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.     

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.     

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.     

β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3

O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.     

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.     

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.     

Ionic complexation properties of per(3,6-anhydro)cyclodextrin derivatives towards lanthanides

Using per(3,6-anhydro)cyclodextrin derivatives [per(3,6-anhydro)CD], it was possible to produce new lanthanide chelates by careful choice of the size and functional groups. Heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose fulfils the best criteria for complexation of lanthanide ions. Nuclear magnetic resonance was used to derive the association constants and the stoichiometries of these new complexes. Finally, a three-dimensional structure of these complexes consistent with the NMR data is proposed, to ascertain the position of lanthanide in the cavity of the per(3,6-anhydro)CD. For the present purposes, heptakis(2-O-acetyl-3,6-anhydro)cyclomaltoheptaose, octakis(2-O-acetyl-3,6-anhydro)cyclomaltooctaose, heptakis(3,6-anhydro-2-O-methyl)cyclomaltoheptaose and octakis(3,6-anhydro-2-O-methyl)cyclomaltooctaose have been synthesized and purified.     

Mapping of two O-GlcNAc modification sites in the capsid protein of the potyvirus Plum pox virus

A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.     

Global Population Genetic Structure of Caenorhabditis remanei Reveals Incipient Speciation

Mating system transitions dramatically alter the evolutionary trajectories of genomes that can be revealed by contrasts of species with disparate modes of reproduction. For such transitions in Caenorhabditis nematodes, some major causes of genome variation in selfing species have been discerned. And yet, we have only limited understanding of species-wide population genetic processes for their outcrossing relatives, which represent the reproductive state of the progenitors of selfing species. Multilocus-multipopulation sequence polymorphism data provide a powerful means to uncover the historical demography and evolutionary processes that shape genomes. Here we survey nucleotide polymorphism across the X chromosome for three populations of the outcrossing nematode Caenorhabditis remanei and demonstrate its divergence from a fourth population describing a closely related new species from China, C. sp. 23. We find high genetic variation globally and within each local population sample. Despite geographic barriers and moderate genetic differentiation between Europe and North America, considerable gene flow connects C. remanei populations. We discovered C. sp. 23 while investigating C. remanei, observing strong genetic differentiation characteristic of reproductive isolation that was confirmed by substantial F(2) hybrid breakdown in interspecific crosses. That C. sp. 23 represents a distinct biological species provides a cautionary example of how standard practice can fail for mating tests of species identity in this group. This species pair permits full application of divergence population genetic methods to obligately outcrossing species of Caenorhabditis and also presents a new focus for interrogation of the genetics and evolution of speciation with the Caenorhabditis model system.     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.     

Secondary structure models of 18S and 28S rRNAs of the true bugs based on complete rDNA sequences of Eurydema maracandica Oshanin, 1871 (Heteroptera,?Pentatomidae)

The sequences of 18S and 28S rDNAs have been used as molecular markers to resolve phylogenetic relationships of Heteroptera for two decades. The complete sequences of 18S rDNAs have been used in many studies, while in most studies only partial sequences of 28S rDNAs have been used due to technical difficulties of amplifying the complete lengths. In this study, we amplified the complete 18S and 28S rDNA sequences of Eurydema maracandica Oshanin, 1871, and reconstructed the secondary structure models of the corresponding rRNAs. In addition, and more importantly, all of the length variable regions of 18S rRNA were compared among 37 families of Heteroptera based on 140 sequences, and the D3 region of 28S rRNA was compared among 51 families based on 84 sequences. It was found that 8 length variable regions could potentially serve as molecular synapomorphies for some monophyletic groups. Therefore discoveries of more molecular synapomorphies for specific clades can be anticipated from amplification of complete 18S and 28S rDNAs of more representatives of Heteroptera.     

Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.     

Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic beta-cell death under hyperglycemic conditions

The objective of this study was to identify proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) in pancreatic beta-cells and to understand their roles in cell death under hyperglycemic conditions. Here we report that heat shock protein 60 (HSP60) is modified with O-GlcNAc. Levels of O-GlcNAcylated HSP60 increased twofold in response to hyperglycemic conditions. HSP60 is a chaperonin known to bind to Bax in the cytoplasm under normoglycemic conditions. Under hyperglycemic conditions, Bax detached from O-GlcNAcylated HSP60 and translocated to mitochondria. Hyperglycemic conditions were also associated with cytochrome c release, caspase-3 activation, and cell death, suggesting that elevated O-GlcNAcylation of HSP60 interferes with HSP60-Bax interactions, leading to pancreatic beta-cell death.     

Membrane-associated Activation of Cholesterol ��-Glucosyltransferase, an Enzyme Responsible for Biosynthesis of Cholesteryl-��-d-Glucopyranoside in Helicobacter pylori Critical for Its Survival

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Modulation of Dynamin-related Protein 1 (DRP1) Function by Increased O-linked-β-N-acetylglucosamine Modification (O-GlcNAc) in Cardiac Myocytes

O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.     

Developing a common bean core collection suitable for association mapping studies

Because of the continuous introduction of germplasm from abroad, some collectionshave a high number of accessions, making it difficult to explore the geneticvariability present in a germplasm bank for conservation and breeding purposes.Therefore, the aim of this study was to quantify and analyze the structure of geneticvariability among 500 common bean accessions to construct a core collection. A totalof 58 SSRs were used for this purpose. The polymorphism information content (PIC) inthe 180 common bean accessions selected to compose the core collection ranged from0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500accessions were clustered into 15 distinct groups and the 180 accessions into fourdistinct groups in the Structure analysis. According to analysis of molecularvariance, the most divergent accessions comprised 97.2% of the observed geneticvariability present within the base collection, confirming the efficiency of theselection criterion. The 180 selected accessions will be used for association mappingin future studies and could be potentially used by breeders to direct new crosses andgenerate elite cultivars that meet current and future global market needs.     

Structural insight into the mechanism of streptozotocin inhibition of O-GlcNAcase

Despite decades of its use in diabetes research, the mechanism of cytotoxicity of streptozotocin (STZ) toward pancreatic β-islet cells has remained a topic of discussion. Although STZ toxicity is likely a function of its capacity to promote DNA alkylation, it has been proposed that STZ induces pancreatic β-cell death through O-GlcNAcase inhibition. In this report, we explore the binding mode of STZ to a close homolog of human O-GlcNAcase, BtGH84 from Bacteroides thetaiotaomicron. Our results show that STZ binds in the enzyme active site in its intact form, without the formation of a covalent adduct, consistent with solution studies on BtGH84 and human O-GlcNAcase, as well as with structural work on a homolog from Clostridium perfringens. The active site of the BtGH84 is considerably deformed upon STZ binding and as a result the catalytic machinery is expelled from the binding cavity.