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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

ER-targeted ChaC1 purges the organelle of its glutathione content.(A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). (B) Trace of time-dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation at 405 nm vs 488 nm (ex 405/488) following introduction of the fully reduced probe into the indicated solutions of oxidized glutathione (GSSG) or PDI and GSSG. (C) Similar trace of reduced Grx1-roGFP2. Note the indifference of roGFP2 and the marked responsiveness of Grx1-roGFP to oxidized glutathione. (D) Graph of the initial velocity of Grx1-roGFP2 oxidation as a function of GSSG concentration, fitted to Linweaver–Burk plot. Half-maximal velocity is observed at 18 µM GSSG. (E) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 and Grx1-roGFP2 probes expressed in the ER of HeLa cells following a brief (1 min) reductive pulse with dithiothreitol (DTT, 2 mM) followed by a washout. (F) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse. Shown are means ±SD (N = 4, *p<0.01). (G) Trace of time-dependent changes in the ratio of oxidized to reduced probes expressed in the ER of HeLa cells alongside active or inactive ChaC1 (tagged at its C-terminus with mCherry to allow visualization of cells co-expressing the redox probes and the glutathione-depleting enzyme) following a brief reductive pulse with dithiothreitol and a washout. Note that the expression of active ChaC1 in the ER eliminates the kinetic advantage of Grx1-roGFP2 over roGFP2 in re-oxidation during the recovery from a DTT reductive pulse. (H) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse in cells co-expressing active or inactive CHaC1. Shown are means ±SEM (N = 20, *p<0.01). (I) Bar diagram of cellular glutathione levels following 36 hr of doxycycline (DOX) induction of cytosolic and ER localized active and inactive ChaC1 in the absence and presence of concomitant exposure to buthionine-sulfoxide (BSO, 50 µM). Also shown is a time course of total cellular glutathione following induction of active and inactive mCherry-KDEL-tagged ChaC1.DOI:http://dx.doi.org/10.7554/eLife.03421.007
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fig3: ER-targeted ChaC1 purges the organelle of its glutathione content.(A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). (B) Trace of time-dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation at 405 nm vs 488 nm (ex 405/488) following introduction of the fully reduced probe into the indicated solutions of oxidized glutathione (GSSG) or PDI and GSSG. (C) Similar trace of reduced Grx1-roGFP2. Note the indifference of roGFP2 and the marked responsiveness of Grx1-roGFP to oxidized glutathione. (D) Graph of the initial velocity of Grx1-roGFP2 oxidation as a function of GSSG concentration, fitted to Linweaver–Burk plot. Half-maximal velocity is observed at 18 µM GSSG. (E) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 and Grx1-roGFP2 probes expressed in the ER of HeLa cells following a brief (1 min) reductive pulse with dithiothreitol (DTT, 2 mM) followed by a washout. (F) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse. Shown are means ±SD (N = 4, *p<0.01). (G) Trace of time-dependent changes in the ratio of oxidized to reduced probes expressed in the ER of HeLa cells alongside active or inactive ChaC1 (tagged at its C-terminus with mCherry to allow visualization of cells co-expressing the redox probes and the glutathione-depleting enzyme) following a brief reductive pulse with dithiothreitol and a washout. Note that the expression of active ChaC1 in the ER eliminates the kinetic advantage of Grx1-roGFP2 over roGFP2 in re-oxidation during the recovery from a DTT reductive pulse. (H) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse in cells co-expressing active or inactive CHaC1. Shown are means ±SEM (N = 20, *p<0.01). (I) Bar diagram of cellular glutathione levels following 36 hr of doxycycline (DOX) induction of cytosolic and ER localized active and inactive ChaC1 in the absence and presence of concomitant exposure to buthionine-sulfoxide (BSO, 50 µM). Also shown is a time course of total cellular glutathione following induction of active and inactive mCherry-KDEL-tagged ChaC1.DOI:http://dx.doi.org/10.7554/eLife.03421.007

Mentions: Measuring the impact of ER-ChaC1CtoS on glutathione levels required an assay that would be selectively sensitive to the ER pool of glutathione. Glutaredoxin (Grx1) has been shown to dramatically accelerate the interaction of a linked redox-sensitive green fluorescent protein (roGFP) with glutathione, both in vivo and in vitro (Gutscher et al., 2008; Birk et al., 2013) (cartooned Figure 3A). We confirmed the reported ability of a linked Grx1 to accelerate the equilibration of roGFP with a glutathione buffer: alone, reduced roGFP2 was only slowly oxidized by glutathione (Figure 3B), but the linked Grx1 markedly accelerated the oxidation of Grx1-roGFP2 (Figure 3, compare the red traces in panels B and C). The rate of probe oxidation by glutathione was concentration-dependent, with half-saturation (Kmax0.5) attained at ∼18 µM GSSG (Figure 3D). Importantly, the presence of oxidized PDI had a minor role in further accelerating the oxidation of Grx1-roGFP2, but dominated the oxidation kinetics of roGFP2 alone (Figure 3B,C).10.7554/eLife.03421.007Figure 3.ER-targeted ChaC1 purges the organelle of its glutathione content.


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)

ER-targeted ChaC1 purges the organelle of its glutathione content.(A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). (B) Trace of time-dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation at 405 nm vs 488 nm (ex 405/488) following introduction of the fully reduced probe into the indicated solutions of oxidized glutathione (GSSG) or PDI and GSSG. (C) Similar trace of reduced Grx1-roGFP2. Note the indifference of roGFP2 and the marked responsiveness of Grx1-roGFP to oxidized glutathione. (D) Graph of the initial velocity of Grx1-roGFP2 oxidation as a function of GSSG concentration, fitted to Linweaver–Burk plot. Half-maximal velocity is observed at 18 µM GSSG. (E) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 and Grx1-roGFP2 probes expressed in the ER of HeLa cells following a brief (1 min) reductive pulse with dithiothreitol (DTT, 2 mM) followed by a washout. (F) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse. Shown are means ±SD (N = 4, *p<0.01). (G) Trace of time-dependent changes in the ratio of oxidized to reduced probes expressed in the ER of HeLa cells alongside active or inactive ChaC1 (tagged at its C-terminus with mCherry to allow visualization of cells co-expressing the redox probes and the glutathione-depleting enzyme) following a brief reductive pulse with dithiothreitol and a washout. Note that the expression of active ChaC1 in the ER eliminates the kinetic advantage of Grx1-roGFP2 over roGFP2 in re-oxidation during the recovery from a DTT reductive pulse. (H) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse in cells co-expressing active or inactive CHaC1. Shown are means ±SEM (N = 20, *p<0.01). (I) Bar diagram of cellular glutathione levels following 36 hr of doxycycline (DOX) induction of cytosolic and ER localized active and inactive ChaC1 in the absence and presence of concomitant exposure to buthionine-sulfoxide (BSO, 50 µM). Also shown is a time course of total cellular glutathione following induction of active and inactive mCherry-KDEL-tagged ChaC1.DOI:http://dx.doi.org/10.7554/eLife.03421.007
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fig3: ER-targeted ChaC1 purges the organelle of its glutathione content.(A) Cartoon contrasting the slow coupling of roGFP2 with the glutathione redox buffer (dashed lines) and the rapid coupling of Grx1-roGFP2 with the glutathione redox buffer (after Gutscher et al., 2008). (B) Trace of time-dependent changes in the ratio of reduced to oxidized roGFP2 detected optically as the ratio between the emission signal (at 535 nm) upon excitation at 405 nm vs 488 nm (ex 405/488) following introduction of the fully reduced probe into the indicated solutions of oxidized glutathione (GSSG) or PDI and GSSG. (C) Similar trace of reduced Grx1-roGFP2. Note the indifference of roGFP2 and the marked responsiveness of Grx1-roGFP to oxidized glutathione. (D) Graph of the initial velocity of Grx1-roGFP2 oxidation as a function of GSSG concentration, fitted to Linweaver–Burk plot. Half-maximal velocity is observed at 18 µM GSSG. (E) Trace of time-dependent changes in the ratio of oxidized and reduced roGFP2 and Grx1-roGFP2 probes expressed in the ER of HeLa cells following a brief (1 min) reductive pulse with dithiothreitol (DTT, 2 mM) followed by a washout. (F) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse. Shown are means ±SD (N = 4, *p<0.01). (G) Trace of time-dependent changes in the ratio of oxidized to reduced probes expressed in the ER of HeLa cells alongside active or inactive ChaC1 (tagged at its C-terminus with mCherry to allow visualization of cells co-expressing the redox probes and the glutathione-depleting enzyme) following a brief reductive pulse with dithiothreitol and a washout. Note that the expression of active ChaC1 in the ER eliminates the kinetic advantage of Grx1-roGFP2 over roGFP2 in re-oxidation during the recovery from a DTT reductive pulse. (H) Bar diagram of the half-time to recovery of oxidized roGFP2 and Grx1-roGFP2 following the reductive DTT pulse in cells co-expressing active or inactive CHaC1. Shown are means ±SEM (N = 20, *p<0.01). (I) Bar diagram of cellular glutathione levels following 36 hr of doxycycline (DOX) induction of cytosolic and ER localized active and inactive ChaC1 in the absence and presence of concomitant exposure to buthionine-sulfoxide (BSO, 50 µM). Also shown is a time course of total cellular glutathione following induction of active and inactive mCherry-KDEL-tagged ChaC1.DOI:http://dx.doi.org/10.7554/eLife.03421.007
Mentions: Measuring the impact of ER-ChaC1CtoS on glutathione levels required an assay that would be selectively sensitive to the ER pool of glutathione. Glutaredoxin (Grx1) has been shown to dramatically accelerate the interaction of a linked redox-sensitive green fluorescent protein (roGFP) with glutathione, both in vivo and in vitro (Gutscher et al., 2008; Birk et al., 2013) (cartooned Figure 3A). We confirmed the reported ability of a linked Grx1 to accelerate the equilibration of roGFP with a glutathione buffer: alone, reduced roGFP2 was only slowly oxidized by glutathione (Figure 3B), but the linked Grx1 markedly accelerated the oxidation of Grx1-roGFP2 (Figure 3, compare the red traces in panels B and C). The rate of probe oxidation by glutathione was concentration-dependent, with half-saturation (Kmax0.5) attained at ∼18 µM GSSG (Figure 3D). Importantly, the presence of oxidized PDI had a minor role in further accelerating the oxidation of Grx1-roGFP2, but dominated the oxidation kinetics of roGFP2 alone (Figure 3B,C).10.7554/eLife.03421.007Figure 3.ER-targeted ChaC1 purges the organelle of its glutathione content.

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