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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

Replacement of cysteines with serines circumvents aberrant disulfide bond formation in ER-localized ChaC1.Immunblot of FLAG-tagged authentic, cytosolic mouse ChaC1, ER-localized ChaC1 and ER-localized cysteine-free ChaC1CtoS in lysates of stably-transfected Flp-In T-REx HEK 293T cells expressing the proteins under the control of a tetracycline-inducible promoter. Shown are samples blocked with N-ethyl maleimide at the time of lysis and resolved by reducing and non-reducing SDS-PAGE. Where indicated the cells had been exposed to doxycycline to induce expression of the heterologous protein.DOI:http://dx.doi.org/10.7554/eLife.03421.006
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fig2s1: Replacement of cysteines with serines circumvents aberrant disulfide bond formation in ER-localized ChaC1.Immunblot of FLAG-tagged authentic, cytosolic mouse ChaC1, ER-localized ChaC1 and ER-localized cysteine-free ChaC1CtoS in lysates of stably-transfected Flp-In T-REx HEK 293T cells expressing the proteins under the control of a tetracycline-inducible promoter. Shown are samples blocked with N-ethyl maleimide at the time of lysis and resolved by reducing and non-reducing SDS-PAGE. Where indicated the cells had been exposed to doxycycline to induce expression of the heterologous protein.DOI:http://dx.doi.org/10.7554/eLife.03421.006

Mentions: To exploit ChaC1 as a tool to purge the ER of glutathione, we targeted expression of this cytosolic enzyme to the ER, by fusing the coding sequence to an N-terminal cleavable signal peptide and a C-terminal KDEL ER retention signal. An N-terminal FLAG-M1 peptide tag was included, to facilitate detection of the enzyme. Cells transfected with a plasmid encoding ER-FLAG-ChaC1 expressed a protein of the expected mobility on reducing SDS-PAGE that reacted with the anti-FLAG antibody (Figure 2A) and resulted in a granular staining pattern that overlapped with that of the ER marker calreticulin (Figure 2B). However, unlike cytosolic ChaC1, which migrates at a position expected of the reduced monomer on non-reducing SDS-PAGE (Figure 2—figure supplement 1), ER-localized ChaC1 migrated as a heterogenous collection of species, consistent with inappropriate disulfide bond formation (compare the reducing and non-reducing SDS-PAGE in Figure 2A and Figure 2—figure supplement 1).10.7554/eLife.03421.005Figure 2.Cysteine-free ChaC1 is an active enzyme that can be targeted to the endoplasmic-reticulum.


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)

Replacement of cysteines with serines circumvents aberrant disulfide bond formation in ER-localized ChaC1.Immunblot of FLAG-tagged authentic, cytosolic mouse ChaC1, ER-localized ChaC1 and ER-localized cysteine-free ChaC1CtoS in lysates of stably-transfected Flp-In T-REx HEK 293T cells expressing the proteins under the control of a tetracycline-inducible promoter. Shown are samples blocked with N-ethyl maleimide at the time of lysis and resolved by reducing and non-reducing SDS-PAGE. Where indicated the cells had been exposed to doxycycline to induce expression of the heterologous protein.DOI:http://dx.doi.org/10.7554/eLife.03421.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2s1: Replacement of cysteines with serines circumvents aberrant disulfide bond formation in ER-localized ChaC1.Immunblot of FLAG-tagged authentic, cytosolic mouse ChaC1, ER-localized ChaC1 and ER-localized cysteine-free ChaC1CtoS in lysates of stably-transfected Flp-In T-REx HEK 293T cells expressing the proteins under the control of a tetracycline-inducible promoter. Shown are samples blocked with N-ethyl maleimide at the time of lysis and resolved by reducing and non-reducing SDS-PAGE. Where indicated the cells had been exposed to doxycycline to induce expression of the heterologous protein.DOI:http://dx.doi.org/10.7554/eLife.03421.006
Mentions: To exploit ChaC1 as a tool to purge the ER of glutathione, we targeted expression of this cytosolic enzyme to the ER, by fusing the coding sequence to an N-terminal cleavable signal peptide and a C-terminal KDEL ER retention signal. An N-terminal FLAG-M1 peptide tag was included, to facilitate detection of the enzyme. Cells transfected with a plasmid encoding ER-FLAG-ChaC1 expressed a protein of the expected mobility on reducing SDS-PAGE that reacted with the anti-FLAG antibody (Figure 2A) and resulted in a granular staining pattern that overlapped with that of the ER marker calreticulin (Figure 2B). However, unlike cytosolic ChaC1, which migrates at a position expected of the reduced monomer on non-reducing SDS-PAGE (Figure 2—figure supplement 1), ER-localized ChaC1 migrated as a heterogenous collection of species, consistent with inappropriate disulfide bond formation (compare the reducing and non-reducing SDS-PAGE in Figure 2A and Figure 2—figure supplement 1).10.7554/eLife.03421.005Figure 2.Cysteine-free ChaC1 is an active enzyme that can be targeted to the endoplasmic-reticulum.

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