Dona C. Love

Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia

Insulin resistance and β cell toxicity are key features of type 2 diabetes. One leading hypothesis suggests that these abnormalities result from excessive flux of nutrients through the UDP–hexosamine biosynthetic pathway leading to “glucose toxicity.” How the products of the hexosamine pathway mediate these effects is not known. Here, we show that transgenic overexpression of an enzyme using UDP-GlcNAc to modify proteins with O-GlcNAc produces the type 2 diabetic phenotype. Even modest overexpression of an isoform of O-GlcNAc transferase, in muscle and fat, leads to insulin resistance and hyperleptinemia. These data support the proposal that O-linked GlcNAc transferase participates in a hexosamine-dependent signaling pathway that is linked to insulin resistance and leptin production.

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. In response to nutrient availability, O-GlcNAcylation is poised to influence X chromosome inactivation and genetic imprinting, as well as embryonic development. The wide range of physiological functions regulated by O-GlcNAc cycling suggests an unexplored nexus between epigenetic regulation in disease and nutrient availability.

Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene.

O-Linked N-acetylglucosamine (GlcNAc) transferase (OGT) mediates a novel hexosamine-dependent signal transduction pathway. Yet, little is known about the regulation of ogt gene expression in mammals. We report the sequence and characterization of the mouse ogt locus and provide a comparison with the human and rat ogt genes. The mammalian ogt genes are similar in structure and exhibit approximately 80% sequence identity. The mouse and human ogt genes contain two potential promoters producing four major transcripts. By analyzing 56 human cDNA clones and other existing expressed sequence tags, we found that at least three protein products differing at their amino terminus result from alternative splicing. We used OGT-specific antisera to demonstrate the presence of these isoforms in HeLa cells. The longest form is a nucleocytoplasmic OGT (ncOGT) with 12 tetratricopeptide repeats (TPRs); a shorter form of OGT encodes a mitochondrially sequestered enzyme with 9 TPRs and an N-terminal mitochondrion-targeting sequence (mOGT). An even shorter form of OGT (sOGT) contains only 2 TPRs. The genomic organization of OGT appears to be highly conserved throughout metazoan evolution. These results provide the basis for a more detailed analysis of the significance and regulation of the nucleocytoplasmic and mitochondrial isoforms of OGT in mammals.

Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase

O-linked GlcNAc transferase (OGT) mediates a novel glycan-dependent signaling pathway, but the intracellular targeting of OGT is poorly understood. We examined the localization of OGT by immunofluorescence microscopy, subcellular fractionation and immunoblotting using highly specific affinity-purified antisera. In addition to the expected nuclear localization, we found that OGT was highly concentrated in mitochondria. Since the mitochondrial OGT (103 kDa) was smaller than OGT found in other compartments (116 kDa) we reasoned that it was one of two predicted splice variants of OGT. The N-termini of these isoforms are unique; the shorter form contains a potential mitochondrial targeting sequence. We found that when epitope-tagged, the shorter form (mOGT; 103 kDa) concentrated in HeLa cell mitochondria, whereas the longer form (ncOGT; 116 kDa) localized to the nucleus and cytoplasm. The N-terminus of mOGT was essential for proper targeting. Although mOGT appears to be an active transferase, O-linked GlcNAc-modified substrates do not accumulate in mitochondria. Using immunoelectron microscopy and mitochondrial fractionation, we found that mOGT was tightly associated with the mitochondrial inner membrane. The differential localization of mitochondrial and nucleocytoplasmic isoforms of OGT suggests that they perform unique intracellular functions.

Cessation of rapid late endosomal tubulovesicular trafficking in Niemann–Pick type C1 disease

Niemann–Pick type C1 (NPC1) disease results from a defect in the NPC1 protein and is characterized by a pathological accumulation of cholesterol and glycolipids in endocytic organelles. We followed the biosynthesis and trafficking of NPC1 with the use of a functional green fluorescent protein-fused NPC1. Newly synthesized NPC1 is exported from the endoplasmic reticulum and requires transit through the Golgi before it is targeted to late endosomes. NPC1-containing late endosomes then move by a dynamic process involving tubulation and fission, followed by rapid retrograde and anterograde migration along microtubules. Cell fusion studies with normal and mutant NPC1 cells show that exchange of contents between late endosomes and lysosomes depends upon ongoing tubulovesicular late endocytic trafficking. In turn, rapid endosomal tubular movement requires an intact NPC1 sterol-sensing domain and is retarded by an elevated endosomal cholesterol content. We conclude that the neuropathology and cellular lysosomal lipid accumulation in NPC1 disease results, at least in part, from striking defects in late endosomal tubulovesicular trafficking.

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. Two highly conserved enzymes (O-GlcNAc transferase and O-GlcNAcase) catalyze the final steps in this nutrient-driven “hexosamine-signaling pathway.” A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes. Here, we show that Caenorhabditis elegans oga-1 encodes an active O-GlcNAcase. We also describe a knockout allele, oga-1(ok1207), that is viable and fertile yet accumulates O-GlcNAc on nuclear pores and other cellular proteins. Interfering with O-GlcNAc cycling with either oga-1(ok1207) or the O-GlcNAc transferase-null ogt-1(ok430) altered Ser- and Thr-phosphoprotein profiles and increased glycogen synthase kinase 3β (GSK-3β) levels. Both the oga-1(ok1207) and ogt-1(ok430) strains showed elevated stores of glycogen and trehalose, and decreased lipid storage. These striking metabolic changes prompted us to examine the insulin-like signaling pathway controlling nutrient storage, longevity, and dauer formation in the C. elegans O-GlcNAc cycling mutants. Indeed, we found that the oga-1(ok1207) knockout augmented dauer formation induced by a temperature sensitive insulin-like receptor (daf-2) mutant under conditions in which the ogt-1(ok430)-null diminished dauer formation. Our findings suggest that the enzymes of O-GlcNAc cycling “fine-tune” insulin-like signaling in response to nutrient flux. The knockout of O-GlcNAcase (oga-1) in C. elegans mimics many of the metabolic and signaling changes associated with human insulin resistance and provides a genetically amenable model of non-insulin-dependent diabetes.

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. Using anti-O-GlcNAc ChIP-on-chip whole-genome tiling arrays on wild-type and mutant strains, we detected over 800 promoters where O-GlcNAc cycling occurs, including microRNA loci and multigene operons. Intriguingly, O-GlcNAc-marked promoters are biased toward genes associated with PIP3 signaling, hexosamine biosynthesis, and lipid/carbohydrate metabolism. These marked genes are linked to insulin-like signaling, metabolism, aging, stress, and pathogen-response pathways in C. elegans. Whole-genome transcriptional profiling of the O-GlcNAc cycling mutants confirmed dramatic deregulation of genes in these key pathways. As predicted, the O-GlcNAc cycling mutants show altered lifespan and UV stress susceptibility phenotypes. We propose that O-GlcNAc cycling at promoters participates in a molecular program impacting nutrient-responsive pathways in C. elegans, including stress, pathogen response, and adult lifespan. The observed impact of O-GlcNAc cycling on both signaling and transcription in C. elegans has important implications for human diseases of aging, including diabetes and neurodegeneration.

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. This review describes the pair of enzymes responsible for the cycling of this post-translational modification: O-GlcNAc transferase (OGT) and beta-N-acetylglucosaminidase (OGA), with a focus on the function of their structural domains. We will also highlight the important processes and substrates regulated by these enzymes, with an emphasis on the role of O-GlcNAc as a nutrient sensor impacting insulin signaling and the cellular stress response. Finally, we will focus attention on the many ways by which O-GlcNAc cycling may affect the cellular machinery in the neuroendocrine and central nervous systems.

Mex67p of Schizosaccharomyces pombe Interacts with Rae1p in Mediating mRNA Export

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

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. Intriguingly, loss of function of O-GlcNAc transferase alleviated, whereas loss of O-GlcNAcase enhanced, the phenotype of multiple neurodegenerative disease models. The O-GlcNAc cycling mutants act in part by altering DAF-16–dependent transcription and modulating the protein degradation machinery. These findings suggest that O-GlcNAc levels may directly influence neurodegenerative disease progression, thus making the enzymes of O-GlcNAc cycling attractive targets for neurodegenerative disease therapies.