BBA - General Subjects (v.1800, #2)

Nucleocytoplasmic Glycosylation by Christopher M. West (47-48).

O-GlcNAc protein modification in plants: Evolution and function by Neil E. Olszewski; Christopher M. West; Slim O. Sassi; Lynn M. Hartweck (49-56).
The role in plants of posttranslational modification of proteins with O-linked N-acetylglucosamine and the evolution and function of O-GlcNAc transferases responsible for this modification are reviewed. Phylogenetic analysis of eukaryotic O-GlcNAc transferases (OGTs) leads us to propose that plants have two distinct OGTs, SEC- and SPY-like, that originated in prokaryotes. Animals and some fungi have a SEC-like enzyme while plants have both. Green algae and some members of the Apicomplexa and amoebozoa have the SPY-like enzyme. Interestingly the progenitor of the Apicomplexa lineage likely had a photosynthetic plastid that persists in a degenerated form in some species, raising the possibility that plant SPY-like OGTs are derived from a photosynthetic endosymbiont. OGTs have multiple tetratricopeptide repeats (TPRs) that within the SEC- and SPY-like classes exhibit evidence of strong selective pressure on specific repeats, suggesting that the function of these repeats is conserved. SPY-like and SEC-like OGTs have both unique and overlapping roles in the plant. The phenotypes of sec and spy single and double mutants indicate that O-GlcNAc modification is essential and that it affects diverse plant processes including response to hormones and environmental signals, circadian rhythms, development, intercellular transport and virus infection. The mechanistic details of how O-GlcNAc modification affects these processes are largely unknown. A major impediment to understanding this is the lack of knowledge of the identities of the modified proteins.
Keywords: O-GlcNAc modification; O-GlcNAc transferase; Arabidopsis; Plants; Posttranslational regulation; Evolution;

The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose–insulin–potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection.
Keywords: Hexosamine biosynthesis; Protein O-glycosylation; O-GlcNAc transferase; Capacitative calcium entry (CCE); STIM1; Orai1;

Dysregulation of the nutrient/stress sensor O-GlcNAcylation is involved in the etiology of cardiovascular disorders, type-2 diabetes and Alzheimer's disease by Tony Lefebvre; Vanessa Dehennaut; Céline Guinez; Stéphanie Olivier; Ludivine Drougat; Anne-Marie Mir; Marlène Mortuaire; Anne-Sophie Vercoutter-Edouart; Jean-Claude Michalski (67-79).
O-GlcNAcylation is widespread within the cytosolic and nuclear compartments of cells. This post-translational modification is likely an indicator of good health since its intracellular level correlates with the availability of extracellular glucose. Apart from its status as a nutrient sensor, O-GlcNAcylation may also act as a stress sensor since it exerts its fundamental effects in response to stress. Several studies report that the cell quickly responds to an insult by elevating O-GlcNAcylation levels and by unmasking a newly described Hsp70–GlcNAc binding property. From a more practical point of view, it has been shown that O-GlcNAcylation impairments contribute to the etiology of cardiovascular diseases, type-2 diabetes and Alzheimer's disease (AD), three illnesses common in occidental societies. Many studies have demonstrated that O-GlcNAcylation operates as a powerful cardioprotector and that by raising O-GlcNAcylation levels, the organism more successfully resists trauma–hemorrhage and ischemia/reperfusion injury. Recent data have also shown that insulin resistance and, more broadly, type-2 diabetes can be controlled by O-GlcNAcylation of the insulin pathway and O-GlcNAcylation of the gluconeogenesis transcription factors FoxO1 and CRCT2. Lastly, the finding that AD may correspond to a type-3 diabetes offers new perspectives into the knowledge of the neuropathology and into the search for new therapeutic avenues.
Keywords: O-GlcNAcylation; Cardiovascular disorders; Type-2 diabetes; Type-3 diabetes; Alzheimer's disease; Stress; OGT; OGA; Hsp70; HGBA; Hsp70–GlcNAc binding activity;

The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine by John A. Hanover; Michael W. Krause; Dona C. Love (80-95).
The enzymes of O-GlcNAc cycling couple the nutrient-dependent synthesis of UDP-GlcNAc to O-GlcNAc modification of Ser/Thr residues of key nuclear and cytoplasmic targets. This series of reactions culminating in O-GlcNAcylation of targets has been termed the hexosamine signaling pathway (HSP). The evolutionarily ancient enzymes of O-GlcNAc cycling have co-evolved with other signaling effecter molecules; they are recruited to their targets by many of the same mechanisms used to organize canonic kinase-dependent signaling pathways. This co-recruitment of the enzymes of O-GlcNAc cycling drives a binary switch impacting pathways of anabolism and growth (nutrient uptake) and catabolic pathways (nutrient sparing and salvage). The hexosamine signaling pathway (HSP) has thus emerged as a versatile cellular regulator modulating numerous cellular signaling cascades influencing growth, metabolism, cellular stress, circadian rhythm, and host–pathogen interactions. In mammals, the nutrient-sensing HSP has been harnessed to regulate such cell-specific functions as neutrophil migration, and activation of B-cells and T-cells. This review summarizes the diverse approaches being used to examine O-GlcNAc cycling. It will emphasize the impact O-GlcNAcylation has upon signaling pathways that may be become deregulated in diseases of the immune system, diabetes mellitus, cancer, cardiovascular disease, and neurodegenerative diseases.
Keywords: Hexosamine; Insulin; Growth factor; O-GlcNAc; Neurodegeneration; Cancer; Diabetes; Cardiovascular disease; mTOR; AMPK; AKT; MAPK;

Since its discovery in the early 1980s, O-linked-β-N-acetylglucosamine (O-GlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc, and β-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of O-GlcNAc's functions has grown rapidly.In this review, the functions of GlcNAcylation in regulating cellular processes, its extensive crosstalk with protein phosphorylation, and regulation of OGT and O-GlcNAcase will be explored.GlcNAcylation rivals phosphorylation in terms of its abundance, protein distribution and its cycling on and off of proteins. GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling, transcription and the cytoskeleton in response to nutrients and stress.Abnormal crosstalk between GlcNAcylation and phosphorylation underlies dysregulation in diabetes, including glucose toxicity, and defective GlcNAcylation is involved in neurodegenerative disease and cancer and most recently in AIDS.
Keywords: O-GlcNAc; GlcNAcylation; Phosphorylation; OGT; OGA; Stress; Glucosamine; Alzheimer's disease; Signaling; Diabetes; O-GlcNAcase; O-GlcNAc transferase;

The O-GlcNAc modification is found on many nucleocytoplasmic proteins. The dynamic nature of O-GlcNAc, which in some ways is reminiscent of phosphorylation, has enabled investigators to modulate the stoichiometry of O-GlcNAc on proteins in order to study its function. Although several genetic and pharmacological methods for manipulating O-GlcNAc levels have been described, one of the most direct approaches of increasing global O-GlcNAc levels is by using small-molecule inhibitors of O-GlcNAcase (OGA). As the interest in increasing O-GlcNAc levels has grown, so too has the number of OGA inhibitors. This review provides an overview of the available methods of increasing O-GlcNAc levels, with a special emphasis on inhibition of OGA by small molecules. Known inhibitors of OGA are discussed with particular attention on those most suitable for cell-based biological studies. Several examples in which OGA inhibitors have been used to study the functional role of the O-GlcNAc modification in biological systems are discussed, highlighting the pros and cons of different inhibitors.
Keywords: O-GlcNAc; Nucleocytoplasmic glycosylation; Glycoprotein; O-GlcNAcase; OGA; Enzyme inhibitor; Glycoside hydrolase; Substrate-assisted catalysis;

Structural analyses of enzymes involved in the O-GlcNAc modification by Carlos Martinez-Fleites; Yuan He; Gideon J. Davies (122-133).
In order to study the O-GlcNAc modification in vivo, it is evident that a range of specific small molecule inhibitors would be a valuable asset. One strategy for the design of such compounds would be to utilise 3-D structural information in tandem with knowledge of catalytic mechanism. The last few years has seen major breakthroughs in our understanding of the 3-D structure of the enzymes involved in the O-GlcNAc modification notably from the study of the tetratricopeptide repeat (TPR) domain of the human O-GlcNAc transferase, of the bacterial homologs of the O-GlcNAc hydrolase and more latterly bacterial homologs of the O-GlcNAc transferase itself. Of particular note are the bacterial O-GlcNAc hydrolase homologs that provide near identical active centres to the human enzyme. These have informed the design and/or subsequent analysis of inhibitors of this enzyme which have found great use in the chemical dissection of the O-GlcNAc in vivo, as described by Macauley and Vocadlo elsewhere in this issue.
Keywords: Structure; O-GlcNAc; Enzyme; Reaction mechanism; Carbohydrate-active enzyme; GH84; GT41; Hydrolase; Transferase;

Bacterial toxin and effector glycosyltransferases by Yury Belyi; Klaus Aktories (134-143).
Clostridial glucosylating cytotoxins, including Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal toxin, are major virulence factors and causative agents of human diseases. These toxins mono-O-glucosylate (or mono-O-GlcNAcylate) a specific threonine residue of Rho/Ras-proteins, which is essential for the function of the molecular switches. Recently, a related group of glucosyltransferases from Legionella pneumophila has been identified. These Legionella glucosyltransferases modify the large GTPase elongation factor eEF1A at a serine residue by mono-O-glucosylation, thereby inhibiting protein synthesis of target cells. Recent results on structures, functions and biological roles of both groups of bacterial toxin glucosyltransferases will be discussed.
Keywords: Glycosyltransferase; Rho protein; Bacterial protein toxin; Glucosylation; Clostridial glucosylating toxin; Clostridium difficile toxin; Legionella pneumophila; Elongation factor 1A;

Plastid peptidoglycan by Hiroyoshi Takano; Katsuaki Takechi (144-151).
It is now widely accepted that an endosymbiotic cyanobacterium evolved into the plastid of the primary photosynthetic eukaryotes: glaucocystophytes, red algae, and green plants. It has been thought that during the evolution of plants, the peptidoglycan wall (or murein) was lost from the endosymbiont immediately after the branching off of the glaucocystophytes, which have peptidoglycan-armed plastids termed cyanelles. However, we found that the moss Physcomitrella patens has all of the genes for peptidoglycan biosynthesis with the exception of one racemase. The aim of the present review is to summarize recent findings on plastid peptidoglycan and to present a hypothesis for the evolution of plastids containing peptidoglycan. Gene knockout experiments for the Mur(ein) genes, including MurE in P. patens, showed that the peptidoglycan synthesis pathway is related to plastid division, although no structure can be detected between the inner and outer envelopes of the chloroplasts by electron microscopy. On the other hand, MurE in Arabidopsis thaliana has a function in plastid gene expression and not in division. Based on data regarding plant genomes and antibiotic treatment experiments of plastid division, we propose that the loss of peptidoglycan occurred independently at least three times during plant evolution: from the lineage of red algae, from the chlorophytes, and during land plant evolution.
Keywords: Arabidopsis; Chlamydia; Evolution; Moss; Plastid division; Peptidoglycan;

Chlorella viruses encode most, if not all, of the machinery to glycosylate their glycoproteins independent of the endoplasmic reticulum and Golgi by James L. Van Etten; James R. Gurnon; Giane M. Yanai-Balser; David D. Dunigan; Michael V. Graves (152-159).
In contrast to all other viruses that use the host machinery located in the endoplasmic reticulum and Golgi to glycosylate their glycoproteins, the large dsDNA-containing chlorella viruses encode most, if not all, of the components to glycosylate their major capsid proteins. Furthermore, all experimental results indicate that glycosylation occurs independent of the endoplasmic reticulum and Golgi.
Keywords: Chlorella viruses; PBCV-1; Virus-encoded glycosyltransferases; Cytoplasmic glycosylation; Virus major capsid protein;

The soil amoeba Dictyostelium is an obligate aerobe that monitors O2 for informational purposes in addition to utilizing it for oxidative metabolism. Whereas low O2 suffices for proliferation, a higher level is required for slugs to culminate into fruiting bodies, and O2 influences slug polarity, slug migration, and cell-type proportioning. Dictyostelium expresses a cytoplasmic prolyl 4-hydroxylase (P4H1) known to mediate O2-sensing in animals, but lacks HIFα, a major hydroxylation target whose accumulation directly induces animal hypoxia-dependent transcriptional changes. The O2-requirement for culmination is increased by P4H1-gene disruption and reduced by P4H1 overexpression. A target of Dictyostelium P4H1 is Skp1, a subunit of the SCF-class of E3-ubiquitin ligases related to the VBC-class that mediates hydroxylation-dependent degradation of animal HIFα. Skp1 is a target of a novel cytoplasmic O-glycosylation pathway that modifies HyPro143 with a pentasaccharide, and glycosyltransferase mutants reveal that glycosylation intermediates have antagonistic effects toward P4H1 in O2-signaling. Current evidence indicates that Skp1 is the only glycosylation target in cells, based on metabolic labeling, biochemical complementation, and enzyme specificity studies. Bioinformatics studies suggest that the HyPro-modification pathway existed in the ancestral eukaryotic lineage and was retained in selected modern day unicellular organisms whose life cycles experience varying degrees of hypoxia. It is proposed that, in Dictyostelium and other protists including the agent for human toxoplasmosis Toxoplasma gondii, prolyl hydroxylation and glycosylation mediate O2-signaling in hierarchical fashion via Skp1 to control the proteome, directly via degradation rather than indirectly via transcription as found in animals.
Keywords: Cytoplasmic glycosylation; Hydroxyproline; Skp1; Hypoxia; Dictyostelium; Toxoplasma;

Lectin-like ERAD players in ER and cytosol by Yukiko Yoshida; Keiji Tanaka (172-180).
Protein quality control in the endoplasmic reticulum (ER) is an elaborate process conserved from yeast to mammals, ensuring that only newly synthesized proteins with correct conformations in the ER are sorted further into the secretory pathway. It is well known that high-mannose type N-glycans are involved in protein-folding events. In the quality control process, proteins that fail to achieve proper folding or proper assembly are degraded in a process known as ER-associated degradation (ERAD). The ERAD pathway comprises multiple steps including substrate recognition and targeting to the retro-translocation machinery, retrotranslocation from the ER into the cytosol, and proteasomal degradation through ubiquitination. Recent studies have documented the important roles of sugar-recognition (lectin-type) molecules for trimmed high-mannose type N-glycans and glycosidases in the ERAD pathways in both ER and cytosol. In this review, we discuss a fundamental system that monitors glycoprotein folding in the ER and the unique roles of the sugar-recognizing ubiquitin ligase and peptide:N-glycanase (PNGase) in the cytosolic ERAD pathway.
Keywords: F-box protein; ERAD; N-glycan; Quality control; Lectin;

Dynamics of galectin-3 in the nucleus and cytoplasm by Kevin C. Haudek; Kimberly J. Spronk; Patricia G. Voss; Ronald J. Patterson; John L. Wang; Eric J. Arnoys (181-189).
This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (Mr ∼ 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH2-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein–protein interactions rather than through protein–carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3's anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein's carbohydrate-binding activity, per se, remains a challenge for future investigations.
Keywords: Carbohydrate-binding protein; Lectin; Nucleo-cytoplasmic transport;

Nucleocytoplasmic plant lectins by Nausicaä Lannoo; Els J.M. Van Damme (190-201).
During the last decade it was unambiguously shown that plants synthesize minute amounts of carbohydrate-binding proteins upon exposure to stress situations like drought, high salt, hormone treatment, pathogen attack or insect herbivory. In contrast to the ‘classical’ plant lectins, which are typically found in storage vacuoles or in the extracellular compartment this new class of lectins is located in the cytoplasm and the nucleus. Based on these observations the concept was developed that lectin-mediated protein–carbohydrate interactions in the cytoplasm and the nucleus play an important role in the stress physiology of the plant cell. Hitherto, six families of nucleocytoplasmic lectins have been identified. This review gives an overview of our current knowledge on the occurrence of nucleocytoplasmic plant lectins. The carbohydrate-binding properties of these lectins and potential ligands in the nucleocytoplasmic compartment are discussed in view of the physiological role of the lectins in the plant cell.
Keywords: Cytosol; Glycosylation; Inducible; Lectin; N-glycan; Nucleus;