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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

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An Ascorbic Acid-Responsive Defect in Type I Procollagen Maturation in Mutant Cells(A) Immunoblot of type I procollagen in lysates of MEFs of the indicated genotypes. Where noted, cells were exposed to brefeldin A (BFA, 2 μg/mL) for 3 hr before harvest to inhibit export of procollagen from the ER. The anti-Tubulin blot (lower panel) serves a loading control. Shown is a representative experiment performed in duplicate. The bar diagram below the immunoblot shows the mean ± SEM of the ratio of the procollagen to tubulin signal expressed in arbitrary units (au) from three such experiments (n = 3, ∗∗p < 0.01).(B) Immunoblot of procollagen from BFA-treated MEFs. Where indicated, disulfides in the sample were reduced with TCEP before exposure to the thiol-modifying agent PEG-MAL-2000. The modified (PEG-MAL) and unmodified (0) species are marked.(C) Nonreducing and reducing immunoblot of protein disulfide isomerase (PDI) following exposure of MEFs to the reducing agent dithiothreitol (DTT pulse) and a washout for the indicated time (chase). The migration of reduced and oxidized forms of PDI is noted.(D) Plot of oxidized PDI as percentage of the total from the experiment shown in (C). Note the subtlety of the kinetic defect in PDI reoxidation following DTT washout in the mutant cells.(E) Procollagen immunoblot, as in (A). Where indicated, the culture media was supplemented with ascorbate (to a concentration of 50 μM or 100 μM).
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fig4: An Ascorbic Acid-Responsive Defect in Type I Procollagen Maturation in Mutant Cells(A) Immunoblot of type I procollagen in lysates of MEFs of the indicated genotypes. Where noted, cells were exposed to brefeldin A (BFA, 2 μg/mL) for 3 hr before harvest to inhibit export of procollagen from the ER. The anti-Tubulin blot (lower panel) serves a loading control. Shown is a representative experiment performed in duplicate. The bar diagram below the immunoblot shows the mean ± SEM of the ratio of the procollagen to tubulin signal expressed in arbitrary units (au) from three such experiments (n = 3, ∗∗p < 0.01).(B) Immunoblot of procollagen from BFA-treated MEFs. Where indicated, disulfides in the sample were reduced with TCEP before exposure to the thiol-modifying agent PEG-MAL-2000. The modified (PEG-MAL) and unmodified (0) species are marked.(C) Nonreducing and reducing immunoblot of protein disulfide isomerase (PDI) following exposure of MEFs to the reducing agent dithiothreitol (DTT pulse) and a washout for the indicated time (chase). The migration of reduced and oxidized forms of PDI is noted.(D) Plot of oxidized PDI as percentage of the total from the experiment shown in (C). Note the subtlety of the kinetic defect in PDI reoxidation following DTT washout in the mutant cells.(E) Procollagen immunoblot, as in (A). Where indicated, the culture media was supplemented with ascorbate (to a concentration of 50 μM or 100 μM).

Mentions: Type I collagen, the abundant species made by MEFs, is synthesized as a precursor, procollagen. The precursor is modified cotranslationally by lumenal proline and lysine hydroxylases, and later trimer-stabilizing disulfide bonds form between the C-terminal propieces. The procollagen trimer traffics rapidly from the ER and is efficiently processed to mature collagen in post-ER compartments (Lamande and Bateman, 1999). Therefore wild-type MEFs have low levels of procollagen. Exposure to brefeldin A (BFA), which interferes with ER trafficking, traps procollagen in the ER and increases its steady-state levels (Figure 4A, lanes 1–4). Both DM and TM MEFs have abnormally elevated steady-state levels of procollagen (Figure 4A). These observations point to a defect in procollagen maturation in the mutant cells.


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)

An Ascorbic Acid-Responsive Defect in Type I Procollagen Maturation in Mutant Cells(A) Immunoblot of type I procollagen in lysates of MEFs of the indicated genotypes. Where noted, cells were exposed to brefeldin A (BFA, 2 μg/mL) for 3 hr before harvest to inhibit export of procollagen from the ER. The anti-Tubulin blot (lower panel) serves a loading control. Shown is a representative experiment performed in duplicate. The bar diagram below the immunoblot shows the mean ± SEM of the ratio of the procollagen to tubulin signal expressed in arbitrary units (au) from three such experiments (n = 3, ∗∗p < 0.01).(B) Immunoblot of procollagen from BFA-treated MEFs. Where indicated, disulfides in the sample were reduced with TCEP before exposure to the thiol-modifying agent PEG-MAL-2000. The modified (PEG-MAL) and unmodified (0) species are marked.(C) Nonreducing and reducing immunoblot of protein disulfide isomerase (PDI) following exposure of MEFs to the reducing agent dithiothreitol (DTT pulse) and a washout for the indicated time (chase). The migration of reduced and oxidized forms of PDI is noted.(D) Plot of oxidized PDI as percentage of the total from the experiment shown in (C). Note the subtlety of the kinetic defect in PDI reoxidation following DTT washout in the mutant cells.(E) Procollagen immunoblot, as in (A). Where indicated, the culture media was supplemented with ascorbate (to a concentration of 50 μM or 100 μM).
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fig4: An Ascorbic Acid-Responsive Defect in Type I Procollagen Maturation in Mutant Cells(A) Immunoblot of type I procollagen in lysates of MEFs of the indicated genotypes. Where noted, cells were exposed to brefeldin A (BFA, 2 μg/mL) for 3 hr before harvest to inhibit export of procollagen from the ER. The anti-Tubulin blot (lower panel) serves a loading control. Shown is a representative experiment performed in duplicate. The bar diagram below the immunoblot shows the mean ± SEM of the ratio of the procollagen to tubulin signal expressed in arbitrary units (au) from three such experiments (n = 3, ∗∗p < 0.01).(B) Immunoblot of procollagen from BFA-treated MEFs. Where indicated, disulfides in the sample were reduced with TCEP before exposure to the thiol-modifying agent PEG-MAL-2000. The modified (PEG-MAL) and unmodified (0) species are marked.(C) Nonreducing and reducing immunoblot of protein disulfide isomerase (PDI) following exposure of MEFs to the reducing agent dithiothreitol (DTT pulse) and a washout for the indicated time (chase). The migration of reduced and oxidized forms of PDI is noted.(D) Plot of oxidized PDI as percentage of the total from the experiment shown in (C). Note the subtlety of the kinetic defect in PDI reoxidation following DTT washout in the mutant cells.(E) Procollagen immunoblot, as in (A). Where indicated, the culture media was supplemented with ascorbate (to a concentration of 50 μM or 100 μM).
Mentions: Type I collagen, the abundant species made by MEFs, is synthesized as a precursor, procollagen. The precursor is modified cotranslationally by lumenal proline and lysine hydroxylases, and later trimer-stabilizing disulfide bonds form between the C-terminal propieces. The procollagen trimer traffics rapidly from the ER and is efficiently processed to mature collagen in post-ER compartments (Lamande and Bateman, 1999). Therefore wild-type MEFs have low levels of procollagen. Exposure to brefeldin A (BFA), which interferes with ER trafficking, traps procollagen in the ER and increases its steady-state levels (Figure 4A, lanes 1–4). Both DM and TM MEFs have abnormally elevated steady-state levels of procollagen (Figure 4A). These observations point to a defect in procollagen maturation in the mutant cells.

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