Molecular Biology and Biochemistry, Department of

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O -GlcNAc and Neurodegeneration: Biochemical Mechanisms and Potential Roles in Alzheimer's Disease and Beyond

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-04-24
Abstract: 

Alzheimer disease (AD) is a growing problem for aging populations worldwide. Despite significant efforts, no therapeutics are available that stop or slow progression of AD, which has driven interest in the basic causes of AD and the search for new therapeutic strategies. Longitudinal studies have clarified that defects in glucose metabolism occur in patients exhibiting Mild Cognitive Impairment (MCI) and glucose hypometabolism is an early pathological change within AD brain. Further, type 2 diabetes mellitus (T2DM) is a strong risk factor for the development of AD. These findings have stimulated interest in the possibility that disrupted glucose regulated signaling within the brain could contribute to the progression of AD. One such process of interest is the addition of O-linked N-acetylglucosamine (O-GlcNAc) residues onto nuclear and cytoplasmic proteins within mammals. O-GlcNAc is notably abundant within brain and is present on hundreds of proteins including several, such as tau and the amyloid precursor protein, which are involved in the pathophysiology AD. The cellular levels of O-GlcNAc are coupled to nutrient availability through the action of just two enzymes. O-GlcNAc transferase (OGT) is the glycosyltransferase that acts to install O-GlcNAc onto proteins and O-GlcNAcase (OGA) is the glycoside hydrolase that acts to remove O-GlcNAc from proteins. Uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) is the donor sugar substrate for OGT and its levels vary with cellular glucose availability because it is generated from glucose through the hexosamine biosynthetic pathway (HBSP). Within the brains of AD patients O-GlcNAc levels have been found to be decreased and aggregates of tau appear to lack O-GlcNAc entirely. Accordingly, glucose hypometabolism within the brain may result in disruption of the normal functions of O-GlcNAc within the brain and thereby contribute to downstream neurodegeneration. While this hypothesis remains largely speculative, recent studies using different mouse models of AD have demonstrated the protective benefit of pharmacologically increased brain O-GlcNAc levels. In this review we summarize the state of knowledge in the area of O-GlcNAc as it pertains to AD while also addressing some of the basic biochemical roles of O-GlcNAc and how these might contribute to protecting against AD and other neurodegenerative diseases.

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O-GlcNAc Modification of tau Directly Inhibits Its Aggregation without Perturbing the Conformational Properties of tau Monomers

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-04-17
Abstract: 

The aggregation of the microtubule-associated protein tau into paired helical filaments to form neurofibrillary tangles constitutes one of the pathological hallmarks of Alzheimer's disease. Tau is post-translationally modified by the addition of N-acetyl-d-glucosamine O-linked to several serine and threonine residues (O-GlcNAc). Previously, increased O-GlcNAcylation of tau has been shown to block the accumulation of tau aggregates within a tauopathy mouse model. Here we show that O-GlcNAc modification of full-length human tau impairs the rate and extent of its heparin-induced aggregation without perturbing its activity toward microtubule polymerization. O-GlcNAcylation, however, does not impact the “global-fold” of tau as measured by a Förster resonance energy transfer assay. Similarly, nuclear magnetic resonance studies demonstrated that O-GlcNAcylation only minimally perturbs the local structural and dynamic features of a tau fragment (residues 353–408) spanning the last microtubule binding repeat to the major GlcNAc-acceptor Ser400. These data indicate that the inhibitory effects of O-GlcNAc on tau aggregation may result from enhanced monomer solubility or the destabilization of fibrils or soluble aggregates, rather than by altering the conformational properties of the monomeric protein. This work further underscores the potential of targeting the O-GlcNAc pathway for potential Alzheimer's disease therapeutics.

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Selective Trihydroxyazepane NagZ Inhibitors Increase Sensitivity of Pseudomonas Aeruginosa to β-lactams

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2013-09-26
Abstract: 

AmpC β-lactamase confers resistance to β-lactam antibiotics in many Gram negative bacteria. Inducible expression of AmpC requires an N-acetylglucosaminidase termed NagZ. Here we describe the synthesis and characterization of hydroxyazepane inhibitors of NagZ. We find that these inhibitors enhance the susceptibility of clinically relevant Pseudomonas aeruginosa to β-lactams.

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Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2013-08-15
Abstract: 

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.

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Reduced Protein O-glycosylation in the Nervous System of the Mutant SOD1 Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2012-05-16
Abstract: 

Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with β-O-linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.

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Article
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Visualizing the Reaction Coordinate of an O-GlcNAc Hydrolase

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2010-01-12
Abstract: 

N-Acetylglucosamine β-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.

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Article
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Probing Synergy between Two Catalytic Strategies in the Glycoside Hydrolase O-GlcNAcase Using Multiple Linear Free Energy Relationships

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-08-28
Abstract: 

Human O-GlcNAcase plays an important role in regulating the post-translational modification of serine and threonine residues with β-O-linked N-acetylglucosamine monosaccharide unit (O-GlcNAc). The mechanism of O-GlcNAcase involves nucleophilic participation of the 2-acetamido group of the substrate to displace a glycosidically linked leaving group. The tolerance of this enzyme for variation in substrate structure has enabled us to characterize O-GlcNAcase transition states using several series of substrates to generate multiple simultaneous free-energy relationships. Patterns revealing changes in mechanism, transition state, and rate-determining step upon concomitant variation of both nucleophilic strength and leaving group abilities are observed. The observed changes in mechanism reflect the roles played by the enzymic general acid and the catalytic nucleophile. Significantly, these results illustrate how the enzyme synergistically harnesses both modes of catalysis; a feature that eludes many small molecule models of catalysis. These studies also suggest the kinetic significance of an oxocarbenium ion intermediate in the O-GlcNAcase-catalyzed hydrolysis of glucosaminides, probing the limits of what may be learned using nonatomistic investigations of enzymic transition-state structure and offering general insights into how the superfamily of retaining glycoside hydrolases act as efficient catalysts.

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Article
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Mislocalization of TDP-43 in the G93A Mutant SOD1 Transgenic Mouse Model of ALS

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-04-18
Abstract: 

Previous evidence demonstrates that TAR DNA binding protein (TDP-43) mislocalization is a key pathological feature of amyotrophic lateral sclerosis (ALS). TDP-43 normally shows nuclear localization, but in CNS tissue from patients who died with ALS this protein mislocalizes to the cytoplasm. Disease specific TDP-43 species have also been reported to include hyperphosphorylated TDP-43, as well as a C-terminal fragment. Whether these abnormal TDP-43 features are present in patients with SOD1-related familial ALS (fALS), or in mutant SOD1 over-expressing transgenic mouse models of ALS remains controversial. Here we investigate TDP-43 pathology in transgenic mice expressing the G93A mutant form of SOD1. In contrast to previous reports we observe redistribution of TDP-43 to the cytoplasm of motor neurons in mutant SOD1 transgenic mice, but this is seen only in mice having advanced disease. Furthermore, we also observe rounded TDP-43 immunoreactive inclusions associated with intense ubiquitin immunoreactivity in lumbar spinal cord at end stage disease in mSOD mice. These data indicate that TDP-43 mislocalization and ubiquitination are present in end stage mSOD mice. However, we do not observe C-terminal TDP-43 fragments nor TDP-43 hyperphosphorylated species in these end stage mSOD mice. Our findings indicate that G93A mutant SOD1 transgenic mice recapitulate some key pathological, but not all biochemical hallmarks, of TDP-43 pathology previously observed in human ALS. These studies suggest motor neuron degeneration in the mutant SOD1 transgenic mice is associated with TDP-43 histopathology.

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Article
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Enzymatic Characterization and Inhibition of the Nuclear Variant of Human O-GlcNAcase

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-06-12
Abstract: 

Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present.

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Molecular Basis for Inhibition of GH84 Glycoside Hydrolases by Substituted Azepanes: Conformational Flexibility Enables Probing of Substrate Distortion

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-03-30
Abstract: 

Here we report the synthesis of a series of polyhydroxylated 3- and 5-acetamido azepanes and detail the molecular basis of their inhibition of family 84 glycoside hydrolases. These family 84 enzymes include human O-GlcNAcase, an enzyme involved in post-translational processing of intracellular proteins modified by O-linked β-N-acetylglucosamine residues. Detailed structural analysis of the binding of these azepanes to BtGH84, a bacterial homologue of O-GlcNAcase, highlights their conformational flexibility. Molecular mechanics and molecular dynamics calculations reveal that binding to the enzyme involves significant conformational distortion of these inhibitors from their preferred solution conformations. The binding of these azepanes provides structural insight into substrate distortion that likely occurs along the reaction coordinate followed by O-GlcNAcase during glycoside hydrolysis. This class of inhibitors may prove to be useful probes for evaluating the conformational itineraries of glycosidases and aid the development of more potent and specific glycosidase inhibitors.

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