Protein O-GlcNAc Transferase - an overview (2023)

4.11.4.3.1 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

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.

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].

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.

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 K_Mof 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 K_Iin 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 K_Iof 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. .

6.2EGOmutation i Adams-Olivers syndrom

Three homozygous mutations forEGOhave been reported to date. All of these mutations reduce the ability to O-GlcNAcylate EGF domains. In addition, an EOGT^R377Qmutant 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.

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).

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 clearK_Mvalues ​​(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.

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.

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.

3 Metabolic pathways