Calnexin Chaperone System Research Articles

Hydrophobicity of protein determinants influences the recognition of substrates by EDEM1 and EDEM2 in human cells.

BackgroundEDEM1 and EDEM2 are crucial regulators of the endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts misfolded glycoproteins from the calnexin chaperone system. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however the precise mechanism of substrate recognition and sorting to the ERAD pathway is still poorly understood. It has previously been demonstrated that EDEM1 and EDEM2 binding does not require the trimming of substrate glycans or even ERAD substrate glycosylation, thus suggesting that both chaperones probably recognize misfolded regions of aberrant proteins.ResultsIn this work, we focused on the substrate recognition by EDEM1 and EDEM2, asking whether hydrophobicity of protein determinants might be important for these interactions in human cells. In the study we used ricin, a protein toxin that utilizes the ERAD pathway in its retrotranslocation from the ER to the cytosol, and a model misfolded protein, the pancreatic isoform of human β-secretase, BACE457. Mutations in the hydrophobic regions of these proteins allowed us to obtain mutated forms with increased and decreased hydrophobicity.ConclusionsOur data provide the first evidence that recognition of ERAD substrates by EDEM1 and EDEM2 might be determined by a sufficiently high hydrophobicity of protein determinants. Moreover, EDEM proteins can bind hydrophobic transmembrane regions of misfolded ERAD substrates. These data contribute to the general understanding of the regulation of ERAD in mammalian cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0047-7) contains supplementary material, which is available to authorized users.

The role of EDEM2 compared with EDEM1 in ricin transport from the endoplasmic reticulum to the cytosol

EDEM1 [ER (endoplasmic reticulum)-degradation-enhancing α-mannosidase I-like protein 1] and EDEM2 are crucial regulators of ERAD (ER-associated degradation) that extracts non-native glycoproteins from the calnexin chaperone system. Ricin is a potent plant cytotoxin composed of an A-chain (RTA) connected by a disulfide bond to a cell-binding lectin B-chain (RTB). After endocytic uptake, the toxin is transported retrogradely to the ER, where the enzymatically active RTA is translocated to the cytosol in a similar manner as misfolded ER proteins. This transport is promoted by EDEM1. In the present study we report that EDEM2 is also involved in ricin retrotranslocation out of the ER. However, the role of EDEM1 and EDEM2in ricin transport to the cytosol seems to differ. EDEM2 promotes ricin retrotranslocation irrespectively of ER translocon accessibility; moreover, co-immunoprecipitation and pull-down studies revealed that more ricin can interact with EDEM2 in comparison with EDEM1. On the other hand, interactions of both lectins with RTA are dependent on the structure of the RTA. Thus our data display a newly discovered role for EDEM2. Moreover, analysis of the involvement of EDEM1 and EDEM2 in ricin retrotranslocation to the cytosol may provide crucial information about general mechanisms of the recognition of ERAD substrates in the ER.

Malectin Participates in a Backup Glycoprotein Quality Control Pathway in the Mammalian ER

Malectin is a conserved, endoplasmic reticulum (ER)-resident lectin that recognizes high mannose oligosaccharides displaying terminal glucose residues. Here we show that Malectin is an ER stress-induced protein that selectively associates with glycopolypeptides without affecting their entry and their retention in the Calnexin chaperone system. Analysis of the obligate Calnexin client influenza virus hemagglutinin (HA) revealed that Calnexin and Malectin associated with different timing to different HA conformers and that Malectin associated with misfolded HA. Analysis of the facultative Calnexin clients NHK and α1-antitrypsin (α1AT) revealed that induction of Malectin expression to simulate conditions of ER stress resulted in persistent association between the ER lectin and the model cargo glycoproteins, interfered with processing of cargo-linked oligosaccharides and reduced cargo secretion. We propose that Malectin intervention is activated upon ER stress to inhibit secretion of defective gene products that might be generated under conditions of aberrant functioning of the ER quality control machinery.

A dual role for EDEM1 in the processing of rod opsin

Mutations in rod opsin, the archetypal G-protein-coupled receptor, cause retinitis pigmentosa. The majority of mutations, e.g. P23H, cause protein misfolding, resulting in ER retention, induction of the unfolded protein response and degradation by ERAD. If misfolded rod opsin escapes degradation, it aggregates and forms intracellular inclusions. Therefore, it is important to identify the chaperones that mediate the folding or degradation of rod opsin. ER degradation enhancing alpha-mannosidase-like 1 (EDEM1) can enhance the release of terminally misfolded glycoproteins from the calnexin chaperone system. Here, we identify EDEM1 as a novel chaperone of rod opsin. EDEM1 expression promoted the degradation of P23H rod opsin and decreased its aggregation. By contrast, shRNA-mediated knockdown of EDEM1 increased both the amount of P23H rod opsin and its aggregation into inclusions. EDEM1 was detected in rod photoreceptor inner segments and EndoH-sensitive rod opsin co-immunoprecipitated with EDEM1 from retina, suggesting that rod opsin is a physiological EDEM1 client. Unexpectedly, EDEM1 binding to rod opsin was independent of mannose trimming and EDEM1 promoted the cell-surface expression of mutant rod opsin. Collectively, the data suggest that EDEM1 is a chaperone for rod opsin and that expression of EDEM1 can be used to promote correct folding, as well as enhanced degradation, of mutant proteins in the ER to combat protein-misfolding disease.

Consequences of Individual N-glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE

BACE is an aspartic protease involved in the production of a toxic peptide accumulating in the brain of Alzheimer's disease patients. After attainment of the native structure in the endoplasmic reticulum (ER), BACE is released into the secretory pathway. To better understand the mechanisms regulating protein biogenesis in the mammalian ER, we determined the fate of five variants of soluble BACE with 4, 3, 2, 1, or 0 N-linked glycans. The number of N-glycans displayed on BACE correlated directly with folding and secretion rates and with the yield of active BACE harvested from the cell culture media. Addition of a single N-glycan was sufficient to recruit the calnexin chaperone system and/or for oligosaccharide de-glucosylation by the ER-resident alpha-glucosidase II. Addition of 1-4 N-glycans progressively enhanced the dissociation rate from BiP and reduced the propensity of newly synthesized BACE to enter aberrant soluble and insoluble aggregates. Finally, inhibition of the proteasome increased the yield of active BACE. This shows that active protein normally targeted for destruction can be diverted for secretion, as if for BACE the quality control system would be acting too stringently in the ER lumen, thus causing loss of functional polypeptides.

Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities

EDEM1 is a crucial regulator of endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts non-native glycopolypeptides from the calnexin chaperone system. Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. We report that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-I-coated vesicles and is rapidly degraded. The rapid turnover of EDEM1 is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides. We define as ERAD tuning the mechanisms operating in the mammalian ER at steady state to offer kinetic advantage to folding over disposal of unstructured nascent chains by selective and rapid degradation of ERAD regulators.

Distinct classes of trafficking rBAT mutants cause the type I cystinuria phenotype

Most mutations in the rBAT subunit of the heterodimeric cystine transporter rBAT-b(0,+)AT cause type I cystinuria. Trafficking of the transporter requires the intracellular assembly of the two subunits. Without its partner, rBAT, but not b(0,+)AT, is rapidly degraded. We analyzed the initial biogenesis of wild-type rBAT and type I cystinuria rBAT mutants. rBAT was degraded, at least in part, via the ERAD pathway. Assembly with b(0,+)AT within the endoplasmic reticulum (ER) blocked rBAT degradation and could be independent of the calnexin chaperone system. This system was, however, necessary for post-assembly maturation of the heterodimer. Without b(0,+)AT, wild-type and rBAT mutants were degraded with similar kinetics. In its presence, rBAT mutants showed strongly reduced (L89P) or no transport activity, failed to acquire complex N-glycosylation and to oligomerize, suggesting assembly and/or folding defects. Most of the transmembrane domain mutant L89P did not heterodimerize with b(0,+)AT and was degraded. However, the few [L89P]rBAT-b(0,+)AT heterodimers were stable, consistent with assembly, but not folding, defects. Mutants of the rBAT extracellular domain (T216M, R365W, M467K and M467T) efficiently assembled with b(0,+)AT but were subsequently degraded. Together with earlier results, the data suggest a two-step biogenesis model, with the early assembly of the subunits followed by folding of the rBAT extracellular domain. Defects on either of these steps lead to the type I cystinuria phenotype.

Substrate-Specific Requirements for UGT1-Dependent Release from Calnexin

Newly synthesized glycoproteins displaying monoglucosylated N-glycans bind to the endoplasmic reticulum (ER) chaperone calnexin, and their maturation is catalyzed by the calnexin-associated oxidoreductase ERp57. Folding substrates are eventually released from calnexin, and terminal glucoses are removed from N-glycans. The UDP-glucose:glycoprotein glucosyltransferase (UGT1, UGGT, GT) monitors the folding state of polypeptides released from calnexin and adds back a glucose residue on N-glycans of nonnative polypeptides, thereby prolonging retention in the calnexin chaperone system for additional folding attempts. Here we show that for certain newly synthesized glycoproteins UGT1 deletion has no effect on binding to calnexin. These proteins must normally complete their folding program in one binding event. Other proteins normally undergo multiple binding events, and UGT1 deletion results in their premature release from calnexin. For other proteins, UGT1 deletion substantially delays release from calnexin, unexpectedly showing that UGT1 activity might be required for a structural maturation needed for substrate dissociation from calnexin and export from the ER.

Glycoprotein folding and the role of EDEM1, EDEM2 and EDEM3 in degradation of folding-defective glycoproteins

Proteins synthesized in the endoplasmic reticulum (ER) lumen are exposed to several dedicated chaperones and folding factors that ensure efficient maturation. Nevertheless, protein folding remains error-prone and mutations in the polypeptide sequence may significantly reduce folding-efficiency. Folding-incompetent proteins carrying N-glycans are extracted from futile folding cycles in the calnexin chaperone system upon intervention of EDEM1, EDEM2 and EDEM3, three ER-stress-induced members of the glycosyl hydrolase 47 family. This review describes current knowledge about mechanisms regulating folding and disposal of glycoproteins.

N-glycan processing in ER quality control

Glycosylation of asparagine residues in Asn-x-Ser/Thr motifs is a common covalent modification of proteins in the lumen of the endoplasmic reticulum (ER). By substantially contributing to the overall hydrophilicity of the polypeptide, pre-assembled core glycans inhibit possible aggregation caused by the inevitable exposure of hydrophobic patches on the as yet unstructured chains. Thereafter, N-glycans are modified by ER-resident enzymes glucosidase I (GI), glucosidase II (GII), UDP-glucose:glycoprotein glucosyltransferase (UGT) and mannosidase(s) and become functional appendices that determine the fate of the associated polypeptide. Recent work has improved our understanding of how the removal of terminal glucose residues from N-glycans allows newly synthesized proteins to access the calnexin chaperone system; how substrate retention in this specialized chaperone system is regulated by de-/re-glucosylation cycles catalyzed by GII and UGT1; and how acceleration of N-glycan dismantling upon induction of EDEM variants promotes ER-associated degradation (ERAD) under conditions of ER stress. In particular, characterization of cells lacking certain ER chaperones has revealed important new information on the mechanisms regulating protein folding and quality control. Tight regulation of N-glycan modifications is crucial to maintain protein quality control, to ensure the synthesis of functional polypeptides and to avoid constipation of the ER with folding-defective polypeptides.

Consequences of ERp57 Deletion on Oxidative Folding of Obligate and Facultative Clients of the Calnexin Cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Persistent Glycoprotein Misfolding Activates the Glucosidase II/UGT1-Driven Calnexin Cycle to Delay Aggregation and Loss of Folding Competence

The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. Here, we compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding.