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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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Low ACEQ1069R protein levels in HEK cells is due to degradation by the ubiquitin proteasome system.A. Analysis of ACE protein levels by western blot in HEK cells stably expressing the WT form or the mutant (Q1069R). Empty vector is used as a negative control. GAPDH was used as a loading control. Quantification of 3 independent experiments is shown on the right. Error bars represent ±SD. P<0.05. B. Relative quantitative analysis of ACE mRNA levels in HEK cells stably expressing WT or ACEQ1069R, normalized to β-actin. Error bar represents ±SD. C. Analysis of ACE protein stability by pulse-chase experiment in HEK cells. Cells were treated with CHX, for the indicated times. Protein levels were assessed by western blot analysis. Quantification of 3 independent experiments is shown. Error bars represent ±SD. P<0.05. D. Western blot analysis of ACEQ1069R HEK cells. Cells in growth medium were treated for 2h with control medium or media with increasing dosages of ALLN. On the right, quantification of three independent experiments is shown. Error bars represent ±SD. P<0.05.
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pone-0020854-g002: Low ACEQ1069R protein levels in HEK cells is due to degradation by the ubiquitin proteasome system.A. Analysis of ACE protein levels by western blot in HEK cells stably expressing the WT form or the mutant (Q1069R). Empty vector is used as a negative control. GAPDH was used as a loading control. Quantification of 3 independent experiments is shown on the right. Error bars represent ±SD. P<0.05. B. Relative quantitative analysis of ACE mRNA levels in HEK cells stably expressing WT or ACEQ1069R, normalized to β-actin. Error bar represents ±SD. C. Analysis of ACE protein stability by pulse-chase experiment in HEK cells. Cells were treated with CHX, for the indicated times. Protein levels were assessed by western blot analysis. Quantification of 3 independent experiments is shown. Error bars represent ±SD. P<0.05. D. Western blot analysis of ACEQ1069R HEK cells. Cells in growth medium were treated for 2h with control medium or media with increasing dosages of ALLN. On the right, quantification of three independent experiments is shown. Error bars represent ±SD. P<0.05.

Mentions: In order to study ACE processing, we established a cellular model to analyze localization, trafficking and turnover of WT and mutant ACE. We generated stable cell lines expressing either WT ACE (ACEWT) or ACE Q1069R (ACEQ1069R) in a human embryonic kidney (HEK) cells. We performed immunoblot analysis to investigate ACE protein levels both cell lines using an anti-ACE antibody that recognizes the denatured form of the protein (anti-ACE 1D8). ACE protein levels were approximately 80% lower in ACEQ1068R cells compared to ACEWT cells (Figure 2A); the lower level of mutant ACE protein in ACEQ1068R cells could be attributed to lower protein expression, decreased protein stability, or both. To discriminate between these possibilities, we performed quantitative RT-PCR analysis on ACEWT and ACEQ1069R cells quantifying ACE mRNA levels and found no significant difference between mRNA levels (Figure 2B). Thus, the marked discrepancy between the mRNA and protein levels of wild type and mutant ACE-expressing cells suggested reduced protein stability for the mutant form. Next, we analyzed the half-life of ACEWT and ACEQ1069R. Cells were treated with cyclohexamide (CHX) to inhibit de novo protein synthesis and protein levels were monitored by immunoblot analysis. While the ACEWT protein showed essentially no decrease in protein levels over a 120 minutes time course, ACEQ1069R protein is more rapidly degraded and is not detected at 120 minutes, suggesting a decreased stability of the mutant protein (Figure 2C).


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)

Low ACEQ1069R protein levels in HEK cells is due to degradation by the ubiquitin proteasome system.A. Analysis of ACE protein levels by western blot in HEK cells stably expressing the WT form or the mutant (Q1069R). Empty vector is used as a negative control. GAPDH was used as a loading control. Quantification of 3 independent experiments is shown on the right. Error bars represent ±SD. P<0.05. B. Relative quantitative analysis of ACE mRNA levels in HEK cells stably expressing WT or ACEQ1069R, normalized to β-actin. Error bar represents ±SD. C. Analysis of ACE protein stability by pulse-chase experiment in HEK cells. Cells were treated with CHX, for the indicated times. Protein levels were assessed by western blot analysis. Quantification of 3 independent experiments is shown. Error bars represent ±SD. P<0.05. D. Western blot analysis of ACEQ1069R HEK cells. Cells in growth medium were treated for 2h with control medium or media with increasing dosages of ALLN. On the right, quantification of three independent experiments is shown. Error bars represent ±SD. P<0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111453&req=5

pone-0020854-g002: Low ACEQ1069R protein levels in HEK cells is due to degradation by the ubiquitin proteasome system.A. Analysis of ACE protein levels by western blot in HEK cells stably expressing the WT form or the mutant (Q1069R). Empty vector is used as a negative control. GAPDH was used as a loading control. Quantification of 3 independent experiments is shown on the right. Error bars represent ±SD. P<0.05. B. Relative quantitative analysis of ACE mRNA levels in HEK cells stably expressing WT or ACEQ1069R, normalized to β-actin. Error bar represents ±SD. C. Analysis of ACE protein stability by pulse-chase experiment in HEK cells. Cells were treated with CHX, for the indicated times. Protein levels were assessed by western blot analysis. Quantification of 3 independent experiments is shown. Error bars represent ±SD. P<0.05. D. Western blot analysis of ACEQ1069R HEK cells. Cells in growth medium were treated for 2h with control medium or media with increasing dosages of ALLN. On the right, quantification of three independent experiments is shown. Error bars represent ±SD. P<0.05.
Mentions: In order to study ACE processing, we established a cellular model to analyze localization, trafficking and turnover of WT and mutant ACE. We generated stable cell lines expressing either WT ACE (ACEWT) or ACE Q1069R (ACEQ1069R) in a human embryonic kidney (HEK) cells. We performed immunoblot analysis to investigate ACE protein levels both cell lines using an anti-ACE antibody that recognizes the denatured form of the protein (anti-ACE 1D8). ACE protein levels were approximately 80% lower in ACEQ1068R cells compared to ACEWT cells (Figure 2A); the lower level of mutant ACE protein in ACEQ1068R cells could be attributed to lower protein expression, decreased protein stability, or both. To discriminate between these possibilities, we performed quantitative RT-PCR analysis on ACEWT and ACEQ1069R cells quantifying ACE mRNA levels and found no significant difference between mRNA levels (Figure 2B). Thus, the marked discrepancy between the mRNA and protein levels of wild type and mutant ACE-expressing cells suggested reduced protein stability for the mutant form. Next, we analyzed the half-life of ACEWT and ACEQ1069R. Cells were treated with cyclohexamide (CHX) to inhibit de novo protein synthesis and protein levels were monitored by immunoblot analysis. While the ACEWT protein showed essentially no decrease in protein levels over a 120 minutes time course, ACEQ1069R protein is more rapidly degraded and is not detected at 120 minutes, suggesting a decreased stability of the mutant protein (Figure 2C).

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