased O-GlcNAc levels in obese mouse livers correlated with increased FASN levels, and interestingly, FASN was found to be O-GlcNAcylated, which reduces proteosomal degradation by interacting with the deubiquitinating enzyme nucleolar and spindle-associated protein 1 (NUSAP1) promoter. in normal liver cell lines and hepatocarcinoma lines[94]. In addition, Sodi et al. shown that O-GlcNAc regulates the master lipid regulator SREBP1 and its transcriptional targets, including FASN, ACLY, ACC and othersviaAMPK regulation in breast cancer cells and lipogenic tissue and that SREBP1 can overcome growth defects caused by reduced O-GlcNAcylation in breast tumors[95].

Rabb et al recently showed that OGT and FASN are co-localized in hepatocellular carcinoma HepG2 lines, O-GlcNAcylation of FASN is dependent on glucose levels, and FASN expression depends on OGT upon serum stimulation in a cell cycle-dependent manner[96]. Importantly, their study reveals regulation of FASN in light of a previously investigated link between O-GlcNAc and another fundamental energy-sensing pathway, such as the mammalian target of rapamycin (mTOR).[57,97]. FASN expression correlated with mTOR activation and increased O-GlcNAcylation in two mouse models of obesity and chronically activated insulin and mTOR, and importantly, inhibition of FASN reduced O-GlcNAcylation and mTOR activation, suggesting a dual regulation of FASN by O -GlcNAc and mTOR suggest, but also a feedback loop between these three nutrient-dependent pathways. O-GlcNAc can also regulateagainlipogenesis in breast cancer cell lines through O-GlcNAcylation of serine/arginine-rich protein kinase 2 (SRPK2) by a nuclear localization signal that activates its nuclear translocation through importin-α and enables phosphorylation of proteins that promote cleavage of lipogenic pre-mRNAs[98]. In hepatocellular carcinoma (HCC), acyl-CoA ligase 4 (ACSL4) levels, which mediate fatty acid synthesis, are elevated, and ACSL4 promotes HCC proliferation through activation of mTOR, glucose transporter (GLUT1) and increased O-GlcNAc and conversely O-GlcNAcylation increases ACSL4 -expression[99]show another example of O-GlcNAc regulation by feedback loops. Thus, O-GlcNAc plays a key role in regulating lipid metabolism in cancer cells.

3.3 Modulation of other signal paths

In recent years, increased O-GlcNAcylation in several cancers has been shown to promote cancer progression through several signaling pathways. Chen et al report that increased O-GlcNAc levels in bladder cancer promote increased NUSAP1 protein levels, a microtubule-binding protein that promotes proliferation and inhibits apoptosis in bladder cancer cells, while decreased O-GlcNAc levels induce down-regulation of NUSAP1 expression and block the development of cancer[100]. Yu et al. report that increased O-GlcNAc levels in colorectal cancer are associated with an increase in integrin 5α (ITGA5) levels and show that ITAG5 is O-GlcNAcyl, which blocks its degradation to promote tumorigenesis[101].

The Laman group demonstrated an association between F-box protein (FBP) encoded by FBXL17 and global increase in O-GlcNAcylation in breast cancer. FBXL17 protein typically functions in the ubiquitination pathway; in breast cancer, FBXL17 is often rearranged and loses the ubiquitous activity of the FBXL17 protein. FBXL17 can interact with UAP1, the last enzyme in HBP, UDPN-acetylhexosamine pyrophosphorylase 1 (UAP1) catalyzes the conversion of UTP and GlcNAc-1-P to UDP-GlcNAc. FBXL17 can regulate UAP1 activityviabinding leads to an inhibition of UAP1 phosphorylation activity, while knockdown of FBXL17 leads to dysregulated UAP1 and increased levels of O-GlcNAcylation in breast cancer cells[102].

Another role of O-GlcNAc in liver cancer was identified through an association between X-active-specific transcript (XIST) and OGT, both of which are upregulated in liver cancer and correlate with poor prognosis in patients[103]and miR-424-5p, a microRNA shown to play a tumor suppressive role in HCC[104-106]and to be the regulator of OGT[107]. In liver cancer cells, XIST silencing reduced migration, invasion, and increased apoptosis and epithelial to mesenchymal transition (EMT) markers, which were honored after miR-424-5p silencing. XIST was found to bind to miR-424-5p, whose expression is inversely correlated with OGT and liver tumorigenesis in patient tissues, and miR-424-5p was found to bind to OGT and negatively regulate O-GlcNAcylation, which is required for RAF1 proto-oncogene serine /threonine kinase stabilization[108], providing a novel insight where miR-424-5p-dependent downregulation of O-GlcNAc levels reduces the pro-oncogenic effect of RAF1[109].

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(Video) 27th Frontiers in Chemistry: The Split Personality of Human O-GlcNAc Transferase


Protein O-GlcNAc Transferase - an overview? ›

Protein O-GlcNAc transferase also known as OGT or O-linked N-acetylglucosaminyltransferase is an enzyme (EC 2.4. 1.255) that in humans is encoded by the OGT gene. OGT catalyzes the addition of the O-GlcNAc post-translational modification to proteins.

What is the role and function of O-GlcNAcylation in cancer? ›

O-GlcNAcylation is associated with the immune surveillance in tumor microenvironment. O-GlcNAcylation regulates the activation and differentiation of T cells and macrophages, thereby exerting signal transduction and immune surveillance.

What is the role of O linked GlcNAc modification in cell signaling? ›

The primary function of O-GlcNAcylation appears to be the modulation of cellular processes in response to nutrients and to cellular stress [3, 17]. Cells dynamically induce O-GlcNAc protein modification in response to numerous forms of cellular stress, and this appears to be a protective response of cells.

What is the importance of GlcNAc? ›

Numerous studies have confirmed that O-GlcNAcylation is involved in the occurrence and progression of cancers in multiple systems throughout the body. It is also involved in regulating multiple cancer hallmarks, such as metabolic reprogramming, proliferation, invasion, metastasis, and angiogenesis.

What is the function of the OGT enzyme? ›

OGT is essential for proliferation of prostate cancer cells. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. silencing of OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation.

What are the roles of TIF1γ in cancer? ›

In some tumors, TIF1γ functions as a tumor promoter and prevents the apoptosis of tumor cells, but it also acts as a tumor suppressor in other tumors and inhibits the growth of tumor cells.

What is the role of PI3K in cancer metabolism? ›

Oncogenic activation of the PI3K-AKT pathway in cancer cells reprograms cellular metabolism by augmenting the activity of nutrient transporters and metabolic enzymes, thereby supporting the anabolic demands of aberrantly growing cells.

What is the significance of O-linked glycosylation? ›

O-Glycosylation in general has impact on a diversity of biological processes covering cellular aspects (targeted transport of glycoproteins), molecular aspects (protein conformation, resistance to proteolysis), and aspects involved in cellular communication (cell-cell and cell-matrix interaction).

Why is O-linked glycosylation important? ›

O-glycans, which are the sugars added to the serine or threonine, have numerous functions throughout the body, including trafficking of cells in the immune system, allowing recognition of foreign material, controlling cell metabolism and providing cartilage and tendon flexibility.

Does inhibition of O-GlcNAcylation decreases the cytotoxic function of natural killer cells? ›

Inhibition of O-GlcNAcylation Decreases the Cytotoxic Function of Natural Killer Cells. Natural killer (NK) cells mediate killing of malignant and virus-infected cells, a property that is explored as a cell therapy approach in the clinic.

What is the function of GlcNAc phosphotransferase and at which location does it function? ›

GlcNAc-1-phosphotransferase helps prepare certain newly made enzymes for transport to lysosomes. Lysosomes are compartments within the cell that use digestive enzymes called hydrolases to break down large molecules into smaller ones that can be reused by cells.

What is the meaning of O-GlcNAcylation? ›

O-GlcNAcylation is an atypical, reversible, and dynamic glycosylation that plays a critical role in maintaining the normal physiological functions of cells by regulating various biological processes such as signal transduction, proteasome activity, apoptosis, autophagy, transcription, and translation.

Where is GlcNAc found? ›

O-GlcNAc is particularly abundant within the nucleus, where it occurs on the transcriptional regulatory machinery including RNA polymerase II catalytic subunit carboxy-terminal domain (CTD) and myriad basal and specialized RNA polymerase II transcription factors.

What is a small molecule that inhibits OGT activity in cells? ›

Hence, we conclude that OSMI-1 inhibits OGT activity in cells.

Where is the OGT gene located? ›

Human O-GlcNAc transferase (OGT), located on the X chromosome (Xq13. 1), encodes a 110 kDa protein [17, 18] that is highly conserved from Caenorhabditis elegans to Homo sapiens [19].

What is the full form of OGT? ›

Ohio Graduation Test, a statewide exit test as a result of the No Child Left Behind Act.

What is the role of glypicans in cancer progression and therapy? ›

Glypicans play important roles in development by modulating morphogen gradient formation5 and cell growth by regulating Wnt68 and other signaling pathways. Glypicans are abnormally expressed in multiples types of cancer and are crucial for cancer cell growth and progression.

What is the goal of oncologic surgery in the management of cancer? ›

Surgical oncology may be used to: Diagnose cancer (diagnostic surgery or biopsy) Remove a tumor or a portion of the cancer (curative or debulking surgery) Determine where the cancer is located, whether it has spread and whether it is affecting the functions of other organs (staging surgery)

What are the roles of integrin αvβ6 in cancer? ›

Integrin αvβ6 seems to promote cell invasion and migration both of which are involved in metastasis.

What happens when PI3K is activated? ›

Activation of PI3K signaling

A variety of signaling proteins, such as kinases AKT and PDK1 can bind to the lipid products of PI3K and thereby localize to the cell membrane to activate cell growth and cell survival pathways [14].

What happens when PI3K is inhibited? ›

Therefore, inhibiting PI3K/AKT signaling suppresses the FOXO phosphorylation, causing FOXO-dependent repression of RTKs leading to derepression of molecules downstream of AKT such as S6K and growth factor receptor-bound protein 10 (GRB10), ultimately resulting in activation of multiple RTKs and partial maintenance of ...

What is the mutation of PI3K in cancer? ›

The PIK3CA mutations are somatic, cancer-specific and heterozygous and can be divided into four classes defined by the four domains of the catalytic subunit in which they occur: the adaptor-binding domain (ABD), C2 domain, helical domain and catalytic domain.

What enzyme removes O-glycosylation? ›

Endoglycosidases are enzymes that catalyze the cleavage of an internal glycoside bond in an oligosaccharide. Exoglycosidases are enzymes that remove terminal carbohydrates from the non-reducing end of a glycan, but do not cleave internal bonds between carbohydrates.

Is glycosylation good or bad? ›

As a general rule, glycoconjugate with exposed core or aberrant terminal glycosylation trees will indicate stress or damage and will immunologically flag cells carrying these abnormal glycoforms. By contrast, healthy cells avert immune recognition by virtue of their normal terminal glycosylation patterns.

Where is O-glycosylation initiated? ›

In contrast to N-linked glycosylation, the biosynthesis of O-linked glycosylation is initiated in the Golgi apparatus and occurs through the stepwise addition of monosaccharide residues.

Does glycosylation determine blood type? ›

The ABO blood type of each person is determined by a single gene. For the A type, there is a gene for GTA, a glycosyltransferase that adds N-acetylgalactosamine. For type B, the gene encodes GTB, a different glycosyltransferase that adds galactose. For type O, neither enzyme is made.

How does glycosylation affect immune response? ›

Glycosylation can alter the structure and function of proteins by steric influences or by mediating interactions with glycan-binding proteins. Changes in the glycome can occur in response to environmental and genetic stimuli and are often associated with the acquisition of altered cellular phenotypes.

What is an example of O-linked glycosylation? ›

Most of O-glycans have N-acetylgalactosamine (GalNAc) as a common core. Several glycoproteins, such as mucins (MUCs), immunoglobulins, and caseins are examples of O-glycosylated structures. These glycans are further elongated with other monosaccharides and sulfate groups.

What is the inhibitor of natural killer cells? ›

Human NK cells express two major classes of inhibitory receptors, the inhibitory members of KIR and the CD94‐NKG2A heterodimer. While NKG2A is expressed in both mouse and human, KIR is expressed in human and not in mouse. KIR is a type I transmembrane receptor possessing extracellular Ig‐like domains.

What suppresses NK cells? ›

TGF‐β impairs NK cell function directly by limiting NK cell antibody‐dependent cellular cytotoxicity (ADCC) and IFN‐γ production through inhibition of the transcription factor SMAD3.

What inhibits natural killer cells? ›

The MHC class I molecules are recognized by NK cell inhibitory receptors and the ligation of these receptors inhibits the activation of NK cells.

Does deficiency in GlcNAc 1 phosphotransferase leads to I-cell disease? ›

I-cell disease is an autosomal recessive disorder caused by a deficiency of GlcNAc phosphotransferase, which phosphorylates mannose residues to mannose-6-phosphate on N-linked glycoproteins in the Golgi apparatus within cells.

What is the cause of I-cell disease? ›

I-cell disease is caused by a mutation in the GNPTA gene that leads to a deficiency in the enzyme UDP-N-acetylglucoseamine-1-phosphotransferase. I-cell disease is inherited as an autosomal recessive genetic trait.

What is the purpose of phosphotransferase system? ›

The phosphotransferase system is involved in transporting many sugars into bacteria, including glucose, mannose, fructose and cellobiose. PTS sugars can differ between bacterial groups, mirroring the most suitable carbon sources available in the environment every group evolved.

Does excessive O-GlcNAcylation cause heart failure? ›

Elevated cardiac O-GlcNAcylation is associated with pathologic hypertrophy and heart failure in animal models and is well described in patients with hypertension and aortic stenosis, conditions of increased left ventricular afterload.

What is the effect of phosphorylation and O-GlcNAcylation on proline rich domains of Tau? ›

Tau undergoes several post-translational modifications of which phosphorylation and O-GlcNAcylation are key chemical modifications. Tau aggregates into paired helical filaments and neurofibrillary tangles upon hyperphosphorylation, whereas O-GlcNAcylation stabilizes the soluble form of Tau.

What is the abbreviation for acetylglucosamine? ›

N-Acetylglucosamine (GlcNAc) is an amide derivative of the monosaccharide glucose.

How do you pronounce O-GlcNAcylation? ›

A common form of post-translational modification in animal cells has the tongue-twisting name of O-GlcNAcylation (pronounced oh-gluck-nakel-ation). It is a process by which certain amino acids in proteins get attached to a sugar molecule, and this modification impacts a wide array of cellular functions.

Where does acetyl glucosamine come from? ›

N-Acetyl-D-glucosamine is a metabolite found in or produced by Escherichia coli (strain K12, MG1655). N-Acetyl-D-Glucosamine is a natural product found in Streptomyces alfalfae, Euglena gracilis, and other organisms with data available.

What is the role of O GlcNAcylation in immune cell activation? ›

In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.

What is a small molecule that decreases the activity of an enzyme by binding to a site other than the catalytic site? ›

A small molecule that decreases the activity of an enzyme by binding to a site other than the catalytic site is termed a(n): allosteric inhibitor.

What are small molecule binding sites? ›

Small-molecule drugs typically function by binding to and modulating the biological activity of their protein targets. Drug-binding sites resemble pockets or grooves on the surface of the target protein, and are generally present even when the drug is not bound.

How big is the OGT protein? ›

Structure. The human OGT gene has 1046 amino acid residues, and is a heterotrimer consisting of two 110 kDa subunits and one 78 kDa subunit.

Where are transcriptionally active genes located? ›

In general, transcriptionally silent domains reside at the nuclear periphery, whereas active domains locate within the interior.

Where is PITX1 gene located? ›

The PITX1 protein is found primarily in the developing legs and feet. The protein acts as a transcription factor, which is a protein that attaches (binds) to specific regions of DNA and helps control the activity of particular genes.

What does OJT mean in medical terms? ›

On the Job TrainingCurrently selected.

What is the full form of OD test? ›

Optical density measurement (OD or OD600) is used in microbiology to estimate the concentration of bacteria or other cells in a liquid.

What is the role of ornithine decarboxylase in cancer? ›

ODC, a key polyamine biosynthesis enzyme, plays a major role in the process of carcinogenesis. An association between high levels of polyamines and cancer was first reported in the late 1960s by Russell and Snyder [42], who measured high levels of ODC activity in regenerated rat liver and in several human cancers.

What are the distinct roles of sialyl transferases in cancer biology and onco immunology? ›

Sialyltransferases are involved in the biosynthesis of tumor-associated sialoglycans, which via recognition by sialic acid-binding proteins, influence tumor progression and the immune response of the host.

How do NK cells induce cytotoxicity? ›

Target cell recognition induces the formation of a lytic immunological synapse between the NK cell and its target. The polarized exocytosis of secretory lysosomes is then activated and these organelles release their cytotoxic contents at the lytic synapse, specifically killing the target cell.

Which mechanism do natural killer NK cells destroy virally infected cells? ›

Upon activation and recruitment to the site of infection, NK cells employ three main strategies to kill virally infected cells: the production of cytokines, the secretion of cytolytic granules, and the use of death receptor-mediated cytolysis [8].

Which drug is ornithine decarboxylase inhibitor? ›

Substances and drugs that inhibit or block the activity of ORNITHINE DECARBOXYLASE. A medication used topically to reduce unwanted facial hair growth in women. The postulated mechanism of action is through irreversible inhibition of ornithine decarboxylase (ODC) in the skin.

Why do we need ornithine? ›

The primary use of L-ornithine in health supplements is to support athletic performance. It is also used to support liver functioning, healthy wound recovery and stress management.

Does ornithine increase growth hormone? ›

Arginine and ornithine supplementation increases growth hormone and insulin-like growth factor-1 serum levels after heavy-resistance exercise in strength-trained athletes.

What is the difference between tau and phosphorylated tau? ›

Cerebro-spinal fluid (CSF) total tau (T-tau) has been suggested as a general marker of neurodegeneration [4], while phosphorylated tau (P-tau) may be a more specific marker for AD because neurofibrillary tangles primarily consist of tau protein in the abnormally hyperphosphorylated state [5].

What happens when tau is phosphorylated? ›

Phosphorylation of tau by GSK-3 promotes the formation of insoluble oligomeric tau species that can constitute both full-length and truncated tau species (62, 63). The majority of insoluble tau in AD brain is intact (13).

Which 3 amino acids get phosphorylated and why? ›

The amino acids most commonly phosphorylated are serine, threonine, tyrosine in eukaryotes, and also histidine in prokaryotes and plants (though it is now known to be common in humans). These phosphorylations play important and well-characterized roles in signaling pathways and metabolism.

What is an important enzyme involved in cancer development? ›

The well-known classic glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; the housekeeping gene) is also implicated in cancer. Overexpression of GAPDH is considered an important feature of numerous types of cancer (28–30).

What are T regulatory cells in cancer immunotherapy? ›

Regulatory T (Treg) cells suppress abnormal/excessive immune responses to self‐ and nonself‐antigens to maintain immune homeostasis. In tumor immunity, Treg cells are involved in tumor development and progression by inhibiting antitumor immunity.

What are cytotoxic T cells in cancer immunotherapy? ›

Cytotoxic CD8+ T cells of the adaptive immune system are the most powerful effectors in the anticancer immune response and constitute the backbone of cancer immunotherapy.


1. Protein O-GlcNAcylation: a sweet “weep & sweep” for helminth expulsion
2. Mysterious World of O GlcNAc: John A. Hanover
3. The O-GlcNAc database
(Stephanie Olivier-Van Stichelen)
4. [Webinar] Protein O-GlcNAcylation: a sweet “weep & sweep” for helminth expulsion (20220830 2nd)
5. Antiviral function of O GlcNAc transferase: immunity plus metabolism
6. Protein O-GlcNAcylation: a sweet "weep & sweep" for helminth expulsion
(kai timmer)


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