Nutrient-Driven -GlcNAcylation at Promoters Impacts Genome-Wide RNA Pol II Distribution.

Nutrient-driven -GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In -GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these -GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of -GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the -GlcNAcase OGA-1.

Nutrient-driven -linked -acetylglucosamine (-GlcNAc) cycling impacts neurodevelopmental timing and metabolism.

Nutrient-driven -GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the -GlcNAcase () gene in the mouse brain to define the role of -GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain -GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation.

Drosophila O-GlcNAcase Deletion Globally Perturbs Chromatin O-GlcNAcylation.

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification.

Conditional knock-out reveals a requirement for O-linked N-Acetylglucosaminase (O-GlcNAcase) in metabolic homeostasis.

O-GlcNAc cycling is maintained by the reciprocal activities of the O-GlcNAc transferase and the O-GlcNAcase (OGA) enzymes. O-GlcNAc transferase is responsible for O-GlcNAc addition to serine and threonine (Ser/Thr) residues and OGA for its removal. Although the Oga gene (MGEA5) is a documented human diabetes susceptibility locus, its role in maintaining insulin-glucose homeostasis is unclear.

Disruption of O-GlcNAc Cycling in C. elegans Perturbs Nucleotide Sugar Pools and Complex Glycans.

The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C.

Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation.

Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase.

Chemical tools to explore nutrient-driven O-GlcNAc cycling.

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.

Versatile O-GlcNAc transferase assay for high-throughput identification of enzyme variants, substrates, and inhibitors.

The dynamic glycosylation of serine/threonine residues on nucleocytoplasmic proteins with a single N-acetylglucosamine (O-GlcNAcylation) is critical for many important cellular processes. Cellular O-GlcNAc levels are highly regulated by two enzymes: O-GlcNAc transferase (OGT) is responsible for GlcNAc addition and O-GlcNAcase (OGA) is responsible for removal of the sugar. The lack of a rapid and simple method for monitoring OGT activity has impeded the efficient discovery of potent OGT inhibitors.

Evaluation of the Fluids Mixing Enclosure System for Life Science Experiments During a Commercial Spaceflight Experiment.

The Student Spaceflight Experiments Program (SSEP) is a United States national science, technology, engineering, and mathematics initiative that aims to increase student interest in science by offering opportunities to perform spaceflight experiments. The experiment detailed here was selected and flown aboard the third SSEP mission and the first SSEP mission to the International Space Station (ISS). is a small, transparent, self-fertilizing hermaphroditic roundworm that is commonly used in biological experiments both on Earth and in Low Earth Orbit.

Optimizing the selectivity of DIFO-based reagents for intracellular bioorthogonal applications.

One of the most commonly employed bioorthogonal reactions with azides is copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC, a 'click' reaction). More recently, the strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction) was developed, in which an alkyne is sufficiently strained to promote rapid cycloaddition with an azide to form a stable triazole conjugate. In this report, we show that an internal alkyne in a strained ring system with two electron-withdrawing fluorine atoms adjacent to the carbon-carbon triple bond reacts to yield covalent adducts not only with azide moieties but also reacts with free sulfhydryl groups abundant in the cytosol.

O-GlcNAc cycling mutants modulate proteotoxicity in Caenorhabditis elegans models of human neurodegenerative diseases.

O-GlcNAcylation is an abundant posttranslational modification in the brain implicated in human neurodegenerative diseases. We have exploited viable null alleles of the enzymes of O-GlcNAc cycling to examine the role of O-GlcNAcylation in well-characterized Caenorhabditis elegans models of neurodegenerative proteotoxicity. O-GlcNAc cycling dramatically modulated the severity of the phenotype in transgenic models of tauopathy, amyloid β-peptide, and polyglutamine expansion.

The signal peptide of mouse mammary tumor virus-env: a phosphoprotein tumor modulator.

Mouse mammary tumor virus (MMTV) is associated primarily with mammary carcinomas and lymphomas. The signal peptide of the MMTV envelope precursor is uniquely targeted to nucleoli of cells that harbor the virus, where it can function as a nuclear export factor for intron-containing transcripts. Antibodies to this signal peptide, which we refer to as p14, were previously shown to label nucleoli in a subset of human breast cancers.

Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation.

O-GlcNAcylation, which is a nutrient-sensitive sugar modification, participates in the epigenetic regulation of gene expression. The enzymes involved in O-linked β-D-N-acetylglucosamine (O-GlcNAc) cycling - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - target key transcriptional and epigenetic regulators including RNA polymerase II, histones, histone deacetylase complexes and members of the Polycomb and Trithorax groups. Thus, O-GlcNAc cycling may serve as a homeostatic mechanism linking nutrient availability to higher-order chromatin organization.

A lipid-droplet-targeted O-GlcNAcase isoform is a key regulator of the proteasome.

Protein-O-linked N-Acetyl-β-D-glucosaminidase (O-GlcNAcase, OGA; also known as hexosaminidase C) participates in a nutrient-sensing, hexosamine signaling pathway by removing O-linked N-acetylglucosamine (O-GlcNAc) from key target proteins. Perturbations in O-GlcNAc signaling have been linked to Alzheimer's disease, diabetes and cancer. Mammalian O-GlcNAcase exists as two major spliced isoforms differing only by the presence (OGA-L) or absence (OGA-S) of a histone-acetyltransferase domain.

O-linked-N-acetylglucosamine cycling and insulin signaling are required for the glucose stress response in Caenorhabditis elegans.

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C.

Blocking O-linked GlcNAc cycling in Drosophila insulin-producing cells perturbs glucose-insulin homeostasis.

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling.

Elevated O-GlcNAc-dependent signaling through inducible mOGT expression selectively triggers apoptosis.

O-linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAc addition to numerous cellular proteins including transcription and nuclear pore complexes and plays a key role in cellular signaling. One differentially spliced isoform of OGT is normally targeted to mitochondria (mOGT) but is quite cytotoxic when expressed in cells compared with the ncOGT isoform. To understand the basis of this selective cytotoxicity, we constructed a fully functional ecdysone-inducible GFP-OGT.

OGA inhibition by GlcNAc-selenazoline.

The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.

O-GlcNAc cycling: emerging roles in development and epigenetics.

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan.

Dynamic O-GlcNAc cycling at promoters of Caenorhabditis elegans genes regulating longevity, stress, and immunity.

Nutrient-driven O-GlcNAcylation of key components of the transcription machinery may epigenetically modulate gene expression in metazoans. The global effects of GlcNAcylation on transcription can be addressed directly in C. elegans because knockouts of the O-GlcNAc cycling enzymes are viable and fertile.

O-GlcNAc cycling: implications for neurodegenerative disorders.

The dynamic post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation, is an important mechanism for modulating cellular signaling pathways. O-GlcNAcylation impacts transcription, translation, organelle trafficking, proteasomal degradation and apoptosis. O-GlcNAcylation has been implicated in the etiology of several human diseases including type-2 diabetes and neurodegeneration.

The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine.

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.

Calmodulin-driven nuclear entry: trigger for sex determination and terminal differentiation.

We originally proposed that Ca(2+)-calmodulin mediates a novel nuclear entry pathway distinct from the canonic Ran-dependent pathway (Sweitzer, T. D., and Hanover, J.

Distinctive inhibition of O-GlcNAcase isoforms by an alpha-GlcNAc thiolsulfonate.

O-GlcNAcase (OGA) promotes O-GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O-GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown.

The High Mobility Group Box Transcription Factor Nhp6Ap enters the nucleus by a calmodulin-dependent, Ran-independent pathway.

A gradient of Ran.GTP typically regulates traffic through the nuclear pore by modulating association of receptors with cargo. However, here we demonstrate that the yeast high mobility group box transcription factor Nhp6Ap enters the nucleus via a novel nuclear localization signal recognized by calcium calmodulin in a process that does not require Ran.