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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

Activity of the unfolded protein response (UPR) as measured by an XBP1-splicing reporter is also unaffected by depletion of ER glutathione.FACScans as in ‘6B’ of CHO cells stably transduced with an XBP1 splicing reporter linked to the expression of the fluorescent protein Venus (Iwawaki et al., 2004). Where indicated cells were co-transfected with expression plasmids for a hyperactive mutant of ERO1 (C104A, C133A; ERO1*) tagged by the cell-surface marker CD2 and wild-type or inactive mutants of ChaC1 fused to mCherry. The axis of the scans is labeled with the cognate signals.DOI:http://dx.doi.org/10.7554/eLife.03421.011
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fig6s1: Activity of the unfolded protein response (UPR) as measured by an XBP1-splicing reporter is also unaffected by depletion of ER glutathione.FACScans as in ‘6B’ of CHO cells stably transduced with an XBP1 splicing reporter linked to the expression of the fluorescent protein Venus (Iwawaki et al., 2004). Where indicated cells were co-transfected with expression plasmids for a hyperactive mutant of ERO1 (C104A, C133A; ERO1*) tagged by the cell-surface marker CD2 and wild-type or inactive mutants of ChaC1 fused to mCherry. The axis of the scans is labeled with the cognate signals.DOI:http://dx.doi.org/10.7554/eLife.03421.011

Mentions: The role of ER glutathione was further explored under conditions in which ER redox balance was perturbed by co-expression of a deregulated allele of the ER oxidase ERO1 (Sevier et al., 2007), which has been shown to hyper-oxidize the mammalian ER (Baker et al., 2008) and modestly activate the UPR (Hansen et al., 2012). Introduction of the C104A; C133A human ERO1L (ERO1*), expressed from a plasmid tagged with the CD2 surface marker indeed modestly activated the UPR, whether measured in cells stably expressing the CHOP::GFP transcriptional reporter (Figure 6B, reflected in the higher GFP levels of cells co-expressing the CD2 marker which tags the ERO1*-expressing cells, panels 2, 4, and 7) or an XBP1::Venus splicing reporter (Iwawaki et al., 2004) (Figure 6—figure supplement 1). However, co-expression of active ChaC1CtoS had no evident synergistic effect with ERO1* on UPR activity beyond that observed with the enzymatically inactive E116Q mutant enzyme. This is evident by first noting that in this co-transfection experiment most ERO1* expressing cells (CD2 positive) are also co-expressing the Cherry-tagged ChaC1CtoS (Figure 6B, panels 3 and 6) and then noting that the relationship between ERO1* expression (for which CD2 is a surrogate) and the CHOP::GFP signal is indistinguishable in cells co-expressing wild-type and enzymatically inactive ChaC1CtoS (Figure 6B panels 4 and 7). Similarly, the co-expression of ERO1* does not impart sensitivity to active ChaC1, as reflected by the observation that CHOP::GFP levels are unaffected by ChaC1CtoS in these double positive cells (Figure 6B panels 5 and 8). These findings indicate that glutathione is dispensable for the function of the ER even under hyperoxidizing conditions.


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)

Activity of the unfolded protein response (UPR) as measured by an XBP1-splicing reporter is also unaffected by depletion of ER glutathione.FACScans as in ‘6B’ of CHO cells stably transduced with an XBP1 splicing reporter linked to the expression of the fluorescent protein Venus (Iwawaki et al., 2004). Where indicated cells were co-transfected with expression plasmids for a hyperactive mutant of ERO1 (C104A, C133A; ERO1*) tagged by the cell-surface marker CD2 and wild-type or inactive mutants of ChaC1 fused to mCherry. The axis of the scans is labeled with the cognate signals.DOI:http://dx.doi.org/10.7554/eLife.03421.011
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4109312&req=5

fig6s1: Activity of the unfolded protein response (UPR) as measured by an XBP1-splicing reporter is also unaffected by depletion of ER glutathione.FACScans as in ‘6B’ of CHO cells stably transduced with an XBP1 splicing reporter linked to the expression of the fluorescent protein Venus (Iwawaki et al., 2004). Where indicated cells were co-transfected with expression plasmids for a hyperactive mutant of ERO1 (C104A, C133A; ERO1*) tagged by the cell-surface marker CD2 and wild-type or inactive mutants of ChaC1 fused to mCherry. The axis of the scans is labeled with the cognate signals.DOI:http://dx.doi.org/10.7554/eLife.03421.011
Mentions: The role of ER glutathione was further explored under conditions in which ER redox balance was perturbed by co-expression of a deregulated allele of the ER oxidase ERO1 (Sevier et al., 2007), which has been shown to hyper-oxidize the mammalian ER (Baker et al., 2008) and modestly activate the UPR (Hansen et al., 2012). Introduction of the C104A; C133A human ERO1L (ERO1*), expressed from a plasmid tagged with the CD2 surface marker indeed modestly activated the UPR, whether measured in cells stably expressing the CHOP::GFP transcriptional reporter (Figure 6B, reflected in the higher GFP levels of cells co-expressing the CD2 marker which tags the ERO1*-expressing cells, panels 2, 4, and 7) or an XBP1::Venus splicing reporter (Iwawaki et al., 2004) (Figure 6—figure supplement 1). However, co-expression of active ChaC1CtoS had no evident synergistic effect with ERO1* on UPR activity beyond that observed with the enzymatically inactive E116Q mutant enzyme. This is evident by first noting that in this co-transfection experiment most ERO1* expressing cells (CD2 positive) are also co-expressing the Cherry-tagged ChaC1CtoS (Figure 6B, panels 3 and 6) and then noting that the relationship between ERO1* expression (for which CD2 is a surrogate) and the CHOP::GFP signal is indistinguishable in cells co-expressing wild-type and enzymatically inactive ChaC1CtoS (Figure 6B panels 4 and 7). Similarly, the co-expression of ERO1* does not impart sensitivity to active ChaC1, as reflected by the observation that CHOP::GFP levels are unaffected by ChaC1CtoS in these double positive cells (Figure 6B panels 5 and 8). These findings indicate that glutathione is dispensable for the function of the ER even under hyperoxidizing conditions.

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