Limits...
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.

Show MeSH

Related in: MedlinePlus

BFA and tunycamicin abolishes the 30°C rescue of ACEQ1069R at the plasma membrane.A. Immunostaining analysis by confocal microscopy of ACEQ1069R HEK cells grown at 30°C on glass coverslips in the presence of 10 µg/ml Tunycamicin for 6h, 10 µg/ml BFA for 6h or control media. Cells were not permeabilized and stained with ACE (green). Scale bars, 20 µm. Quantification of number of cell which display ACE membrane signal. Quantification of 3 independent experiments. Error bars represent ±SD. B. Immunostain analysis by confocal microscopy of ACEQ1069R HEK cells grown in the same conditions as in A. Cells were fixed, permeabilized, stained for ACE (green) and Golgi (red). Nuclei are stained blue with DAPI. Insets represent enlarged images in the boxed regions. Scale bars, 20 µm. Quantification of the green signal overlapping with red signal was done using ImageJ software. Quantification of 3 independent experiments. Erros bars represent ±SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111453&req=5

pone-0020854-g005: BFA and tunycamicin abolishes the 30°C rescue of ACEQ1069R at the plasma membrane.A. Immunostaining analysis by confocal microscopy of ACEQ1069R HEK cells grown at 30°C on glass coverslips in the presence of 10 µg/ml Tunycamicin for 6h, 10 µg/ml BFA for 6h or control media. Cells were not permeabilized and stained with ACE (green). Scale bars, 20 µm. Quantification of number of cell which display ACE membrane signal. Quantification of 3 independent experiments. Error bars represent ±SD. B. Immunostain analysis by confocal microscopy of ACEQ1069R HEK cells grown in the same conditions as in A. Cells were fixed, permeabilized, stained for ACE (green) and Golgi (red). Nuclei are stained blue with DAPI. Insets represent enlarged images in the boxed regions. Scale bars, 20 µm. Quantification of the green signal overlapping with red signal was done using ImageJ software. Quantification of 3 independent experiments. Erros bars represent ±SD.

Mentions: Incubation of cells at 30°C increases the interaction of misfolded proteins with molecular chaperones, facilitating their proper folding and trafficking. Thus, we analyzed the subcellular localization of ACEWT and ACEQ1069R by flow cytometry using the i2H5 antibody and cells (non-permeabilized) grown at 30°C for 4 days in order to determine if ACEQ1069R is misfolded. The proportion of i2H5+ cells in the population of ACEQ1069R cells grown at 30°C increased 9-fold in contrast to cells grown at 37°C (Figure 4A); WT ACE was not affected (Figure 4A). We confirmed these results by fluorescence microscopy with ACEQ1069R and ACEWT cells grown at 30°C for 4 days. ACEQ1069R cells grown at 30°C had more positively stained cells compared to cells grown at 37°C (Figure 4B). We examined ACE protein levels at the two different temperatures in order to exclude the possibility that the rescue of mutant ACE localization to the plasma membrane at 30°C was due to alteration in protein levels. Cells grown at 30°C did not affect protein levels of either WT or mutant proteins as compared to 37°C (Figure S4). Since growing cells at 30°C can augment the amount of the mutant protein present at the plasma membrane and this effect is not increased in the presence of proteasome inhibitor, we suspected that protein trafficking from the ER to Golgi was rescued at 30°C. To test this, HEK cells stably expressing ACEQ1069R or ACEWT were permeabilized and stained with anti-ACE i2H5 antibody and markers for calnexin and giantin; cells were then visualized by confocal microscopy (Figure 4C and Figure S5). Interestingly, there was an increase in co-localization of ACEQ1069R with the Golgi, suggesting an improvement in ACE protein trafficking from the ER to Golgi (Figure 4C and Figure S5) resulting in partially restored surface expression of the mutant protein. Our results suggest that the point mutation in ACE gene is responsible for protein misfolding and retention in the ER leading to its degradation by the UPS; moreover, this defect can be partially overcome by growing cells at 30°C. To further confirm our findings we blocked protein trafficking through the secretory pathway using Brefeldin A (BFA) and tunicamycin. BFA interferes with protein transport from the ER to the Golgi apparatus, while retrograde transport from the Golgi to the ER stays unaffected, resulting in the partial redistribution of the Golgi resident protein to the ER. Furthermore, BFA also affects Golgi stacking, leading to dispersion of the Golgi; as a consequence, proteins are unable to reach the plasma membrane. Tunicamycin is an inhibitor of N-linked protein glycosylation affecting protein folding in the ER, which leads to an increase in ER protein retention time. If rescue at 30°C is indeed due to facilitated trafficking through the secretory pathway, treatment of cells with these drugs should oppose the rescue effect. Indeed, treatment of ACEQ1069R cells with BFA and tunicamycin at 30°C, resulted in 50% lower protein surface expression as observed by immunofluorescence (Figure 5A). Moreover, tunicamycin decreased ACEQ1069R colocalization with Golgi markers while BFA showed no colocalization and caused dispersion of the organelle (Figure 5B). Our results indicate that impaired trafficking through the secretory pathway causes reduced plasma membrane levels of ACEQ1069R protein.


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)

BFA and tunycamicin abolishes the 30°C rescue of ACEQ1069R at the plasma membrane.A. Immunostaining analysis by confocal microscopy of ACEQ1069R HEK cells grown at 30°C on glass coverslips in the presence of 10 µg/ml Tunycamicin for 6h, 10 µg/ml BFA for 6h or control media. Cells were not permeabilized and stained with ACE (green). Scale bars, 20 µm. Quantification of number of cell which display ACE membrane signal. Quantification of 3 independent experiments. Error bars represent ±SD. B. Immunostain analysis by confocal microscopy of ACEQ1069R HEK cells grown in the same conditions as in A. Cells were fixed, permeabilized, stained for ACE (green) and Golgi (red). Nuclei are stained blue with DAPI. Insets represent enlarged images in the boxed regions. Scale bars, 20 µm. Quantification of the green signal overlapping with red signal was done using ImageJ software. Quantification of 3 independent experiments. Erros bars represent ±SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020854-g005: BFA and tunycamicin abolishes the 30°C rescue of ACEQ1069R at the plasma membrane.A. Immunostaining analysis by confocal microscopy of ACEQ1069R HEK cells grown at 30°C on glass coverslips in the presence of 10 µg/ml Tunycamicin for 6h, 10 µg/ml BFA for 6h or control media. Cells were not permeabilized and stained with ACE (green). Scale bars, 20 µm. Quantification of number of cell which display ACE membrane signal. Quantification of 3 independent experiments. Error bars represent ±SD. B. Immunostain analysis by confocal microscopy of ACEQ1069R HEK cells grown in the same conditions as in A. Cells were fixed, permeabilized, stained for ACE (green) and Golgi (red). Nuclei are stained blue with DAPI. Insets represent enlarged images in the boxed regions. Scale bars, 20 µm. Quantification of the green signal overlapping with red signal was done using ImageJ software. Quantification of 3 independent experiments. Erros bars represent ±SD.
Mentions: Incubation of cells at 30°C increases the interaction of misfolded proteins with molecular chaperones, facilitating their proper folding and trafficking. Thus, we analyzed the subcellular localization of ACEWT and ACEQ1069R by flow cytometry using the i2H5 antibody and cells (non-permeabilized) grown at 30°C for 4 days in order to determine if ACEQ1069R is misfolded. The proportion of i2H5+ cells in the population of ACEQ1069R cells grown at 30°C increased 9-fold in contrast to cells grown at 37°C (Figure 4A); WT ACE was not affected (Figure 4A). We confirmed these results by fluorescence microscopy with ACEQ1069R and ACEWT cells grown at 30°C for 4 days. ACEQ1069R cells grown at 30°C had more positively stained cells compared to cells grown at 37°C (Figure 4B). We examined ACE protein levels at the two different temperatures in order to exclude the possibility that the rescue of mutant ACE localization to the plasma membrane at 30°C was due to alteration in protein levels. Cells grown at 30°C did not affect protein levels of either WT or mutant proteins as compared to 37°C (Figure S4). Since growing cells at 30°C can augment the amount of the mutant protein present at the plasma membrane and this effect is not increased in the presence of proteasome inhibitor, we suspected that protein trafficking from the ER to Golgi was rescued at 30°C. To test this, HEK cells stably expressing ACEQ1069R or ACEWT were permeabilized and stained with anti-ACE i2H5 antibody and markers for calnexin and giantin; cells were then visualized by confocal microscopy (Figure 4C and Figure S5). Interestingly, there was an increase in co-localization of ACEQ1069R with the Golgi, suggesting an improvement in ACE protein trafficking from the ER to Golgi (Figure 4C and Figure S5) resulting in partially restored surface expression of the mutant protein. Our results suggest that the point mutation in ACE gene is responsible for protein misfolding and retention in the ER leading to its degradation by the UPS; moreover, this defect can be partially overcome by growing cells at 30°C. To further confirm our findings we blocked protein trafficking through the secretory pathway using Brefeldin A (BFA) and tunicamycin. BFA interferes with protein transport from the ER to the Golgi apparatus, while retrograde transport from the Golgi to the ER stays unaffected, resulting in the partial redistribution of the Golgi resident protein to the ER. Furthermore, BFA also affects Golgi stacking, leading to dispersion of the Golgi; as a consequence, proteins are unable to reach the plasma membrane. Tunicamycin is an inhibitor of N-linked protein glycosylation affecting protein folding in the ER, which leads to an increase in ER protein retention time. If rescue at 30°C is indeed due to facilitated trafficking through the secretory pathway, treatment of cells with these drugs should oppose the rescue effect. Indeed, treatment of ACEQ1069R cells with BFA and tunicamycin at 30°C, resulted in 50% lower protein surface expression as observed by immunofluorescence (Figure 5A). Moreover, tunicamycin decreased ACEQ1069R colocalization with Golgi markers while BFA showed no colocalization and caused dispersion of the organelle (Figure 5B). Our results indicate that impaired trafficking through the secretory pathway causes reduced plasma membrane levels of ACEQ1069R protein.

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