Molecular Biology and Biochemistry, Department of

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Alpine Snow Algae Microbiome Diversity in the Coast Range of British Columbia

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-07-28
Abstract: 

Snow algae blooms contain bacteria, fungi, and other microscopic organisms. We surveyed 55 alpine snow algae blooms, collecting a total of 68 samples, from 12 mountains in the Coast Range of British Columbia, Canada. We used microscopy and rDNA metabarcoding to document biodiversity and query species and taxonomic associations. Across all samples, we found 173 algal, 2,739 bacterial, 380 fungal, and 540 protist/animalia operational taxonomic units (OTUs). In a previous study, we reported that most algal species were distributed along an elevational gradient. In the current study, we were surprised to find no corresponding distribution in any other taxa. We also tested the hypothesis that certain bacterial and fungal taxa co-occur with specific algal taxa. However, despite previous evidence that particular genera co-occur, we found no significant correlations between taxa across our 68 samples. Notably, seven bacterial, one fungal, and two cercozoan OTUs were widely distributed across our study regions. Taken together, these data suggest that any mutualisms with algae may not be taxon specific. We also report evidence of snow algae predation by rotifers, tardigrades, springtails, chytrid fungi, and ciliates, establishing the framework for a complex food web.

Document type: 
Article
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Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex

Peer reviewed: 
Yes, item is peer reviewed.
Date created: 
2020-07-22
Abstract: 

Membrane contact sites between the cortical endoplasmic reticulum (ER) and the plasma membrane (PM) provide a direct conduit for small molecule transfer and signaling between the two largest membranes of the cell. Contact is established through ER integral membrane proteins that physically tether the two membranes together, though the general mechanism is remarkably non-specific given the diversity of different tethering proteins. Primary tethers including VAMP-associated proteins (VAPs), Anoctamin/TMEM16/Ist2p homologs, and extended synaptotagmins (E-Syts), are largely conserved in most eukaryotes and are both necessary and sufficient for establishing ER-PM association. In addition, other species-specific ER-PM tether proteins impart unique functional attributes to both membranes at the cell cortex. This review distils recent functional and structural findings about conserved and species-specific tethers that form ER-PM contact sites, with an emphasis on their roles in the coordinate regulation of lipid metabolism, cellular structure, and responses to membrane stress.

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Article
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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.

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Article
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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
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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
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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
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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.

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Article
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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.

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Article
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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