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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

Analysis of the substrate binding properties of ChaC1.(A) Absorbance spectrum of ChaC1-mCherry, Oregon Green (OG), and Oregon green-labeled ChaC1-mCherry. (B) Absorbance profile of size-exclusion chromatogram of Oregon green-labeled ChaC1 mCherry. Note the coincidence of the absorption peak for protein (280 nm), Oregon Green (496 nm) and mCherry (587 nm) (C) FRET signal of an enzymatically inactive OG-ChaC1E116Q-Cherry probe upon exposure to varying concentrations of reduced glutathione. Note the mono-phasic change in FRET signal, consistent with inability of the mutant enzyme to break down glutathione and the contrast with wild-type OG-ChaC1-mCherry (Figure 1D). (D) Graph of the relationship between the steady-state FRET signal of the OG-ChaC1E116Q-Cherry probe and the concentration of reduced glutathione (GSH).DOI:http://dx.doi.org/10.7554/eLife.03421.004
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fig1s1: Analysis of the substrate binding properties of ChaC1.(A) Absorbance spectrum of ChaC1-mCherry, Oregon Green (OG), and Oregon green-labeled ChaC1-mCherry. (B) Absorbance profile of size-exclusion chromatogram of Oregon green-labeled ChaC1 mCherry. Note the coincidence of the absorption peak for protein (280 nm), Oregon Green (496 nm) and mCherry (587 nm) (C) FRET signal of an enzymatically inactive OG-ChaC1E116Q-Cherry probe upon exposure to varying concentrations of reduced glutathione. Note the mono-phasic change in FRET signal, consistent with inability of the mutant enzyme to break down glutathione and the contrast with wild-type OG-ChaC1-mCherry (Figure 1D). (D) Graph of the relationship between the steady-state FRET signal of the OG-ChaC1E116Q-Cherry probe and the concentration of reduced glutathione (GSH).DOI:http://dx.doi.org/10.7554/eLife.03421.004

Mentions: Kumar et al. recently reported that the mammalian pro-apoptotic gene ChaC1 encodes a glutathione-specific γ-glutamyl cyclotransferase that efficiently degrades glutathione (Kumar et al., 2012). We confirmed their observations by measuring the ability of purified murine ChaC1 (expressed in E. coli) to degrade glutathione in vitro: At submicromolar enzyme concentrations, recombinant ChaC1 was able to degrade a 10 mM solution of reduced glutathione within 1 hr (Figure 1A). The enzymatic activity was selective towards reduced glutathione (GSH) (Figure 1B). The inability of oxidized glutathione (GSSG) to serve as a substrate for degradation correlated with its inability to bind a Chac1-based optical probe whose fluorescent resonance energy transfer (FRET) signal reflects substrate binding (Figure 1C–D and Figure 1—figure supplement 1). An E116Q mutation abolished all enzymatic activity (Figure 1E), as observed previously (Kumar et al., 2012).10.7554/eLife.03421.003Figure 1.ChaC1 efficiently and selectively degrades reduced 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)

Analysis of the substrate binding properties of ChaC1.(A) Absorbance spectrum of ChaC1-mCherry, Oregon Green (OG), and Oregon green-labeled ChaC1-mCherry. (B) Absorbance profile of size-exclusion chromatogram of Oregon green-labeled ChaC1 mCherry. Note the coincidence of the absorption peak for protein (280 nm), Oregon Green (496 nm) and mCherry (587 nm) (C) FRET signal of an enzymatically inactive OG-ChaC1E116Q-Cherry probe upon exposure to varying concentrations of reduced glutathione. Note the mono-phasic change in FRET signal, consistent with inability of the mutant enzyme to break down glutathione and the contrast with wild-type OG-ChaC1-mCherry (Figure 1D). (D) Graph of the relationship between the steady-state FRET signal of the OG-ChaC1E116Q-Cherry probe and the concentration of reduced glutathione (GSH).DOI:http://dx.doi.org/10.7554/eLife.03421.004
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109312&req=5

fig1s1: Analysis of the substrate binding properties of ChaC1.(A) Absorbance spectrum of ChaC1-mCherry, Oregon Green (OG), and Oregon green-labeled ChaC1-mCherry. (B) Absorbance profile of size-exclusion chromatogram of Oregon green-labeled ChaC1 mCherry. Note the coincidence of the absorption peak for protein (280 nm), Oregon Green (496 nm) and mCherry (587 nm) (C) FRET signal of an enzymatically inactive OG-ChaC1E116Q-Cherry probe upon exposure to varying concentrations of reduced glutathione. Note the mono-phasic change in FRET signal, consistent with inability of the mutant enzyme to break down glutathione and the contrast with wild-type OG-ChaC1-mCherry (Figure 1D). (D) Graph of the relationship between the steady-state FRET signal of the OG-ChaC1E116Q-Cherry probe and the concentration of reduced glutathione (GSH).DOI:http://dx.doi.org/10.7554/eLife.03421.004
Mentions: Kumar et al. recently reported that the mammalian pro-apoptotic gene ChaC1 encodes a glutathione-specific γ-glutamyl cyclotransferase that efficiently degrades glutathione (Kumar et al., 2012). We confirmed their observations by measuring the ability of purified murine ChaC1 (expressed in E. coli) to degrade glutathione in vitro: At submicromolar enzyme concentrations, recombinant ChaC1 was able to degrade a 10 mM solution of reduced glutathione within 1 hr (Figure 1A). The enzymatic activity was selective towards reduced glutathione (GSH) (Figure 1B). The inability of oxidized glutathione (GSSG) to serve as a substrate for degradation correlated with its inability to bind a Chac1-based optical probe whose fluorescent resonance energy transfer (FRET) signal reflects substrate binding (Figure 1C–D and Figure 1—figure supplement 1). An E116Q mutation abolished all enzymatic activity (Figure 1E), as observed previously (Kumar et al., 2012).10.7554/eLife.03421.003Figure 1.ChaC1 efficiently and selectively degrades reduced 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