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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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Impaired Proline 4 Hydroxylation of Procollagen in Mutant Fibroblasts(A) 4-hydroxyproline content of lysates of brefeldin A-treated MEFs, normalized to the procollagen content of the cells. The upper bar diagram shows the mean ± SEM of the normalized values, expressed in arbitrary units (au) (n = 3, ∗∗p < 0.001, ∗p < 0.05). The lower bar diagram depicts the 4-hydroxyproline content in the individual samples. The procollagen content of each sample was quantified from the fluorescent signal of the immunoblot and is presented under the procollagen blot (Q) in arbitrary units (au).(B) Ascorbic acid content of skin of the indicated mice. Shown are the values obtained in individual animals and the mean for each group (n = 4, ∗∗p < 0.01).
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fig5: Impaired Proline 4 Hydroxylation of Procollagen in Mutant Fibroblasts(A) 4-hydroxyproline content of lysates of brefeldin A-treated MEFs, normalized to the procollagen content of the cells. The upper bar diagram shows the mean ± SEM of the normalized values, expressed in arbitrary units (au) (n = 3, ∗∗p < 0.001, ∗p < 0.05). The lower bar diagram depicts the 4-hydroxyproline content in the individual samples. The procollagen content of each sample was quantified from the fluorescent signal of the immunoblot and is presented under the procollagen blot (Q) in arbitrary units (au).(B) Ascorbic acid content of skin of the indicated mice. Shown are the values obtained in individual animals and the mean for each group (n = 4, ∗∗p < 0.01).

Mentions: Ascorbic acid maintains the activity of ER-localized proline 4-hydroxylases. Failure of proline 4 hydroxylation blocks procollagen secretion and leads to its retention in the ER (Juva et al., 1966). To determine if the procollagen retained in the ER of the mutant cells had abnormally low levels of 4-hydroxyproline, we used BFA to trap procollagen in the ER of MEFs of all three genotypes and assayed the cellular content of 4-hydroxyproline by hydrolysing proteins to amino acids and converting 4-hydroxyproline to a pyrrole by sequential oxidation and decarboxylation, in a colorigenic reaction (Prockop and Udenfriend, 1960). To validate this approach to trapping procollagen in wild-type cells, we confirmed that the 4-hydroxyproline content of wild-type MEFs increased following BFA treatment (Figure S3A). Procollagen 4-hydroxyproline content was 3-fold higher in the BFA-treated wild-type MEFs compared to the DM and 6-fold higher than in the TM cells (Figure 5A).


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)

Impaired Proline 4 Hydroxylation of Procollagen in Mutant Fibroblasts(A) 4-hydroxyproline content of lysates of brefeldin A-treated MEFs, normalized to the procollagen content of the cells. The upper bar diagram shows the mean ± SEM of the normalized values, expressed in arbitrary units (au) (n = 3, ∗∗p < 0.001, ∗p < 0.05). The lower bar diagram depicts the 4-hydroxyproline content in the individual samples. The procollagen content of each sample was quantified from the fluorescent signal of the immunoblot and is presented under the procollagen blot (Q) in arbitrary units (au).(B) Ascorbic acid content of skin of the indicated mice. Shown are the values obtained in individual animals and the mean for each group (n = 4, ∗∗p < 0.01).
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Related In: Results  -  Collection

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fig5: Impaired Proline 4 Hydroxylation of Procollagen in Mutant Fibroblasts(A) 4-hydroxyproline content of lysates of brefeldin A-treated MEFs, normalized to the procollagen content of the cells. The upper bar diagram shows the mean ± SEM of the normalized values, expressed in arbitrary units (au) (n = 3, ∗∗p < 0.001, ∗p < 0.05). The lower bar diagram depicts the 4-hydroxyproline content in the individual samples. The procollagen content of each sample was quantified from the fluorescent signal of the immunoblot and is presented under the procollagen blot (Q) in arbitrary units (au).(B) Ascorbic acid content of skin of the indicated mice. Shown are the values obtained in individual animals and the mean for each group (n = 4, ∗∗p < 0.01).
Mentions: Ascorbic acid maintains the activity of ER-localized proline 4-hydroxylases. Failure of proline 4 hydroxylation blocks procollagen secretion and leads to its retention in the ER (Juva et al., 1966). To determine if the procollagen retained in the ER of the mutant cells had abnormally low levels of 4-hydroxyproline, we used BFA to trap procollagen in the ER of MEFs of all three genotypes and assayed the cellular content of 4-hydroxyproline by hydrolysing proteins to amino acids and converting 4-hydroxyproline to a pyrrole by sequential oxidation and decarboxylation, in a colorigenic reaction (Prockop and Udenfriend, 1960). To validate this approach to trapping procollagen in wild-type cells, we confirmed that the 4-hydroxyproline content of wild-type MEFs increased following BFA treatment (Figure S3A). Procollagen 4-hydroxyproline content was 3-fold higher in the BFA-treated wild-type MEFs compared to the DM and 6-fold higher than in the TM cells (Figure 5A).

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