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Gerald W Hart

Publications and source records attributed to Gerald W Hart.

At least 19 recordsLinked to original sources
PubMed2006

Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats.

Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide beta-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto-Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.

Acetylglucosamine↗
PubMed2006

Reciprocal keratin 18 Ser48 O-GlcNAcylation and Ser52 phosphorylation using peptide analysis.

Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.

Acylation↗
PubMed2006

Cell signaling, the essential role of O-GlcNAc!

An increasing body of evidence points to a central regulatory role for glucose in mediating cellular processes and expands the role of glucose well beyond its traditional role(s) in energy metabolism. Recently, it has been recognized that one downstream effector produced from glucose is UDP-GlcNAc. Levels of UDP-GlcNAc, and the subsequent addition of O-linked beta-N-acetylglucosamine (O-GlcNAc) to Ser/Thr residues, is involved in regulating nuclear and cytoplasmic proteins in a manner analogous to protein phosphorylation. O-GlcNAc protein modification is essential for life in mammalian cells, highlighting the importance of this simple post-translational modification in basic cellular regulation. Recent research has highlighted key roles for O-GlcNAc serving as a nutrient sensor in regulating insulin signaling, the cell cycle, and calcium handling, as well as the cellular stress response.

Acetylglucosamine↗
PubMed2006

O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks.

O-GlcNAc is an ubiquitous post-translational protein modification consisting of a single N-acetlyglucosamine moiety linked to serine or threonine residues on nuclear and cytoplasmic proteins. Recent work has begun to uncover the functional roles of O-GlcNAc in cellular processes. O-GlcNAc modified proteins are involved in sensing the nutrient status of the surrounding cellular environment and adjusting the activity of cellular proteins accordingly. O-GlcNAc regulates cellular responses to hormones such as insulin, initiates a protective response to stress, modulates a cell's capacity to grow and divide, and regulates gene transcription. This review will focus on recent work involving O-GlcNAc in sensing the environment and regulating signaling cascades.

Acetylglucosamine↗
PubMed2006

Fine-tuning ER-beta structure with PTMs.

Estrogen receptor beta is differentially regulated by alternative O-GlcNAcylation/O-phosphorylation at Ser(16). NMR, CD, and molecular dynamics analyses of model peptides show that these alternative modifications induce different peptide conformations, providing a molecular basis for their differential regulation of protein function.

Acetylglucosamine↗
PubMed2006

Identification of O-GlcNAc sites on proteins.

O-linked N-acetylglucosamine (O-GlcNAc) is a monosaccharide posttranslational modification that modifies serine/threonine residues of nucleocytoplasmic proteins in metazoans. O-GlcNAc, like phosphorylation, is dynamic and responsive to numerous stimuli in diverse regulatory pathways. O-GlcNAc may also be found adjacent to or at the same sites as phosphorylation, demonstrating the potential for a reciprocal function on some of these proteins. Like most posttranslational modifications, O-GlcNAc is substoichiometric and may be found at multiple sites with other posttranslational modifications present. Additionally, there is no consensus sequence defining the addition of O-GlcNAc to the peptide backbone, further complicating identification and site mapping. This chapter describes several strategies to confirm that proteins are O-GlcNAc modified and provide subsequent determination of O-GlcNAc attachment sites. We have listed the strengths and limitations of each protocol to allow readers to decide which suits their system and availability of resources. These protocols include galactosyltransferase labeling, immunoblotting, using mass spectrometry based on beta-elimination followed by Michael addition with dithiothreitol, and chemoenzymatic labeling, enrichment, and detection.

Acetylglucosamine↗
PubMed2006

Posttranslational, reversible O-glycosylation is stimulated by high glucose and mediates plasminogen activator inhibitor-1 gene expression and Sp1 transcriptional activity in glomerular mesangial cells.

Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG-stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG-induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral beta-N-acetylglucosaminidase, and by knockdown of O-linked beta-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the beta-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG-induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells.

Acetylglucosamine↗
PubMed2005

Perturbations in O-linked beta-N-acetylglucosamine protein modification cause severe defects in mitotic progression and cytokinesis.

The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification that is rapidly responsive to morphogens, hormones, nutrients, and cellular stress. Here we show that O-GlcNAc is an important regulator of the cell cycle. Increased O-GlcNAc (pharmacologically or genetically) results in growth defects linked to delays in G2/M progression, altered mitotic phosphorylation, and cyclin expression. Overexpression of O-GlcNAcase, the enzyme that removes O-GlcNAc, induces a mitotic exit phenotype accompanied by a delay in mitotic phosphorylation, altered cyclin expression, and pronounced disruption in nuclear organization. Overexpression of the O-GlcNAc transferase, the enzyme that adds O-GlcNAc, results in a polyploid phenotype with faulty cytokinesis. Notably, O-GlcNAc transferase is concentrated at the mitotic spindle and midbody at M phase. These data suggest that dynamic O-GlcNAc processing is a pivotal regulatory component of the cell cycle, controlling cell cycle progression by regulating mitotic phosphorylation, cyclin expression, and cell division.

3T3-L1 Cells↗
PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗
PubMed2005

O-GlcNAc modification of nucleocytoplasmic proteins and diabetes.

Nuclear and cytosolic proteins are glycosylated on serine or threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification is one of various posttranslational modifications and seems to be involved in the modulation of transcription and signal transduction. Accumulating data suggest a role for O-GlcNAc-modified proteins in diabetes, acting as a glucose sensor. It has been suggested that the hexosamine biosynthetic pathway is involved in the mechanism causing insulin resistance and diabetic complications. Excess glucose entering into the hexosamine biosynthetic pathway might cause elevated O-GlcNAc modification of various proteins. In this article, we review the current data regarding the relationship between O-GlcNAc modification and diabetes.

Acetylglucosamine↗
PubMed2004

O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer's disease.

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains of individuals with Alzheimer's disease (AD) and other tauopathies. Tau pathology is critical to pathogenesis and correlates to the severity of dementia. However, the mechanisms leading to abnormal hyperphosphorylation are unknown. Here, we demonstrate that human brain tau was modified by O-GlcNAcylation, a type of protein O-glycosylation by which the monosaccharide beta-N-acetylglucosamine (GlcNAc) attaches to serine/threonine residues via an O-linked glycosidic bond. O-GlcNAcylation regulated phosphorylation of tau in a site-specific manner both in vitro and in vivo. At most of the phosphorylation sites, O-GlcNAcylation negatively regulated tau phosphorylation. In an animal model of starved mice, low glucose uptake/metabolism that mimicked those observed in AD brain produced a decrease in O-GlcNAcylation and consequent hyperphosphorylation of tau at the majority of the phosphorylation sites. The O-GlcNAcylation level in AD brain extracts was decreased as compared to that in controls. These results reveal a mechanism of regulation of tau phosphorylation and suggest that abnormal hyperphosphorylation of tau could result from decreased tau O-GlcNAcylation, which probably is induced by deficient brain glucose uptake/metabolism in AD and other tauopathies.

Acetylglucosamine↗
PubMed2004

O-GlcNAc transferase is in a functional complex with protein phosphatase 1 catalytic subunits.

A hallmark of signal transduction is the dynamic and inducible post-translational modification of proteins. In addition to the well characterized phosphorylation of proteins, other modifications have been shown to be regulatory, including O-linked beta-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modifies serine and threonine residues on a myriad of nuclear and cytosolic proteins, and for several proteins there appears to be a reciprocal relationship between phosphorylation and O-GlcNAc modification. Here we report further evidence of this yin-yang relationship by demonstrating that O-GlcNAc transferase, the enzyme that adds O-GlcNAc to proteins, exists in stable and active complexes with the serine/threonine phosphatases PP1beta and PP1gamma, enzymes that remove phosphate from proteins. The existence of this complex highlights the importance of understanding the dynamic relationship between O-GlcNAc and phosphate in modulating protein function in many cellular processes and disease states such as Alzheimer's disease and type II diabetes.

Animals↗
PubMed2004

O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.

Myriad nuclear and cytoplasmic proteins in metazoans are modified on Ser and Thr residues by the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc). The rapid and dynamic change in O-GlcNAc levels in response to extracellular stimuli, morphogens, the cell cycle and development suggests a key role for O-GlcNAc in signal transduction pathways. Modulation of O-GlcNAc levels has profound effects on the functioning of cells, in part mediated through a complex interplay between O-GlcNAc and O-phosphate. In many well-studied proteins, the O-GlcNAc modification and phosphorylation are reciprocal. That is, they occur on different subsets of the protein population, as the site of attachment occurs on the same or adjacent Ser/Thr residues. Recently, O-GlcNAc has been implicated in the etiology of type II diabetes, the regulation of stress response pathways, and in the regulation of the proteasome.

Acetylglucosamine↗
PubMed2004

Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells.

Cellular response to environmental, physiological, or chemical stress is key to survival following injury or disease. Here we describe a unique signaling mechanism by which cells detect and respond to stress in order to survive. A wide variety of stress stimuli rapidly increase nucleocytoplasmic protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc), an essential post-translational modification of Ser and Thr residues of metazoans. Blocking this post-translational modification, or reducing it, renders cells more sensitive to stress and results in decreased cell survival; and increasing O-GlcNAc levels protects cells. O-GlcNAc regulates both the rates and extent of the stress-induced induction of heat shock proteins, providing a molecular basis for these findings.

Acetylglucosamine↗
PubMed2004

O-GlcNAc modification: a nutritional sensor that modulates proteasome function.

The addition of O-linked beta-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues is a post-translational modification of nucleocytoplasmic proteins that is thought to act in a manner analogous to protein phosphorylation. Recent work shows that many proteins of the metazoan proteasome are modified by O-GlcNAc and that the level of glycosylation is responsive to the nutritional state of the cell. Moreover, increased glycosylation of the 19S (or PA700) regulatory subcomplex has been correlated with decreased proteasomal activity, suggesting a new model of proteasomal regulation.

Acetylglucosamine↗
PubMed2004

Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.

The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling pathways.

Animals↗