Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca²⁺. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. hexosamine biosynthesis; calcium; protein O-glycosylation     

Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca²⁺ entry into the cells; however, a direct link between HBP and intracellular Ca²⁺ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP-N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine (O-GlcNAc) on ANG II-mediated changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We found that glucosamine blocked ANG II-induced [Ca²⁺]_i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca²⁺]_i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca²⁺]_i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca²⁺]_i homeostasis in the heart. hypertrophy; left ventricle; calcium channels; calcium signaling     

Impact of Type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart

Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes. hexosamine biosynthesis; protein O-glycosylation; O-linked N-acetylglucosamine transferase     

Up-regulation of O-GlcNAc Transferase with Glucose Deprivation in HepG2 Cells Is Mediated by Decreased Hexosamine Pathway Flux

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have previously shown a significant induction of O-GlcNAc modification under conditions of glucose deprivation. Increased O-GlcNAc modification was mediated by increased mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT). We have investigated the mechanism mediating ncOGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase protein levels or phosphorylation were observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose (1 mm) of glucosamine blocked the induction of ncOGT mRNA and subsequent increase in O-GlcNAc protein modification, suggesting that decreased hexosamine flux is the signal for ncOGT up-regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamat) completely prevented the subsequent up-regulation of ncOGT. Glucosamine treatment also resulted in a 40% rescue of the down-regulation of glycogen synthase activity normally seen after glucose deprivation. We conclude that deglycosylation of proteins within the first few hours of glucose deprivation promotes ncOGT induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation.Dynamic O-linked N-acetylglucosamine (O-GlcNAc)² modification is a critical modulator of the fate and function of diverse nuclear and cytoplasmic proteins. O-GlcNAcylation of target proteins is dependent upon substrate synthesis in the hexosamine biosynthetic pathway (HBP) coupled with O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein modification. The HBP converts a portion of imported glucose to uridine 5′-diphospho (UDP)-GlcNAc. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-GlcNAcase catalyzes O-GlcNAc removal (1). HBP flux is known to parallel substrate (glucose) availability, making the HBP a nutrient sensor (2–5).O-GlcNAcylation is regulated principally by substrate availability. Previous work has indicated that protein O-GlcNAcylation is proportional to substrate (glucose) availability (8). However, we have shown that human hepatocellular carcinoma (HepG2) cells demonstrate a robust O-GlcNAc increase when deprived of glucose, and this O-GlcNAc induction is mediated not by substrate-driven HBP flux increase but instead by increased OGT expression and O-GlcNAcase down-regulation (6). It has subsequently been shown that glucose deprivation of Neuro-2a neuroblastoma cells also results in OGT and O-GlcNAc induction (7). We have therefore investigated the mechanism for regulation of OGT in HepG2 cells and determined that the signal responsible for the induction of OGT mRNA in glucose deprivation is an early decrease in HBP flux and O-GlcNAc modification of proteins. Thus, the levels of O-GlcNAc in these cells are maintained through a feedback mechanism responsive to the degree of protein O-GlcNAc modification.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.     

Native, not nitrated, cytochrome c and mitochondria-derived hydrogen peroxide drive osteoclast apoptosis

Two unresolved aspects of the role of mitochondria-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts, because they are rich in mitochondria and because osteoclast apoptosis is critical in bone metabolism regulation. H₂O₂ production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. With the addition of a mitochondrial uncoupler, H₂O₂ production and apoptosis were blocked, indicating the prominent role of mitochondria-derived H₂O₂. Trapping H₂O₂-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis, and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and Bc-xL were decreased before apoptosis, whereas expression of wild-type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical in initiating apoptosis. Cytosolic cytochrome c participated in activating caspase-3 and -9, both required for apoptosis. Collectively, our data indicate that the mitochondria-dependent apoptotic pathway is one of the major routes operating in osteoclasts. reactive oxygen species; nitric oxide; free radicals; caspase     

Targeting O-Glycosyltransferase (OGT) to Promote Healing of Diabetic Skin Wounds

Non-healing wounds are a significant source of morbidity. This is particularly true for diabetic patients, who tend to develop chronic skin wounds. O-GlcNAc modification of serine and threonine residues is a common regulatory post-translational modification analogous to protein phosphorylation; increased intracellular protein O-GlcNAc modification has been observed in diabetic and hyperglycemic states. Two intracellular enzymes, UDP-N-acetylglucosamine-polypeptide β-N-acetylglucosaminyl transferase (OGT) and O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (OGA), mediate addition and removal, respectively, of N-acetylglucosamine (GlcNAc) from intracellular protein substrates. Alterations in O-GlcNAc modification of intracellular proteins is linked to diabetes, and the increased levels of protein O-GlcNAc modification observed in diabetic tissues may in part explain some of the observed underlying pathophysiology that contributes to delayed wound healing. We have previously shown that increasing protein O-GlcNAc modification by overexpression of OGT in murine keratinocytes results in elevated protein O-GlcNAc modification and a hyperadhesive phenotype. This study was undertaken to explore the hypothesis that increased O-GlcNAc modification of cellular proteins in diabetic skin could contribute to the delayed wound healing observed in patients with diabetic skin ulcers. In the present study, we show that human keratinocytes cultured under hyperglycemic conditions display increased levels of O-GlcNAc modification as well as a delay in the rate of wound closure in vitro. We further show that specific knockdown of OGT by RNA interference (RNAi) reverses this effect, thereby opening up the opportunity for OGT-targeted therapies to promote wound healing in diabetic patients.     

Survey of <Emphasis Type="Italic">O</Emphasis>-GlcNAc level variations in <Emphasis Type="Italic">Xenopus laevis</Emphasis> from oogenesis to early development

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I–V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.     

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.     

Enhanced Transfer of a Photocross-linking N-Acetylglucosamine (GlcNAc) Analog by an O-GlcNAc Transferase Mutant with Converted Substrate Specificity

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins in multicellular organisms. O-GlcNAc modification is catalyzed by the O-GlcNAc transferase (OGT), which transfers N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor UDP-GlcNAc to serine or threonine residues of protein substrates. Recently, we reported a novel metabolic labeling method to introduce the diazirine photocross-linking functional group onto O-GlcNAc residues in mammalian cells. In this method, cells are engineered to produce diazirine-modified UDP-GlcNAc (UDP-GlcNDAz), and the diazirine-modified GlcNAc analog (GlcNDAz) is transferred to substrate proteins by endogenous OGT, producing O-GlcNDAz. O-GlcNDAz-modified proteins can be covalently cross-linked to their binding partners, providing information about O-GlcNAc-dependent interactions. The utility of the method was demonstrated by cross-linking highly O-GlcNAc-modified nucleoporins to proteins involved in nuclear transport. For practical application of this method to a broader range of O-GlcNAc-modified proteins, efficient O-GlcNDAz production is critical. Here we examined the ability of OGT to transfer GlcNDAz and found that the wild-type enzyme (wtOGT) prefers the natural substrate, UDP-GlcNAc, over the unnatural UDP-GlcNDAz. This competition limits O-GlcNDAz production in cells and the extent of O-GlcNDAz-dependent cross-linking. Here we identified an OGT mutant, OGT(C917A), that efficiently transfers GlcNDAz and, surprisingly, has altered substrate specificity, preferring to transfer GlcNDAz rather than GlcNAc to protein substrates. We confirmed the reversed substrate preference by determining the Michaelis-Menten parameters describing the activity of wtOGT and OGT(C917A) with both UDP-GlcNAc and UDP-GlcNDAz. Use of OGT(C917A) enhances O-GlcNDAz production, yielding improved cross-linking of O-GlcNDAz-modified molecules both in vitro and in cells.     

Chemical tools to explore nutrient-driven O-GlcNAc cycling

Abstract

Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the “central dogma” of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.     

Extracellular O-linked β-N-acetylglucosamine: Its biology and relationship to human disease

The O-linked β-N-acetylglucosamine(O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase(OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine(EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.     

Substrate and product analogues as human <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase inhibitors

Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.     

Dual functionality of O-GlcNAc transferase is required for Drosophila development

Post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc) catalysed by O-GlcNAc transferase (OGT) has been linked to regulation of diverse cellular functions. OGT possesses a C-terminal glycosyltransferase catalytic domain and N-terminal tetratricopeptide repeats that are implicated in protein–protein interactions. Drosophila OGT (DmOGT) is encoded by super sex combs (sxc), mutants of which are pupal lethal. However, it is not clear if this phenotype is caused by reduction of O-GlcNAcylation. Here we use a genetic approach to demonstrate that post-pupal Drosophila development can proceed with negligible OGT catalysis, while early embryonic development is OGT activity-dependent. Structural and enzymatic comparison between human OGT (hOGT) and DmOGT informed the rational design of DmOGT point mutants with a range of reduced catalytic activities. Strikingly, a severely hypomorphic OGT mutant complements sxc pupal lethality. However, the hypomorphic OGT mutant-rescued progeny do not produce F2 adults, because a set of Hox genes is de-repressed in F2 embryos, resulting in homeotic phenotypes. Thus, OGT catalytic activity is required up to late pupal stages, while further development proceeds with severely reduced OGT activity.     

Structural insights into mechanism and specificity of O-GlcNAc transferase

Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.     

Interaction between O-GlcNAc Modification and Tyrosine Phosphorylation of Prohibitin: Implication for a Novel Binary Switch

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.