Elevation of Global O-GlcNAc in rodents using a selective O-GlcNAcase inhibitor does not cause insulin resistance or perturb glucohomeostasis

The O-GlcNAc modification is proposed to be a nutrient sensor with studies suggesting that global increases in O-GlcNAc levels cause insulin resistance and impaired glucohomeostasis. We address this hypothesis by using a potent and selective inhibitor of O-GlcNAcase, known as NButGT, in a series of in vivo studies. Treatment of rats and mice with NButGT, [...]

September 24th, 2010|Tags: , |

Inhibition of O-GlcNAcase using a potent and cell-permeable inhibitor does not induce insulin resistance in 3T3-L1 adipocytes

To probe increased O-GlcNAc levels as an independent mechanism governing insulin resistance in 3T3-L1 adipocytes, a new class of O-GlcNAcase (OGA) inhibitor was studied. 6-Acetamido-6-deoxy-castanospermine (6-Ac-Cas) is a potent inhibitor of OGA. The structure of 6-Ac-Cas bound in the active site of an OGA homolog reveals structural features contributing to its potency. Treatment of 3T3-L1 [...]

September 24th, 2010|Tags: , |

Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-D: -glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. [...]

August 13th, 2010|Tags: , , , |

Visualizing the reaction coordinate of an O-GlcNAc hydrolase

N-Acetylglucosamine beta-O-linked to serine and threonine residues of nucleocytoplasmic proteins (O-GlcNAc) has been linked to neurodegeneration, cellular stress response, and transcriptional regulation. Removal of O-GlcNAc is catalyzed by O-GlcNAcase (OGA) using a substrate-assisted catalytic mechanism. Here we define the reaction coordinate using chemical approaches and directly observe both a Michaelis complex and the oxazoline intermediate. [...]

February 17th, 2010|Tags: , |

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance

The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated [...]

October 8th, 2008|Tags: , |

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer’s disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. [...]

June 29th, 2008|Tags: , , , , |

Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation

N-Acetylglucosamine (O-GlcNAc) modification of proteins provides a mechanism for the control of diverse cellular processes through a dynamic interplay with phosphorylation. UDP-GlcNAc:polypeptidyl transferase (OGT) catalyzes O-GlcNAc addition. The structure of an intact OGT homolog and kinetic analysis of human OGT variants reveal a contiguous superhelical groove that directs substrates to the active site. Source: Martinez-Fleites, [...]

June 8th, 2008|Tags: , |

Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise

O-GlcNAcase catalyzes the cleavage of beta-O-linked 2-acetamido-2-deoxy-beta-d-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Two potent inhibitors of this enzyme are O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) and 1,2-dideoxy-2′-methyl-alpha-d-glucopyranoso[2,1-d]-Delta2′-thiazoline (NAG-thiazoline). Derivatives of these inhibitors differ in their selectivity for human O-GlcNAcase over the functionally related human lysosomal beta-hexosamindases, with PUGNAc derivatives showing modest selectivities and NAG-thiazoline [...]

January 24th, 2007|Tags: , |

Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity

O-GlcNAc is an abundant post-translational modification of serine and threonine residues of nucleocytoplasmic proteins. This modification, found only within higher eukaryotes, is a dynamic modification that is often reciprocal to phosphorylation. In a manner analogous to phosphatases, a glycoside hydrolase termed O-GlcNAcase cleaves O-GlcNAc from modified proteins. Enzymes with high sequence similarity to human O-GlcNAcase [...]

March 26th, 2006|Tags: , |

Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants

O-GlcNAcase is a family 84 beta-N-acetylglucosaminidase catalyzing the hydrolytic cleavage of beta-O-linked 2-acetamido-2-deoxy-d-glycopyranose (O-GlcNAc) from serine and threonine residues of posttranslationally modified proteins. O-GlcNAcases use a double-displacement mechanism involving formation and breakdown of a transient bicyclic oxazoline intermediate. The key catalytic residues of any family 84 enzyme facilitating this reaction, however, are unknown. Two mutants [...]

March 21st, 2006|Tags: , |