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1.
Glycobiology ; 29(7): 530-542, 2019 07 01.
Article in English | MEDLINE | ID: mdl-30976784

ABSTRACT

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.


Subject(s)
Arabinose/pharmacology , Endoplasmic Reticulum/enzymology , Glycoside Hydrolase Inhibitors/pharmacology , Imino Furanoses/pharmacology , Pyrrolizidine Alkaloids/pharmacology , Sugar Alcohols/pharmacology , alpha-Glucosidases/metabolism , Animals , Arabinose/chemistry , Dogs , Glycoside Hydrolase Inhibitors/chemistry , Humans , Imino Furanoses/chemistry , Mice , Microsomes/drug effects , Microsomes/metabolism , Pyrrolizidine Alkaloids/chemistry , Sugar Alcohols/chemistry
2.
PLoS One ; 8(3): e59590, 2013.
Article in English | MEDLINE | ID: mdl-23533635

ABSTRACT

BACKGROUND: The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate. METHODOLOGY AND PRINCIPAL FINDINGS: BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61ß, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6. SIGNIFICANCE: On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates.


Subject(s)
Carrier Proteins/metabolism , Membrane Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Membrane Proteins/genetics , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
3.
J Biol Chem ; 279(18): 18861-9, 2004 Apr 30.
Article in English | MEDLINE | ID: mdl-14871899

ABSTRACT

ERp57 is a member of the protein disulfide isomerase (PDI) family that is located in the endoplasmic reticulum (ER) and characterized by its specificity for glycoproteins. Substrate selection by ERp57 is dependent upon its formation of discrete complexes with two ER resident lectins, soluble calreticulin and membrane-bound calnexin. It is these two lectins that directly associate with glycoproteins bearing correctly trimmed oligosaccharide side chains. Thus, ERp57 is presented with a preselected set of substrates upon which it can act, and the specific binding of calreticulin and calnexin to ERp57 is pivotal to the functions of the resulting complexes. To gain further insights into the formation of these ERp57-ER lectin complexes, we have investigated the regions of ERp57 that are specifically required for its binding to calreticulin. Using a quantitative pull-down assay to investigate the binding of ERp57/PDI chimeras to calreticulin, we define the b and b' domains of ERp57 as the minimal elements that are sufficient for complex formation. This analysis further identifies a novel role for the distinctive C-terminal extension of ERp57 in reconstituting complex formation to wild type levels. Using our understanding of substrate binding to the b' domain of PDI as a paradigm, we show that alterations to specific residues in the b' domain of ERp57 dramatically reduce or completely abolish its binding to calreticulin. On the basis of these data, we propose a model where the region of ERp57 equivalent to the primary substrate binding site of archetypal PDI is occupied by calreticulin and suggest that the ER lectins act as adaptor molecules that define the substrate specificity of ERp57.


Subject(s)
Endoplasmic Reticulum/metabolism , Heat-Shock Proteins/chemistry , Isomerases/chemistry , Lectins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Calreticulin/metabolism , Endoplasmic Reticulum/chemistry , Heat-Shock Proteins/genetics , Humans , Isomerases/genetics , Protein Binding , Protein Disulfide-Isomerases , Protein Structure, Tertiary , Recombinant Fusion Proteins , Sequence Alignment , Substrate Specificity
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