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Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

Tsunoda S, Avezov E, Zyryanova A, Konno T, Mendes-Silva L, Pinho Melo E, Harding HP, Ron D - Elife (2014)

Bottom Line: Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins.ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin.Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Wellcome Trust MRC Institute of Metabolic Science, Cambridge, United Kingdom NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Clearance of misfolded  Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing or non-reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 5) or after an additional chase period (indicated). The mobility of the labeled NHK-A1AT, and the ER-chaperone BiP that co-purifies with it, are indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitate. (B) Autoradiograph of samples recovered from 293T cells in an experimental design as in ‘A’. (C) Autoradiograph of samples recovered from HeLa cells in an experimental design as in ‘A’. Where indicated, cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D–F) Plot of time-dependent change in NHK-A1AT (monomer) signal from the reducing gels A–C above. Shown are representative experiments reproduced twice with similar outcome.DOI:http://dx.doi.org/10.7554/eLife.03421.009
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fig5: Clearance of misfolded Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing or non-reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 5) or after an additional chase period (indicated). The mobility of the labeled NHK-A1AT, and the ER-chaperone BiP that co-purifies with it, are indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitate. (B) Autoradiograph of samples recovered from 293T cells in an experimental design as in ‘A’. (C) Autoradiograph of samples recovered from HeLa cells in an experimental design as in ‘A’. Where indicated, cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D–F) Plot of time-dependent change in NHK-A1AT (monomer) signal from the reducing gels A–C above. Shown are representative experiments reproduced twice with similar outcome.DOI:http://dx.doi.org/10.7554/eLife.03421.009

Mentions: The degradation of misfolded NHK-A1AT requires the action of the reduced form of a specialized PDI, ERdj5 (Ushioda et al., 2008; Hagiwara et al., 2011). To determine if an ER pool of glutathione contributes to this process, we compared the half-life of C-terminally FLAG-tagged NHK-A1AT in cells expressing active ER-ChaC1CtoS and the catalytically inactive ER-ChaC1CtoS;E116Q. The stability of NHK-A1AT was unaffected by ChaC1 both in HeLa cells (Figure 5A,D) and in 293T cells (Figure 5B,E). Furthermore, depletion of total cellular pools of glutathione by coincidental exposure to BSO had no effect on NHK-A1AT half-life (Figure 5C,F), complementing the evidence for the dispensability of glutathione for ER-associated degradation of this redox-dependent substrate.10.7554/eLife.03421.009Figure 5.Clearance of misfolded Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.


Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants.

Tsunoda S, Avezov E, Zyryanova A, Konno T, Mendes-Silva L, Pinho Melo E, Harding HP, Ron D - Elife (2014)

Clearance of misfolded  Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing or non-reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 5) or after an additional chase period (indicated). The mobility of the labeled NHK-A1AT, and the ER-chaperone BiP that co-purifies with it, are indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitate. (B) Autoradiograph of samples recovered from 293T cells in an experimental design as in ‘A’. (C) Autoradiograph of samples recovered from HeLa cells in an experimental design as in ‘A’. Where indicated, cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D–F) Plot of time-dependent change in NHK-A1AT (monomer) signal from the reducing gels A–C above. Shown are representative experiments reproduced twice with similar outcome.DOI:http://dx.doi.org/10.7554/eLife.03421.009
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fig5: Clearance of misfolded Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.(A) Autoradiograph of metabolically labeled NHK-A1AT immunopurified from HeLa cells co-expressing C-terminally FLAG-tagged NHK-A1AT and ER-localized enzymatically active (ER-ChaC1CtoS) or inactive ChaC1 (ER-ChaC1CtoS;E116Q) and resolved on a reducing or non-reducing SDS-PAGE. Cells were lysed at the end of a 30-min labeling pulse (lanes 1 and 5) or after an additional chase period (indicated). The mobility of the labeled NHK-A1AT, and the ER-chaperone BiP that co-purifies with it, are indicated, as is the labeled ChaC1, which is also recovered in this anti-FLAG immunoprecipitate. (B) Autoradiograph of samples recovered from 293T cells in an experimental design as in ‘A’. (C) Autoradiograph of samples recovered from HeLa cells in an experimental design as in ‘A’. Where indicated, cells were exposed to the glutathione synthesis inhibitor buthionine-sulfoxide (BSO, 100 µM, 20 hr) before the pulse-chase labeling. (D–F) Plot of time-dependent change in NHK-A1AT (monomer) signal from the reducing gels A–C above. Shown are representative experiments reproduced twice with similar outcome.DOI:http://dx.doi.org/10.7554/eLife.03421.009
Mentions: The degradation of misfolded NHK-A1AT requires the action of the reduced form of a specialized PDI, ERdj5 (Ushioda et al., 2008; Hagiwara et al., 2011). To determine if an ER pool of glutathione contributes to this process, we compared the half-life of C-terminally FLAG-tagged NHK-A1AT in cells expressing active ER-ChaC1CtoS and the catalytically inactive ER-ChaC1CtoS;E116Q. The stability of NHK-A1AT was unaffected by ChaC1 both in HeLa cells (Figure 5A,D) and in 293T cells (Figure 5B,E). Furthermore, depletion of total cellular pools of glutathione by coincidental exposure to BSO had no effect on NHK-A1AT half-life (Figure 5C,F), complementing the evidence for the dispensability of glutathione for ER-associated degradation of this redox-dependent substrate.10.7554/eLife.03421.009Figure 5.Clearance of misfolded Hong Kong mutant alpha 1 anti-trypsin (NHK-A1AT) is unaffected by depletion of ER glutathione.

Bottom Line: Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins.ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin.Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom Wellcome Trust MRC Institute of Metabolic Science, Cambridge, United Kingdom NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.

No MeSH data available.


Related in: MedlinePlus