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Expression of K6W-ubiquitin in lens epithelial cells leads to upregulation of a broad spectrum of molecular chaperones.

Bian Q, Fernandes AF, Taylor A, Wu M, Pereira P, Shang F - Mol. Vis. (2008)

Bottom Line: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases.It appears that the upregulation of these chaperones is related to the elevated levels of abnormal proteins in the cells.The upregulation of molecular chaperones in response to the expression of a dominant negative ubiquitin may compensate for the impairment of the UPP in the degradation of abnormal proteins.

View Article: PubMed Central - PubMed

Affiliation: USDA HNRCA at Tufts University, Boston, Massachusetts, USA.

ABSTRACT

Purpose: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases. The ubiquitin-proteasome pathway (UPP) is one of the protein quality control mechanisms that selectively degrade damaged or obsolete proteins. The other arm of the protein quality control mechanism is molecular chaperones, which bind to and help refold unfolded or misfolded proteins. We previously showed that the molecular chaperones and the UPP work in a competitive manner in eliminating the denatured proteins. To further investigate the interaction between the two protein quality control mechanisms, we determined the effects of the impairment of the UPP on the expression of molecular chaperones in human lens epithelial cells (HLEC).

Methods: K6W-ubiquitin, a dominant negative inhibitor of the UPP, was expressed in confluent HLEC via an adenoviral vector. The mRNA levels of cytoplasmic and endoplasmic reticulum (ER) chaperones were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Protein levels for these chaperones were determined by western blotting.

Results: Expression of K6W-ubiquitin in HLEC increased the expression of a broad spectrum of molecular chaperones. Among the heat-shock proteins, mRNA for alphaB-crystallin, Hsp70, and Hsp90 increased 27 fold, 21 fold, and twofold, respectively, in response to K6W-ubiquitin expression. Among the ER chaperones and ER stress related factors, mRNA levels of protein disulfide isomerase, Grp75, Grp78, Grp94, and the CAAT/enhancer binding protein homologous protein (CHOP) increased from 1.7 fold to 3.7 fold. The mRNA for Hsp60 also increased 1.6 fold in response to the expression of K6W-ubiquitin. The expression pattern of these chaperones in response to the expression of K6W ubiquitin is similar to that obtained when cells were treated with proteasome inhibitors or heat-shock.

Conclusions: It appears that the upregulation of these chaperones is related to the elevated levels of abnormal proteins in the cells. These findings support our hypothesis that the molecular chaperones and the UPP may back each other up in the process of protein quality control. The upregulation of molecular chaperones in response to the expression of a dominant negative ubiquitin may compensate for the impairment of the UPP in the degradation of abnormal proteins.

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Proteasome inhibition upregulates cytoplasmic, endoplasmic reticulum, and mitochondrial chaperones. Lens epithelial cells were treated with or without 10 µM MG132 for 6 h. Total RNA was extracted, and the level of mRNA for these chaperones was determined by real-time RT–PCR using GAPDH a reference. The level of mRNA for these chaperones in the cells not treated with MG132 (the control) was arbitrarily designated as 1. The levels of mRNAs for these chaperones in MG132-treated cells were normalized with the control and expressed as relative levels. Panel A shows the expression of cytoplasmic chaperones. Panel B shows the expression of ER chaperones, an ER stress-related factor, and a mitochondrial chaperone.
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f2: Proteasome inhibition upregulates cytoplasmic, endoplasmic reticulum, and mitochondrial chaperones. Lens epithelial cells were treated with or without 10 µM MG132 for 6 h. Total RNA was extracted, and the level of mRNA for these chaperones was determined by real-time RT–PCR using GAPDH a reference. The level of mRNA for these chaperones in the cells not treated with MG132 (the control) was arbitrarily designated as 1. The levels of mRNAs for these chaperones in MG132-treated cells were normalized with the control and expressed as relative levels. Panel A shows the expression of cytoplasmic chaperones. Panel B shows the expression of ER chaperones, an ER stress-related factor, and a mitochondrial chaperone.

Mentions: To confirm that the upregulation of molecular chaperones in response to the expression of K6W-ubiquitin is due to the impairment of the UPP, we determined the effect of the proteasome inhibitor on the expression of these chaperones. We found that proteasome inhibition also increased the expression of these cytoplasmic and ER chaperones, and the extents of increase are more dramatic than that caused by the expression of K6W-ubiquitin. The mRNA levels for αB-crystallin and Hsp70 increased 42 and 242 fold, respectively, after incubation with MG132 for 6 h (Figure 2A). The mRNA levels for αA-crystallin, Hsp27, Hsc70, and Hsp90 increased from 2.4 to 12-fold in response to proteasome inhibition, but the mRNA level for Hsp40 did not change (Figure 2A). Among the ER chaperones, mRNA levels for Grp75, Grp78, and Grp94 increased from 1.46 to 2.8 fold, but the mRNA level for PDI did not change in response to proteasome inhibition (Figure 2B). The ER stress-related transcription factor, CHOP, and the mitochondrial chaperone, Hsp60, also increased 12.8 and 6.9 fold, respectively, after incubation with MG132 (Figure 2B). These data confirmed that impairment of the UPP leads to upregulation of a broad spectrum of cytoplasmic, ER, and mitochondrial chaperones.


Expression of K6W-ubiquitin in lens epithelial cells leads to upregulation of a broad spectrum of molecular chaperones.

Bian Q, Fernandes AF, Taylor A, Wu M, Pereira P, Shang F - Mol. Vis. (2008)

Proteasome inhibition upregulates cytoplasmic, endoplasmic reticulum, and mitochondrial chaperones. Lens epithelial cells were treated with or without 10 µM MG132 for 6 h. Total RNA was extracted, and the level of mRNA for these chaperones was determined by real-time RT–PCR using GAPDH a reference. The level of mRNA for these chaperones in the cells not treated with MG132 (the control) was arbitrarily designated as 1. The levels of mRNAs for these chaperones in MG132-treated cells were normalized with the control and expressed as relative levels. Panel A shows the expression of cytoplasmic chaperones. Panel B shows the expression of ER chaperones, an ER stress-related factor, and a mitochondrial chaperone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268857&req=5

f2: Proteasome inhibition upregulates cytoplasmic, endoplasmic reticulum, and mitochondrial chaperones. Lens epithelial cells were treated with or without 10 µM MG132 for 6 h. Total RNA was extracted, and the level of mRNA for these chaperones was determined by real-time RT–PCR using GAPDH a reference. The level of mRNA for these chaperones in the cells not treated with MG132 (the control) was arbitrarily designated as 1. The levels of mRNAs for these chaperones in MG132-treated cells were normalized with the control and expressed as relative levels. Panel A shows the expression of cytoplasmic chaperones. Panel B shows the expression of ER chaperones, an ER stress-related factor, and a mitochondrial chaperone.
Mentions: To confirm that the upregulation of molecular chaperones in response to the expression of K6W-ubiquitin is due to the impairment of the UPP, we determined the effect of the proteasome inhibitor on the expression of these chaperones. We found that proteasome inhibition also increased the expression of these cytoplasmic and ER chaperones, and the extents of increase are more dramatic than that caused by the expression of K6W-ubiquitin. The mRNA levels for αB-crystallin and Hsp70 increased 42 and 242 fold, respectively, after incubation with MG132 for 6 h (Figure 2A). The mRNA levels for αA-crystallin, Hsp27, Hsc70, and Hsp90 increased from 2.4 to 12-fold in response to proteasome inhibition, but the mRNA level for Hsp40 did not change (Figure 2A). Among the ER chaperones, mRNA levels for Grp75, Grp78, and Grp94 increased from 1.46 to 2.8 fold, but the mRNA level for PDI did not change in response to proteasome inhibition (Figure 2B). The ER stress-related transcription factor, CHOP, and the mitochondrial chaperone, Hsp60, also increased 12.8 and 6.9 fold, respectively, after incubation with MG132 (Figure 2B). These data confirmed that impairment of the UPP leads to upregulation of a broad spectrum of cytoplasmic, ER, and mitochondrial chaperones.

Bottom Line: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases.It appears that the upregulation of these chaperones is related to the elevated levels of abnormal proteins in the cells.The upregulation of molecular chaperones in response to the expression of a dominant negative ubiquitin may compensate for the impairment of the UPP in the degradation of abnormal proteins.

View Article: PubMed Central - PubMed

Affiliation: USDA HNRCA at Tufts University, Boston, Massachusetts, USA.

ABSTRACT

Purpose: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases. The ubiquitin-proteasome pathway (UPP) is one of the protein quality control mechanisms that selectively degrade damaged or obsolete proteins. The other arm of the protein quality control mechanism is molecular chaperones, which bind to and help refold unfolded or misfolded proteins. We previously showed that the molecular chaperones and the UPP work in a competitive manner in eliminating the denatured proteins. To further investigate the interaction between the two protein quality control mechanisms, we determined the effects of the impairment of the UPP on the expression of molecular chaperones in human lens epithelial cells (HLEC).

Methods: K6W-ubiquitin, a dominant negative inhibitor of the UPP, was expressed in confluent HLEC via an adenoviral vector. The mRNA levels of cytoplasmic and endoplasmic reticulum (ER) chaperones were determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Protein levels for these chaperones were determined by western blotting.

Results: Expression of K6W-ubiquitin in HLEC increased the expression of a broad spectrum of molecular chaperones. Among the heat-shock proteins, mRNA for alphaB-crystallin, Hsp70, and Hsp90 increased 27 fold, 21 fold, and twofold, respectively, in response to K6W-ubiquitin expression. Among the ER chaperones and ER stress related factors, mRNA levels of protein disulfide isomerase, Grp75, Grp78, Grp94, and the CAAT/enhancer binding protein homologous protein (CHOP) increased from 1.7 fold to 3.7 fold. The mRNA for Hsp60 also increased 1.6 fold in response to the expression of K6W-ubiquitin. The expression pattern of these chaperones in response to the expression of K6W ubiquitin is similar to that obtained when cells were treated with proteasome inhibitors or heat-shock.

Conclusions: It appears that the upregulation of these chaperones is related to the elevated levels of abnormal proteins in the cells. These findings support our hypothesis that the molecular chaperones and the UPP may back each other up in the process of protein quality control. The upregulation of molecular chaperones in response to the expression of a dominant negative ubiquitin may compensate for the impairment of the UPP in the degradation of abnormal proteins.

Show MeSH