Molecular Biology and Biochemistry, Department of

Receive updates for this collection

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2014-06-20
Abstract: 

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNActransferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomicelectronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues andenforce a front-face SNi-type reaction in which the substrate Nacetyl sugar substituent coordinates efficient glycosyl transfer.

Document type: 
Article
File(s): 

Multivalency To Inhibit and Discriminate Hexosaminidases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-05-26
Abstract: 

A set of multivalent polyhydroxylated acetamidoazepanes based on ethylene glycol, glucoside, or cyclodextrin scaffolds was prepared. The compounds were assessed against plant, mammalian, and therapeutically relevant hexosaminidases. Multimerization was shown to improve the inhibitory potency with synergy, and to fine tune the selectivity profile between related hexosaminidases.

Document type: 
Article
File(s): 

A Divergent Synthesis to Generate Targeted Libraries of Inhibitors for Endo-N-Acetylglucosaminidases

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-12-18
Abstract: 

Cell active inhibitors of glycoside processing enzymes are valuable research tools that help us understand the physiological roles of this diverse class of enzymes. endo-N-Acetylglucosaminidases have gained increased attention for their important roles in both mammals and human pathogens; however, metabolically stable cell active inhibitors of these enzymes are lacking. Here, we describe a divergent synthetic strategy involving elaboration of a thiazoline core scaffold. We illustrate the potential of this approach by using the copper catalysed azide-alkyne click (CuAAC) reaction, in combination with a suitable catalyst to avoid poisoning by the thiazoline moiety, to generate a targeted panel of candidate inhibitors of endo-N-acetylglucosaminidases and chitinases

Document type: 
Article
File(s): 

A Convenient Approach to Stereoisomeric Iminocyclitols: Generation of Potent Brain‐Permeable OGA Inhibitors

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2015-11-06
Abstract: 

Pyrrolidine‐based iminocyclitols are a promising class of glycosidase inhibitors. Reported herein is a convenient epimerization strategy that provides direct access to a range of stereoisomeric iminocyclitol inhibitors of O‐GlcNAcase (OGA), the enzyme responsible for catalyzing removal of O‐GlcNAc from nucleocytoplasmic proteins. Structural details regarding the binding of these inhibitors to a bacterial homologue of OGA reveal the basis for potency. These compounds are orally available and permeate into rodent brain to increase O‐GlcNAc, and should prove useful tools for studying the role of OGA in health and disease.

Document type: 
Article
File(s): 

A Selective Inhibitor Gal‐PUGNAc of Human Lysosomal β‐Hexosaminidases Modulates Levels of the Ganglioside GM2 in Neuroblastoma Cells

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2009-01-28
Abstract: 

Gal‐PUGNAc (see picture), a highly selective inhibitor for β‐hexosaminidases HEXA and HEXB is cell‐permeable and modulates the activity of HEXA and HEXB in tissue culture, increasing ganglioside GM2 levels. Gal‐PUGNAc should allow the role of these enzymes to be studied at the cellular level without generating a complex chemical phenotype from concomitant inhibition of O‐GlcNAcase.

Document type: 
Article
File(s): 

Pharmacological Inhibition of O-GlcNAcase Enhances Autophagy in Brain through an mTOR-Independent Pathway

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-02-20
Abstract: 

The glycosylation of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is conserved among metazoans and is particularly abundant within brain. O-GlcNAc is involved in diverse cellular processes ranging from the regulation of gene expression to stress response. Moreover, O-GlcNAc is implicated in various diseases including cancers, diabetes, cardiac dysfunction, and neurodegenerative diseases. Pharmacological inhibition of O-GlcNAcase (OGA), the sole enzyme that removes O-GlcNAc, reproducibly slows neurodegeneration in various Alzheimer’s disease (AD) mouse models manifesting either tau or amyloid pathology. These data have stimulated interest in the possibility of using OGA-selective inhibitors as pharmaceuticals to alter the progression of AD. The mechanisms mediating the neuroprotective effects of OGA inhibitors, however, remain poorly understood. Here we show, using a range of methods in neuroblastoma N2a cells, in primary rat neurons, and in mouse brain, that selective OGA inhibitors stimulate autophagy through an mTOR-independent pathway without obvious toxicity. Additionally, OGA inhibition significantly decreased the levels of toxic protein species associated with AD pathogenesis in the JNPL3 tauopathy mouse model as well as the 3×Tg-AD mouse model. These results strongly suggest that OGA inhibitors act within brain through a mechanism involving enhancement of autophagy, which aids the brain in combatting the accumulation of toxic protein species. Our study supports OGA inhibition being a feasible therapeutic strategy for hindering the progression of AD and other neurodegenerative diseases. Moreover, these data suggest more targeted strategies to stimulate autophagy in an mTOR-independent manner may be found within the O-GlcNAc pathway. These findings should aid the advancement of OGA inhibitors within the clinic.

Document type: 
Article
File(s): 

Selective Fluorogenic β-Glucocerebrosidase Substrates for Convenient Analysis of Enzyme Activity in Cell and Tissue Homogenates

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-02-28
Abstract: 

Within mammals, there are often several functionally related glycoside hydrolases, which makes monitoring their activities problematic. This problem is particularly acute for the enzyme β-glucocerebrosidase (GCase), the malfunction of which is a key driver of Gaucher’s disease (GD) and a major risk factor for Parkinson’s disease (PD). Humans harbor two other functionally related β-glucosidases known as GBA2 and GBA3, and the currently used fluorogenic substrates are not selective, which has driven the use of complicated subtractive assays involving the use of detergents and inhibitors. Here we describe the preparation of fluorogenic substrates based on the widely used nonselective substrate resorufin β-d-glucopyranoside. Using recombinant enzymes, we show that these substrates are highly selective for GCase. We also demonstrate their value through the analysis of GCase activity in brain tissue homogenates from transgenic mice expressing mutant human GCase and patient fibroblasts expressing mutant GCase. This approach simplifies the analysis of cell and tissue homogenates and should facilitate the analysis of clinical and laboratory tissues and samples.

Document type: 
Article

Precision Mapping of O-Linked N-Acetylglucosamine Sites in Proteins Using Ultraviolet Photodissociation Mass Spectrometry

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-06-08
Abstract: 

Despite its central importance as a regulator of cellular physiology, identification and precise mapping of O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (PTM) sites in proteins by mass spectrometry (MS) remains a considerable technical challenge. This is due in part to cleavage of the glycosidic bond occurring prior to the peptide backbone during collisionally activated dissociation (CAD), which leads to generation of characteristic oxocarbenium ions and impairs glycosite localization. Herein, we leverage CAD-induced oxocarbenium ion generation to trigger ultraviolet photodissociation (UVPD), an alternate high-energy deposition method that offers extensive fragmentation of peptides while leaving the glycosite intact. Upon activation using UV laser pulses, efficient photodissociation of glycopeptides is achieved with production of multiple sequence ions that enable robust and precise localization of O-GlcNAc sites. Application of this method to tryptic peptides originating from O-GlcNAcylated proteins TAB1 and Polyhomeotic confirmed previously reported O-GlcNAc sites in TAB1 (S395 and S396) and uncovered new sites within both proteins. We expect this strategy will complement existing MS/MS methods and be broadly useful for mapping O-GlcNAcylated residues of both proteins and proteomes.

Document type: 
Article

Quinolinic Acid Amyloid-like Fibrillar Assemblies Seed α-Synuclein Aggregation

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2018-08-08
Abstract: 

Quinolinic acid (QA), a downstream neurometabolite in the kynurenine pathway, the biosynthetic pathway of tryptophan, is associated with neurodegenerative diseases pathology. Mutations in genes encoding kynurenine pathway enzymes, which control the level of QA production, are linked with elevated risk of developing Parkinson's disease. Recent findings have revealed the accumulation and deposition of QA in post-mortem samples, as well as in cellular models of Alzheimer's disease and related disorders. Furthermore, intrastriatal inoculation of mice with QA results in increased levels of phosphorylated α-synuclein and neurodegenerative pathological and behavioral characteristics. However, the cellular and molecular mechanisms underlying the involvement of QA accumulation in protein aggregation and neurodegeneration remain elusive. We recently established that self-assembled ordered structures are formed by various metabolites and hypothesized that these “metabolite amyloids” may seed amyloidogenic proteins. Here we demonstrate the formation of QA amyloid-like fibrillar assemblies and seeding of α-synuclein aggregation by these nanostructures both in vitro and in cell culture. Notably, α-synuclein aggregation kinetics was accelerated by an order of magnitude. Additional amyloid-like properties of QA assemblies were demonstrated using thioflavin T assay, powder X-ray diffraction and cell apoptosis analysis. Moreover, fluorescently labeled QA assemblies were internalized by neuronal cells and co-localized with α-synuclein aggregates. In addition, we observed cell-to-cell propagation of fluorescently labeled QA assemblies in a co-culture of treated and untreated cells. Our findings suggest that excess QA levels, due to mutations in the kynurenine pathway, for example, may lead to the formation of metabolite assemblies that seed α-synuclein aggregation, resulting in neuronal toxicity and induction of Parkinson's disease.

Document type: 
Article
File(s): 

Selective Trihydroxylated Azepane Inhibitors of NagZ, a Glycosidase Involved in Pseudomonas Aeruginosa Resistance to β-lactam Antibiotics

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2017-05-09
Abstract: 

The synthesis of a series of D-gluco-like configured 4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated acetamide moiety at C-3 is described. These synthetic derivatives have been tested for their ability to selectively inhibit the muropeptide recycling glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas aeruginosa to β-lactams, a pathway with substantial therapeutic potential. While introduction of triazole and sulfamide groups failed to lead to glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective inhibitor of NagZ over other glucosaminidases including human OGA and lysosomal hexosaminidases HexA and B.

The synthesis of a series of D-gluco-like configured

4,5,6-trihydroxyazepanes bearing a triazole, a sulfonamide or a fluorinated

acetamide moiety at C-3 is described. These synthetic derivatives have been

tested for their ability to selectively inhibit the muropeptide recycling

glucosaminidase NagZ and to thereby increase sensitivity of Pseudomonas

aeruginosa to β-lactams, a pathway with substantial therapeutic potential.

While introduction of triazole and sulfamide groups failed to lead to

glucosaminidase inhibitors, the NHCOCF3 analog proved to be a selective

inhibitor of NagZ over other glucosaminidases including human OGA and

lysosomal hexosaminidases HexA and B

 

Document type: 
Article
File(s):