Biomedical subjects

Dona C Love

Publications and source records attributed to Dona C Love.

10 recordsLinked to original sources

PubMed2006

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 3beta (GSK-3beta) 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.

Acetylglucosamine↗

PubMed2006

Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate.

A highly sensitive fluorogenic hexosaminidase substrate, fluorescein di(N-acetyl-beta-D-glucosaminide) (FDGlcNAc), was prepared essentially as described previously [Chem. Pharm. Bull. 1993, 41, 314] with some modifications. The fluorescent analog is a substrate for a number of hexosaminidases but here we have focused on the cytoplasmic O-GlcNAcase isoforms. Kinetic analysis using purified O-GlcNAcase and its splice variant (v-O-GlcNAcase) expressed in Escherichia coli suggests that FDGlcNAc is a much more efficient substrate (Km = 84.9 microM) than the conventional substrate, para-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (pNP-beta-GlcNAc, Km = 1.1 mM) and a previously developed fluorogenic substrate, 4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside [MUGlcNAc, Km = 0.43 mM; J. Biol. Chem. 2005, 280, 25313] for O-GlcNAcase. The variant O-GlcNAcase, a protein lacking the C-terminal third of the full-length O-GlcNAcase, exhibited a Km of 2.1 mM with respect to FDGlcNAc. This shorter isoform was not previously thought to exhibit O-GlcNAcase activity based on in vitro studies with pNP-beta-GlcNAc. However, both O-GlcNAcase isoforms reduced O-GlcNAc protein levels extracted from HeLa and HT-29 cells in vitro, indicating that the splice variant is a bona fide O-GlcNAcase. Fluorescein di-N-acetyl-beta-D-galactosaminide (FDGalNAc) is not cleaved by these enzymes, consistent with previous findings that the O-GlcNAcase has substrate specificity toward O-GlcNAc but not O-GalNAc. The enzymatic activity of the shorter isoform of O-GlcNAcase was first detected by using highly sensitive fluorogenic FDGlcNAc substrate. The finding that O-GlcNAcase exists as two distinct isoforms has a number of important implications for the role of O-GlcNAcase in hexosamine signaling.

Acetylglucosaminidase↗

PubMed2006

Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates.

O-linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine or threonine residues of a variety of substrate proteins, including nuclear pore proteins, transcription factors, and proteins implicated in diabetes and neurodegenerative disorders. We have identified two nucleocytoplasmic isoforms of OGT (ncOGT and sOGT) and one isoform that localizes to the mitochondria (mOGT). These three isoforms contain identical catalytic regions but differ in the number of tetratricopeptide repeat motifs found at the N-terminus of each enzyme. We expressed each of these OGT isoforms in a soluble form in Escherichia coli and have used them to identify novel targets including the Src-family tyrosine kinase yes and O-GlcNAc-ase. We demonstrate that some substrate proteins, such as Nup62 and casein kinase II, are glycosylated by both ncOGT and mOGT, while others such as O-GlcNAcase and tau are specifically modified by ncOGT. The yes kinase was specifically modified by mOGT. The short isoform of OGT (sOGT) did not glycosylate any of the substrates tested, although it retains a potentially active catalytic domain. Our findings demonstrate the potential utility of recombinant OGT in identifying new targets and illustrate the necessity to examine all active isoforms of the enzyme. The identification of a tyrosine kinase and O-GlcNAcase as OGT targets suggests the potential for OGT participation in numerous signal transduction cascades.

Acetylglucosaminidase↗

PubMed2005

The hexosamine signaling pathway: deciphering the "O-GlcNAc code".

A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.

Acetylglucosamine↗

PubMed2005

Mouse mammary tumor virus Env-derived peptide associates with nucleolar targets in lymphoma, mammary carcinoma, and human breast cancer.

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

Amino Acid Sequence↗

PubMed2005

A Caenorhabditis elegans model of insulin resistance: altered macronutrient storage and dauer formation in an OGT-1 knockout.

O-linked N-acetylglucosamine (O-GlcNAc) is an evolutionarily conserved modification of nuclear pore proteins, signaling kinases, and transcription factors. The O-GlcNAc transferase (OGT) catalyzing O-GlcNAc addition is essential in mammals and mediates the last step in a nutrient-sensing "hexosamine-signaling pathway." This pathway may be deregulated in diabetes and neurodegenerative disease. To examine the function of O-GlcNAc in a genetically amenable organism, we describe a putative null allele of OGT in Caenorhabditis elegans that is viable and fertile. We demonstrate that, whereas nuclear pore proteins of the homozygous deletion strain are devoid of O-GlcNAc, nuclear transport of transcription factors appears normal. However, the OGT mutant exhibits striking metabolic changes manifested in a approximately 3-fold elevation in trehalose levels and glycogen stores with a concomitant approximately 3-fold decrease in triglycerides levels. In nematodes, a highly conserved insulin-like signaling cascade regulates macronutrient storage, longevity, and dauer formation. The OGT knockout suppresses dauer larvae formation induced by a temperature-sensitive allele of the insulin-like receptor gene daf-2. Our findings demonstrate that OGT modulates macronutrient storage and dauer formation in C. elegans, providing a unique genetic model for examining the role of O-GlcNAc in cellular signaling and insulin resistance.

Animals↗

PubMed2003

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.

Alternative Splicing↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia.

Insulin resistance and beta 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.

Adipose Tissue↗

PubMed2002

Crp79p, like Mex67p, is an auxiliary mRNA export factor in Schizosaccharomyces pombe.

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

Active Transport, Cell Nucleus↗