Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.     

O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Short repeats in the heme oxygenase 1 gene promoter is associated with increased levels of inflammation,ferritin and higher risk of type-2 diabetes mellitus

ObjectiveWe evaluated the relationship between the HO1 genotype, ferritin levels and the risk of type-2 diabetes and inflammation.Research methodsEight hundred thirty-five individuals were evaluated and classified according to their nutritional status and the presence of type-2 diabetes: 153 overweight (OW); 62 obese (OB); 55 type-2 diabetes mellitus (DM); 202 OWDM; 239 OBDM and 124 controls (C). We studied biochemical (glycemia, insulin, lipid profile, liver enzyme, creatinine, hsCRP), hematological (hemoglobin, free erythrocyte protoporphyrin, transferrin receptor and serum Fe and ferritin) and oxidative stress (SOD, GHS and TBARS) parameters. We determined heme oxygenase activity and the (GT)_n polymorphism in its gene promoter.ResultsIndividuals with diabetes, independent of nutritional status, showed high levels of ferritin and HO activity compared to control subjects. Allelic frequency was not different between the groups (Chi², NS) however, genotypes were different (Chi², P < 0.001). The SS (short-short) genotype was higher in all DM individuals compared to controls and MM was higher in controls. SM (short-medium) genotype was an independent risk factor for DM in logistic regression analysis. We observed high risk for type-2 diabetes mellitus in the presence of SM genotype and high levels of ferritin (OR adjusted: 2.7; 1.9–3.6; p < 0.001; compared to control group). It was also significantly related to inflammation.ConclusionThe SM genotype in HO1 gene promoter and ferritin levels were associated with higher risk for type-2 diabetes and for having a higher marker of inflammation, which is the main risk factor for the development of chronic diseases.     

Diminished O-GlcNAcylation in Alzheimer's disease is strongly correlated with mitochondrial anomalies

Uncover the initial cause(s) underlying Alzheimer's disease (AD) pathology is imperative for the development of new therapeutic interventions to counteract AD-related symptomatology and neuropathology in a timely manner. The early stages of AD are characterized by a brain hypometabolic state as denoted by faulty glucose uptake and utilization and abnormal mitochondrial function and distribution which, ultimately, culminates in synaptic “starvation” and neuronal degeneration. Importantly, it was recently recognized that the post-translational modification β-N-acetylglucosamine (O-GlcNAc) modulates mitochondrial function, motility and distribution being proposed to act as a nutrient sensor that links glucose and the metabolic status to neuronal function. Using post-mortem human brain tissue, brain samples from the triple transgenic mouse model of AD (3xTg-AD) and in vitro models of AD (differentiated SH-SY5Y cells exposed to AD-mimicking conditions), the present study is aimed to clarify whether O-GlcNAcylation, the posttranslational modification of intracellular proteins by O-GlcNAc, contributes to “mitochondrial pathology” in AD and its potential as a therapeutic target. A reduction in global O-GlcNAcylation levels was observed in the brain cortex and hippocampus of AD subjects. Moreover, GlcNAcylation levels are higher in mature mice but the levels of this posttranslational modification are lower in 3xTg-AD mice when compared to control mice. The in vitro models of AD also exhibited a marked reduction in global O-GlcNAcylation levels, which was strongly correlated with hampered mitochondrial bioenergetic function, disruption of the mitochondrial network and loss of cell viability. Conversely, the pharmacological modulation of O-GlcNAcylation levels with Thiamet-G restored O-GlcNAcylation levels and cell viability in the in vitro models of AD. Overall, these results suggest that O-GlcNAcylation is involved in AD pathology functioning as a potential link between mitochondrial energetic crisis and synaptic and neuronal degeneration. This posttranslational modification represents a promising therapeutic target to tackle this devastating neurodegenerative disease.     

Excessive O-GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells

Increased modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) has been implicated in the development of diabetic cardiomyopathy. We used the well-characterized ES cells (Nkx2.5GFP knock-in ES cells), to investigate the role of O-GlcNAcylation in cardiomyocyte development. O-GlcNAcylation decreased in differentiating ES cells, as did the expression of O-GlcNAc transferase. Increasing O-GlcNAcylation with glucosamine or by inhibiting N-acetylglucosaminidase (streptozotocin or PUGNAc) decreased the number of cardiomyocyte precursors and cardiac-specific gene expression. On the other hand, decreasing O-GlcNAcylation with an inhibitor of glutamine fructose-6-phosphate amidotransferase (6-diazo-5-oxo-norleucine) increased cardiomyocyte precursors. These results suggest that excessive O-GlcNAcylation impairs cardiac cell differentiation in ES cells.     

Tracking of fast moving neuronal vesicles with ageladine A

O-Linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins is a ubiquitous post-translational modification in multicellular organisms studied so far. Since aberrant O-GlcNAcylation has a link with insulin resistance, it is important to establish the status of O-GlcNAcylation in differentiation of mesenchymal cells such as preadipocytes. In this study, we found a differentiation-dependent drastic increase in the level of O-GlcNAcylation in mouse 3T3-L1 preadipocytes. The occurrence of the increase in O-GlcNAcylation, which correlated with the expression of C/EBPα, was in part due to increased expression of O-GlcNAc transferase. In addition to the well-known O-GlcNAcylated proteins such as nucleoporins and vimentin, pyruvate carboxylase, long chain fatty acid-CoA ligase 1, and Ewing sarcoma protein were identified as the proteins which are heavily O-GlcNAcylated with the adipocyte differentiation. Both adipocyte differentiation and the differentiation-dependent increase in O-GlcNAcylation were blocked by 6-diazo-5-oxo-norleucine. These results suggest that O-GlcNAcylation particilates, at least in part, in adipogenesis.     

Murine platelets are not regulated by O-linked beta-N-acetylglucosamine

It is generally appreciated that platelets derived from diabetic patients display increased responsiveness to low levels of agonists. O-GlcNAcylation has been linked to hyperglycemia-related effects in other tissues; therefore we examined this modification in platelets to determine if O-GlcNAcylation affects platelet function. This post-translational modification consists of an N-acetylglucosamine attached to serine and/or threonine residues. We examined O-GlcNAc levels in platelets from a hyperglycemic murine model of Type I diabetes with known hypersensitivity to agonists and a Type II diabetes model (ob/ob) lacking detectable alterations in the aggregation profile. Neither model showed marked increases in protein O-GlcNAcylation. Treatment of platelets with multiple O-GlcNAcase inhibitors led to O-GlcNAc accumulation on multiple platelet proteins. However, the inhibitor-induced accumulation of this modification does not correlate with any gross alterations in platelet aggregation. These data suggest that while the modification occurs in platelets, their activity is not globally sensitive to O-GlcNAc levels.     

Altered O-GlcNAc modification and phosphorylation of mitochondrial proteins in myoblast cells exposed to high glucose

Hyperglycemia induced increased posttranslational modification of proteins by O-linked-β-N-acetyl glucosamine (O-GlcNAcylation) and mitochondrial dysfunction has been independently implicated in the development of insulin resistance. It is not known whether repertoire of O-GlcNAcylated proteins includes mitochondrial proteins and their altered O-GlcNAcylation impinges on their phosphorylation mediated normal functioning thus contribute to mitochondrial dysfunction and insulin resistance. We have explored the O-GlcNAcylation of mitochondrial proteins from myoblast cells under basal (4 mM) and high glucose (30 mM) conditions using a combination of proteomic approaches. Furthermore, we have assessed the accompanied changes in the phosphorylation of mitochondrial proteins. We report that a number of mitochondrial proteins are O-GlcNAcylated under basal condition which is altered under high glucose condition. In addition, we report that exposure to high glucose not only changes the O-GlcNAcylation of mitochondrial proteins but also changes their phosphorylation profiles. The dynamic and complex interplay between O-GlcNAcylation and phosphorylation of mitochondrial proteins was further validated by immunoblot analysis of HSP60, prohibitin, and voltage-dependent anion channel 1 as candidate proteins. O-GlcNAcylation of mitochondrial proteins may play a role in normal functioning of mitochondria. High glucose induced changes in O-GlcNAcylation and phosphorylation of mitochondrial proteins may be associated with mitochondrial dysfunction and insulin resistance.     

Consumption of a high fat diet promotes protein O-GlcNAcylation in mouse retina via NR4A1-dependent GFAT2 expression

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise in developed countries due to overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-diabetes, we evaluated the impact of a diet high in saturated fat (HFD) on O-GlcNAcylation of retinal proteins, as dysregulated O-GlcNAcylation contributes to diabetic complications and evidence supports a role in DR. Protein O-GlcNAcylation was increased in the retina of mice fed a HFD as compared to littermates receiving control chow. Similarly, O-GlcNAcylation was elevated in retinal Müller cells in culture exposed to the saturated fatty acid palmitate or the ceramide analog Cer6. One potential mechanism responsible for elevated O-GlcNAcylation is increased flux through the hexosamine biosynthetic pathway (HBP). Indeed, inhibition of the pathway's rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT) prevented Cer6-induced O-GlcNAcylation. Importantly, expression of the mRNA encoding GFAT2, but not GFAT1 was elevated in both the retina of mice fed a HFD and in retinal cells in culture exposed to palmitate or Cer6. Notably, expression of nuclear receptor subfamily 4 group A member 1 (NR4A1) was increased in the retina of mice fed a HFD and NR4A1 expression was sufficient to promote GFAT2 mRNA expression and O-GlcNAcylation in retinal cells in culture. Whereas palmitate or Cer6 addition to culture medium enhanced NR4A1 and GFAT2 expression, chemical inhibition of NR4A1 transactivation repressed Cer6-induced GFAT2 mRNA expression. Overall, the results support a model wherein HFD increases retinal protein O-GlcNAcylation by promoting NR4A1-dependent GFAT2 expression.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (O-GlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced O-GlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer’s and Parkinson’s diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.