Muscle-specific overexpression of NCOATGK, splice variant of O-GlcNAcase, induces skeletal muscle atrophy.

The protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification plays an important role in skeletal muscle development and physiological function. In this study, bitransgenic mice were generated that overexpressed NCOAT(GK), an O-GlcNAcase-inactive spliced variant of the O-GlcNAcase gene, specifically in skeletal muscle using the muscle creatine kinase promoter. Expression of the chimeric enhanced green fluorescent protein-NCOAT(GK) transgene caused an increase of cellular O-GlcNAc levels, along with the accumulation and activation of proapoptotic factors in muscles of bitransgenic mice.

O-GlcNAcylation enhances FOXO4 transcriptional regulation in response to stress.

The FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response.

Increased O-GlcNAc causes disrupted lens fiber cell differentiation and cataracts.

Diminished proteolytic functionality in the lens may cause cataracts. We have reported that O-GlcNAc is an endogenous inhibitor of the proteasome. We hypothesize that in the lens there is a cause-and-effect relationship between proteasome inhibition by O-GlcNAc, and cataract formation.

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain.

Metabolic control of proteasome function.

Proteasomes are major cellular proteases that are important for protein turnover and cell survival. Dysregulation of proteasome is related to many major human diseases. Regulation of the proteasome is beginning to be understood by the recent findings that proteasomes are modified and regulated by metabolic factors O-GlcNAcylation and PKA phosphorylation.

Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6.

Dysregulation of the proteasome has been documented in a variety of human diseases such as Alzheimer, muscle atrophy, cataracts etc. Proteolytic activity of 26 S proteasome is ATP- and ubiquitin-dependent. O-GlcNAcylation of Rpt2, one of the AAA ATPases in the 19 S regulatory cap, shuts off the proteasome through the inhibition of ATPase activity.

Induction of macrophage glutamine: fructose-6-phosphate amidotransferase expression by hypoxia and by picolinic acid.

We studied the expression of glutamine: fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme in the hexosamine biosynthetic pathway controlling protein glycosylation. We obtained the first evidence that the GFAT mRNA and protein are constitutively expressed in murine mononuclear phagocytes (Mf) and inducible by picolinic acid (PA), a catabolite of tryptophan, hypoxia and desferrioxamine (DFX). These stimuli share the property to transactivate gene expression through the Hypoxia Responsive Element (HRE).

O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptors.

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors.

Post-translational modification by O-GlcNAc: another way to change protein function.

Modification of intracellular proteins by the beta-linkage of the monosaccharide, N-acetylglucosamine to serine or threonine hydroxyls (O-GlcNAc) is abundant and reversible. Although many proteins bear this post-translational covalent modification, the changes in function of the proteins as a result of this modification are only starting to be understood. In this article, we describe how aspects of the flux from the glucose backbone to this modification are modified and how the cellular activity and content of the GC-box binding transcription factor, Sp1, is altered by O-glycosylation.

Location and characterization of the O-GlcNAcase active site.

NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GlcNAcase and histone acetyltransferase domains. The O-GlcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GlcNAc suggests that the O-GlcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases.

Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development.

Although the knowledge that nuclear and cytoplasmic proteins are modified with N-acetylglucosamine has existed for decades, little has been shown as to its function until recently. There are now substantial data highlighting the significance of proper regulation of this modification in multiple cellular processes. Currently, only two enzymes are known that regulate this modification.

Streptozotocin inhibits O-GlcNAcase via the production of a transition state analog.

Streptozotocin (STZ) is a 2-deoxy-d-glucopyranose derivative of a class of drugs known as alkylnitrosoureas, and is an established diabetogenic agent whose cytotoxic affects on pancreatic beta-cells has been partially explained by the presence of its N-methyl-N-nitrosourea side chain, which has the ability to release nitric oxide as well as donate methyl groups to nucleotides in DNA. It has also been observed that STZ administration results in a rise in the level of O-GlcNAcylated proteins within beta-cells. Not coincidentally, STZ has also been shown to directly inhibit the O-GlcNAcase activity of the enzyme NCOAT in vitro, which is the only enzyme that possesses the ability to remove O-GlcNAc modifications on proteins in the nucleus and cytosol.

The histone acetyltransferase NCOAT contains a zinc finger-like motif involved in substrate recognition.

Nuclear cytoplasmic O-GlcNAcase and acetyltransferase (NCOAT) is a bifunctional enzyme with both glycoside hydrolase and alkyltransferase activity. Its O-GlcNAcase active site lies in the N terminus of the enzyme and its histone acetyltransferase (HAT) domain lies in the C terminus. Whereas the HAT domain of the enzyme is catalytically and structurally similar to other acetyltransferases across subfamilies, NCOAT has a motif resembling a zinc finger-like domain unique to the MYST family of HATs.

Proteolytic processing of TGFalpha redirects its mitogenic activity: the membrane-anchored form is autocrine, the secreted form is paracrine.

Wild-type transforming growth factor alpha (TGFalpha) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFalpha-mediated phenotype, we bred TGFalpha mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor.

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells.

Characterization of the histone acetyltransferase (HAT) domain of a bifunctional protein with activable O-GlcNAcase and HAT activities.

Histones and transcription factors are regulated by a number of post-translational modifications that in turn regulate the transcriptional activity of genes. These modifications occur in large, multisubunit complexes. We have reported previously that mSin3A can recruit O-GlcNAc transferase (OGT) along with histone deacetylase into such a corepressor complex.

Health-care charges generated by patients with diabetes.

For assessment of health-care charges generated by patients with diabetes, 50 patients (25 who were older than 65 years and 25 who were age 65 or younger) whose primary care had been delivered between 1990 and 1995 by a single diabetologist were randomly selected. Institutional health-care bills were accessed from all but three medical center billing offices (dermatology, psychiatry, and radiation oncology areas did not respond). For each dollar billed by endocrinology, the hospital billed 25.

Accumulation of protein O-GlcNAc modification inhibits proteasomes in the brain and coincides with neuronal apoptosis in brain areas with high O-GlcNAc metabolism.

All tissues contain the enzymes that modify and remove O-GlcNAc dynamically from nucleocytoplasmic proteins. These enzymes have been shown to play a role in the control of transcription, vesicular trafficking and, more recently, proteasome function. Modification by O-GlcNAc of the 19S cap of the proteasome inhibits proteasomal function.

Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity.

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity.

Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein.

Three mouse lines expressing Cre recombinase under the control of the human K14 promoter induced specific deletion of loxP flanked target sequences in the epidermis, in tongue, and thymic epithelium of the offspring where the Cre allele was inherited from the father. Where the mother carried the Cre allele, loxP flanked sequences were completely deleted in all tissues of the offspring, even in littermates that did not inherit the Cre allele. This maternally inherited phenotype indicates that the human K14 promoter is transcriptionally active in murine oocytes and that the enzyme remains active until after fertilization, even when the Cre allele becomes transmitted to the polar bodies during meiosis.

O-GlcNAc modification is an endogenous inhibitor of the proteasome.

The ubiquitin proteasome system classically selects its substrates for degradation by tagging them with ubiquitin. Here, we describe another means of controlling proteasome function in a global manner. The 26S proteasome can be inhibited by modification with the enzyme, O-GlcNAc transferase (OGT).

The role of O-linked protein glycosylation in beta-cell dysfunction.

Although only recently described, the pathway of O-linked protein glycosylation is already being implicated in diseases as diverse as cancer and Alzheimer's. Unlike the better known N-linked pathway, O-linked protein glycosylation is a dynamic and regulated event, much like tyrosine phosphorylation. During the process of O-glycosylation, the enzyme O-GlcNAc transferase (OGT) uses the substrate UDP-N-acetylglucosamine (UDP-GlcNAc) to attach a single O-linked N-acetylglucosamine (O-GlcNAc) to nuclear and cytosolic proteins on serine or threonine residues.

Streptozotocin, an O-GlcNAcase inhibitor, blunts insulin and growth hormone secretion.

Type 2 diabetes mellitus results from a complex interaction between nutritional excess and multiple genes. Whereas pancreatic beta-cells normally respond to glucose challenge by rapid insulin release (first phase insulin secretion), there is a loss of this acute response in virtually all of the type 2 diabetes patients with significant fasting hyperglycemia. Our previous studies demonstrated that irreversible intracellular accumulation of a glucose metabolite, protein O-linked N-acetylglucosamine modification (O-GlcNAc), is associated with pancreatic beta-cell apoptosis.