α1-Antitrypsin as model to assess glycan function in endoplasmic reticulum

Termine, Daniel J.; Wang, Ying; Liu, Yan; Sifers, Richard N.

doi:10.1016/j.ymeth.2004.10.006

Cited by 7 publications

(5 citation statements)

References 35 publications

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“…Importantly, both cycloheximide and kifunensine were able to independently ablate the discrete electrophoretic mobility shift of radiolabeled null(Hong Kong) in SDS-PAGE (Fig. 5B, compare CHX, Kif, and Co), implying that both treatments had blocked the removal of mannose from asparagine-linked oligosaccharides (15)(16)(17)(18). Taken together, the results strengthened the conclusion that endogenous mouse ER mannosidase I is a short-lived intracellular protein.…”

Section: Rapid Intracellular Turnover Of Transfected Recombinantsupporting

confidence: 64%

Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis

Wang

Termine

Swulius

et al. 2007

Journal of Biological Chemistry

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In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.Genetic information is directly transformed into biological activity in response to the correct conformational maturation and deployment of encoded proteins (1, 2). Arguably, this dual intention is best exemplified in the endoplasmic reticulum (ER) 2 into which nascent secretory and membrane proteins are translocated. The adoption of native protein structure, facilitated by transient physical engagement with one or more molecular chaperones, precedes productive export from the ER (3). Rather than clogging the secretory pathway, molecules incapable of structural maturation are eliminated by a process coined "ER-associated degradation" (ERAD). The associated multiple requisite steps culminate in the dislocation of misfolded proteins into the cytosol for degradation by 26 S proteasomes (4, 5). Polyubiquitination functions as the signal that mediates both dislocation and proteolysis ( Fig. 1) (6, 7).As with glycoprotein folding (8), the earliest events of ERAD are orchestrated by the covalent modification of asparaginelinked oligosaccharides (Fi...

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Section: Rapid Intracellular Turnover Of Transfected Recombinantsupporting

confidence: 64%

Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis

Wang

Termine

Swulius

et al. 2007

Journal of Biological Chemistry

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“…To determine whether the Derlin-1–p97/VCP interaction is essential for dislocation of a soluble ERAD substrate, we assessed the effect of S-tagged wild-type Derlin-1 (Derlin-1 WT ) or mutant Derlin-1 1–240 lacking the SHP box on the degradation of coexpressed NHK, a constitutively degraded truncated variant of α-1 antitrypsin 4,42 . Coexpression of HA-tagged NHK with Derlin-1 1–240 or Derlin-1 1–193 lacking the entire cytoplasmic domain led to the appearance of a nonglycosylated form of NHK that comigrated with NHK from lysates treated with Endo H ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum

Greenblatt

Olzmann

Kopito

2011

Nat Struct Mol Biol

168

216

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The degradation of misfolded secretory proteins is ultimately mediated by the ubiquitin-proteasome system in the cytoplasm, therefore endoplasmic reticulum–associated degradation (ERAD) substrates must be dislocated across the ER membrane through a process driven by the AAA ATPase p97/VCP. Derlins recruit p97/VCP and have been proposed to be part of the dislocation machinery. Here we report that Derlins are inactive members of the rhomboid family of intramembrane proteases and bind p97/VCP through C-terminal SHP boxes. Human Derlin-1 harboring mutations within the rhomboid domain stabilized mutant α-1 antitrypsin (NHK) at the cytosolic face of the ER membrane without disrupting the p97/VCP interaction. We propose that substrate interaction and p97/VCP recruitment are separate functions that are essential for dislocation and can be assigned respectively to the rhomboid domain and the C terminus of Derlin-1. These data suggest that intramembrane proteolysis and protein dislocation share unexpected mechanistic features.

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“…We have previously shown that the IDR of EDEM1 located at its N-terminus is required for accelerating the degradation of tyrosinase and its mutants [19]; therefore, we hypothesised that IDR-deficient EDEM1 would also impair the degradation of other reported ERAD clients. To test this hypothesis, we used previously characterized glycosylated ERAD substrates: α1-antitrypsin (α-1AT), Null Hong Kong (NHK), Ribophorin (Ri)-332, and beta-secretase (BACE)-476 [38][39][40]. We first confirmed the EDEM1 dependency for the proteasomal degradation of these substrates and found that the inhibition of proteasome activity almost entirely rescued the EDEM1-induced degradation of the ERAD clients (Figure 2A-C and Figure A1A-D).…”

Section: The Id Region Of Edem1 Is Required For Proteasomal Degradation Of Erad Clientsmentioning

confidence: 99%

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

Chirițoiu

Munteanu

et al. 2020

IJMS

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Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

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α1-Antitrypsin as model to assess glycan function in endoplasmic reticulum

Cited by 7 publications

References 35 publications

Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis

Human Endoplasmic Reticulum Mannosidase I Is Subject to Regulated Proteolysis

Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

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