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Impaired proteostasis contributes to renal tubular dysgenesis.

de Oliveira RM, Marijanovic Z, Carvalho F, Miltényi GM, Matos JE, Tenreiro S, Oliveira S, Enguita FJ, Stone R, Outeiro TF - PLoS ONE (2011)

Bottom Line: Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell.Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels.In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal.

ABSTRACT
Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.

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ACEQ1069R activity is normal in HEK cells at 30°C.A. Western blot analyses of media from ACEWT and ACEQ1069R HEK cells grown at 37°C and 30°C. Equal amounts of total protein were loaded. Quantification of 3 independent experiments. Error bars represent ±SD. P<0.05. B. ACE activity in the media from ACEQ1069R HEK cells grown at 37°C and 30°C. Activity was determined with a synthetic substrate (HHL) for 15 minutes incubation Activity was normalized by the amount of protein present in the media (quantification in A, using arbitrary units).
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pone-0020854-g006: ACEQ1069R activity is normal in HEK cells at 30°C.A. Western blot analyses of media from ACEWT and ACEQ1069R HEK cells grown at 37°C and 30°C. Equal amounts of total protein were loaded. Quantification of 3 independent experiments. Error bars represent ±SD. P<0.05. B. ACE activity in the media from ACEQ1069R HEK cells grown at 37°C and 30°C. Activity was determined with a synthetic substrate (HHL) for 15 minutes incubation Activity was normalized by the amount of protein present in the media (quantification in A, using arbitrary units).

Mentions: ACEQ1069R is not able to reach its proper localization at the plasma membrane due to impaired trafficking (Figure 3A and D). We hypothesized that, once ACEQ1069R reaches the cell surface, the mutant protein will be secreted into the extracellular fluids and its activity should be restored; this hypothesis is further supported by the modeling of the 3-D structure of the ACE protein, which suggests that the ACEQ1069R point mutation should not affect the enzymatic activity of the protein (Figure 1B). To directly test our hypothesis, media from ACEQ1069R and ACEWT cells grown at 30°C for 4 days were harvested and tested by western blot and for ACE activity (Figure 6A and B). Interestingly, in the ACEQ1069R cells ACE activity doubled when cells were grown at 30°C (Figure 6B), indicating that higher ACE levels at the plasma membrane and in the media is coupled with increased enzymatic activity in the media. In summary, our results suggest that if the ACEQ1069R mutant protein reaches its proper localization in the cell, it will be secreted to the extracellular fluids where it can perform its normal function.


Impaired proteostasis contributes to renal tubular dysgenesis.

de Oliveira RM, Marijanovic Z, Carvalho F, Miltényi GM, Matos JE, Tenreiro S, Oliveira S, Enguita FJ, Stone R, Outeiro TF - PLoS ONE (2011)

ACEQ1069R activity is normal in HEK cells at 30°C.A. Western blot analyses of media from ACEWT and ACEQ1069R HEK cells grown at 37°C and 30°C. Equal amounts of total protein were loaded. Quantification of 3 independent experiments. Error bars represent ±SD. P<0.05. B. ACE activity in the media from ACEQ1069R HEK cells grown at 37°C and 30°C. Activity was determined with a synthetic substrate (HHL) for 15 minutes incubation Activity was normalized by the amount of protein present in the media (quantification in A, using arbitrary units).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111453&req=5

pone-0020854-g006: ACEQ1069R activity is normal in HEK cells at 30°C.A. Western blot analyses of media from ACEWT and ACEQ1069R HEK cells grown at 37°C and 30°C. Equal amounts of total protein were loaded. Quantification of 3 independent experiments. Error bars represent ±SD. P<0.05. B. ACE activity in the media from ACEQ1069R HEK cells grown at 37°C and 30°C. Activity was determined with a synthetic substrate (HHL) for 15 minutes incubation Activity was normalized by the amount of protein present in the media (quantification in A, using arbitrary units).
Mentions: ACEQ1069R is not able to reach its proper localization at the plasma membrane due to impaired trafficking (Figure 3A and D). We hypothesized that, once ACEQ1069R reaches the cell surface, the mutant protein will be secreted into the extracellular fluids and its activity should be restored; this hypothesis is further supported by the modeling of the 3-D structure of the ACE protein, which suggests that the ACEQ1069R point mutation should not affect the enzymatic activity of the protein (Figure 1B). To directly test our hypothesis, media from ACEQ1069R and ACEWT cells grown at 30°C for 4 days were harvested and tested by western blot and for ACE activity (Figure 6A and B). Interestingly, in the ACEQ1069R cells ACE activity doubled when cells were grown at 30°C (Figure 6B), indicating that higher ACE levels at the plasma membrane and in the media is coupled with increased enzymatic activity in the media. In summary, our results suggest that if the ACEQ1069R mutant protein reaches its proper localization in the cell, it will be secreted to the extracellular fluids where it can perform its normal function.

Bottom Line: Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell.Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels.In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.

View Article: PubMed Central - PubMed

Affiliation: Cell and Molecular Neuroscience Unit, Instituto de Medicina Molecular, Lisboa, Portugal.

ABSTRACT
Protein conformational disorders are associated with the appearance, persistence, accumulation, and misprocessing of aberrant proteins in the cell. The etiology of renal tubular dysgenesis (RTD) is linked to mutations in the angiotensin-converting enzyme (ACE). Here, we report the identification of a novel ACE mutation (Q1069R) in an RTD patient. ACE Q1069R is found sequestered in the endoplasmic reticulum and is also subject to increased proteasomal degradation, preventing its transport to the cell surface and extracellular fluids. Modulation of cellular proteostasis by temperature shift causes an extension in the processing time and trafficking of ACE Q1069R resulting in partial rescue of the protein processing defect and an increase in plasma membrane levels. In addition, we found that temperature shifting causes the ACE Q1069R protein to be secreted in an active state, suggesting that the mutation does not affect the enzyme's catalytic properties.

Show MeSH
Related in: MedlinePlus