Limits...
Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Zito E, Hansen HG, Yeo GS, Fujii J, Ron D - Mol. Cell (2012)

Bottom Line: These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins.In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system.Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. ez235@medschl.cam.ac.uk

Show MeSH

Related in: MedlinePlus

Elevated TGF-β Signature in Mutant MEFs and Mouse Tissue(A) Immunoblot of ERO1α, PRDX4, GRP94, BiP, PDI, and Actin in extracts of primary MEFs from embryos of the indicated genotypes.(B) Photomicrographs of fixed primary MEFs of the indicated genotype stained with an antibody to the KDEL-COOH peptide (to delineate the ER) and DAPI to outline the nucleus. Note the flattened morphology and enlarged nuclei of the TM cells.(C) Fluorescence-activated cell scanning (FACS) profiles of MEFs stained with the DNA-binding dye, propidium iodide. Note the prominent population of TM cells with abnormally high DNA content.(D) Relative abundance of TGF-β pathway mRNAs measured by quantitative real-time PCR in cDNA from skin. Shown are the values obtained in individual mice and the mean for each genotype (n = 4, ∗p < 0.05, ∗∗p < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3473360&req=5

fig3: Elevated TGF-β Signature in Mutant MEFs and Mouse Tissue(A) Immunoblot of ERO1α, PRDX4, GRP94, BiP, PDI, and Actin in extracts of primary MEFs from embryos of the indicated genotypes.(B) Photomicrographs of fixed primary MEFs of the indicated genotype stained with an antibody to the KDEL-COOH peptide (to delineate the ER) and DAPI to outline the nucleus. Note the flattened morphology and enlarged nuclei of the TM cells.(C) Fluorescence-activated cell scanning (FACS) profiles of MEFs stained with the DNA-binding dye, propidium iodide. Note the prominent population of TM cells with abnormally high DNA content.(D) Relative abundance of TGF-β pathway mRNAs measured by quantitative real-time PCR in cDNA from skin. Shown are the values obtained in individual mice and the mean for each genotype (n = 4, ∗p < 0.05, ∗∗p < 0.01).

Mentions: The aforementioned observations indicated that the connective tissue of mice with compromised ER thiol oxidases was defective. To explore this problem, we turned to MEFs, a cell culture model for connective tissue. Immunoblot confirmed the presence of ERO1α and PRDX4 in the control population (ERO1β is not detectable in MEFs, Zito et al., 2010a) and their absence in the DM and TM populations. Levels of the ER chaperones BiP and GRP94 were only minimally elevated in the mutant cells, indicating comparable levels of unfolded protein stress in the ER of MEFs of the three genotypes (Figure 3A).


Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice.

Zito E, Hansen HG, Yeo GS, Fujii J, Ron D - Mol. Cell (2012)

Elevated TGF-β Signature in Mutant MEFs and Mouse Tissue(A) Immunoblot of ERO1α, PRDX4, GRP94, BiP, PDI, and Actin in extracts of primary MEFs from embryos of the indicated genotypes.(B) Photomicrographs of fixed primary MEFs of the indicated genotype stained with an antibody to the KDEL-COOH peptide (to delineate the ER) and DAPI to outline the nucleus. Note the flattened morphology and enlarged nuclei of the TM cells.(C) Fluorescence-activated cell scanning (FACS) profiles of MEFs stained with the DNA-binding dye, propidium iodide. Note the prominent population of TM cells with abnormally high DNA content.(D) Relative abundance of TGF-β pathway mRNAs measured by quantitative real-time PCR in cDNA from skin. Shown are the values obtained in individual mice and the mean for each genotype (n = 4, ∗p < 0.05, ∗∗p < 0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473360&req=5

fig3: Elevated TGF-β Signature in Mutant MEFs and Mouse Tissue(A) Immunoblot of ERO1α, PRDX4, GRP94, BiP, PDI, and Actin in extracts of primary MEFs from embryos of the indicated genotypes.(B) Photomicrographs of fixed primary MEFs of the indicated genotype stained with an antibody to the KDEL-COOH peptide (to delineate the ER) and DAPI to outline the nucleus. Note the flattened morphology and enlarged nuclei of the TM cells.(C) Fluorescence-activated cell scanning (FACS) profiles of MEFs stained with the DNA-binding dye, propidium iodide. Note the prominent population of TM cells with abnormally high DNA content.(D) Relative abundance of TGF-β pathway mRNAs measured by quantitative real-time PCR in cDNA from skin. Shown are the values obtained in individual mice and the mean for each genotype (n = 4, ∗p < 0.05, ∗∗p < 0.01).
Mentions: The aforementioned observations indicated that the connective tissue of mice with compromised ER thiol oxidases was defective. To explore this problem, we turned to MEFs, a cell culture model for connective tissue. Immunoblot confirmed the presence of ERO1α and PRDX4 in the control population (ERO1β is not detectable in MEFs, Zito et al., 2010a) and their absence in the DM and TM populations. Levels of the ER chaperones BiP and GRP94 were only minimally elevated in the mutant cells, indicating comparable levels of unfolded protein stress in the ER of MEFs of the three genotypes (Figure 3A).

Bottom Line: These severe abnormalities were associated with an unexpectedly modest delay in disulfide bond formation in secreted proteins but a profound, 5-fold lower procollagen 4-hydroxyproline content and enhanced cysteinyl sulfenic acid modification of ER proteins.In vitro, the presence of a sulfenic acid donor accelerated the oxidative inactivation of ascorbate by an H(2)O(2)-generating system.Compromised ER disulfide relay thus exposes protein thiols to competing oxidation to sulfenic acid, resulting in depletion of ascorbic acid, impaired procollagen proline 4-hydroxylation, and a noncanonical form of scurvy.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Cambridge, UK. ez235@medschl.cam.ac.uk

Show MeSH
Related in: MedlinePlus