OGT functions as a catalytic chaperone under heat stress response: a unique defense role of OGT in hyperthermia

Protein O-GlcNAcylation is proceeded by O-linked GlcNAc transferase (OGT) in nucleocytoplasm and is involved in many biological processes although its physiological role is not clearly defined. To identify the functional significance of O-GlcNAcylation, we investigated heat stress effects on protein O-GlcNAcylation. Here, we found that protein O-GlcNAcylation was significantly increased in vivo during acute heat stress in mammalian cells and simultaneously, the enhanced protein O-GlcNAcylation was closely associated with cell survival in hyperthermia. Our results demonstrate that hyperthermal cytotoxicity may considerably be facilitated under the condition of insufficient level of protein O-GlcNAcylation inside cells. Furthermore, OGT reaction might be crucial for triggering thermotolerance to recover hyperthermal sensitivity without particular induction of heat shock proteins (hsps). Thus, we propose that OGT can respond rapidly to heat stress through the enhancement of nucleocytoplasmic protein O-GlcNAcylation for a rescue from the early phase of hyperthermal cytotoxicity.     

A convenient synthesis of the C-1-phosphonate analogue of UDP-GlcNAc and its evaluation as an inhibitor of O-linked GlcNAc transferase (OGT)

The C-1-phosphonate analogue of UDP-GlcNAc has been synthesized using an alpha-configured C-1-aldehyde as a key intermediate. Addition of the anion of diethyl phosphate to the aldehyde produced the hydroxyphosphonate. The configuration of this key intermediate was determined by X-ray crystallography. Deoxygenation, coupling of the resulting phosphonic acid with UMP and deprotection gave the target molecule as a di-sodium salt. This analogue had no detectable activity as an inhibitor of (OGT).     

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

Inhibition of mTOR affects protein stability of OGT

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.     

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.     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

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.     

Active Expression of the ubiA Gene From E. coli in Tobacco: Influence of Plant ER-Specific Signal Peptides on the Expression of a Membrane-Bound Prenyltransferase in Plant Cells

The ubiA gene from E. coli codes for 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, an integral membrane protein involved in ubiquinone biosynthesis. This prokaryotic membrane protein was stably expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Transgenic lines containing a direct fusion of the ubiA structural gene to a 35S-derived promoter gave very low enzyme activity levels (average 0.16pkat/mg). Inclusion of an N-terminal ER-specific signal peptide from a lectin gene from Phaseolus vulgaris resulted in an average activity of 1.08pkat/mg in the transgenic tobacco lines. The additional inclusion of a C-terminal HDEL tetrapeptide, responsible for the retention of proteins in the endoplasmic reticulum of eukaryotic cells, increased the activity to 18.6pkat/mg. When the promotor of this construct was changed from the 35S derivative to the recently described very strong plant promoter (ocs)₃mas, the activity increased further to 128.6pkat/mg. The most active tobacco line showed activities of the introduced enzyme which exceeded those of wild-type E. coli (the source of ubiA) by a factor of 1100. These results demonstrate the efficacy of plant ER-specific signal peptides for the active expression of a prokaryotic membrane protein in plants.     

Synthesis and Antiaggregative Activity of a New RGDF Mimetic

m-[4-Oxo-4-(piperazin-1-yl)butyrylamino)benzoyl-D,L--(3,4-methylenedioxyphenyl)--alanine, a new mimetic of the peptide RGDF, was synthesized. This compound inhibited ADP-induced platelet aggregation in human blood plasma enriched with platelets with IC₅₀ = 3.5 × 10^–8 M.     

Kinetics and Mechanism of the Peptide Synthesis in Solution

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: = kC _N ^a C _AE ^b , where k is the reaction rate constant, C _N is the concentration of leucinamide, and C _AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.     

Denitrosylation of S-nitrosylated OGT is triggered in LPS-stimulated innate immune response

O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein O-GlcNAcylation has been revealing various aspects of functional significance in biological processes, such as cellular signaling and activation of immune system. We found that OGT is maintained as S-nitrosylated form in resting cells, and its denitrosylation is triggered in innate immune response of lipopolysaccharide (LPS)-treated macrophage cells. S-nitrosylation of OGT strongly inhibits its catalytic activity up to more than 80% of native OGT, and denitrosylation of OGT leads to protein hyper-O-GlcNAcylation. Furthermore, blockage of increased protein O-GlcNAcylation results in significant loss of nitric oxide and cytokine production. We propose that denitrosylation of S-nitrosylated OGT is a direct mechanism for upregulation of OGT activity by which immune defense is critically controlled in LPS-stimulated innate immune response.     

Cloning and Characterization of a Novel Mouse Short-Chain Dehydrogenases/Reductases cDNA, mHsdl2, Encoding a Protein with an SDR Domain and an SCP2 Domain

The short-chain dehydrogenases/reductases (SDRs) play an important role in the body's metabolism. We have cloned a novel mouse SDR cDNA, which encodes a deduced HSD-like protein with a conserved SDR domain and an SCP2 domain. The 1.8 kb cDNA consists of 11 exons and is mapped to mouse chromosome 4B3. The corresponding gene is widely expressed in normal mouse tissues and its expression level in the liver increases after inducement with cholesterol food. The predicted mouse HSDL2 protein, which has a peroxisomal target signal, is localized in the cytoplasm of NIH 3T3 cells.     

Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain

The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT.     

Functional Characterization of a Drosophila Mitochondrial Uncoupling Protein

Sequence alignment of conserved signature motifs predicts the existence of the uncoupling protein 5 (UCP5)/brain mitochondrial carrier protein (BMCP1) homologue in Drosophila melanogaster. Here we demonstrate the functional characterization of the Drosophila melanogaster UCP5 protein (DmUCP5) in the heterologous yeast system, the first insect UCP reported to date. We show that physiological levels of DmUCP5 expression are responsible for an increase in state 4 respiration rates and a decrease in mitochondrial membrane potential. Furthermore, similar to UCP1, UCP2, and UCP3, the uncoupling activity of DmUCP5 is augmented by fatty acids and inhibited by the purine nucleotide GDP. Thus, DmUCP5 shares the mechanisms known to regulate the UCPs characterized to date. A lack of growth inhibition observed in DmUCP5 expressing yeast is consistent with the notion that physiological uncoupling has a minimal effect on cell growth. Finally, semiquantitative RT-PCR analysis shows a distinctive pattern of DmUCP5 expression predominantly localized in the adult head, similar to the expression pattern of its mammalian homologues. The conserved regulation of the expression of this gene from mammals to fruit flies suggests a role for UCP5 in the brain.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .