ARS | Publication request: Expression of K6w-Ubiquitin in Lens Epithelial Cells Leads to Up-Regulation of a Broad Spectrum of Molecular Chaperones

Submitted to: Molecular Vision
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 27, 2008
Publication Date: March 4, 2008
Publisher's URL: http://www.molvis.org/molvis/v14/a49/
Citation: Bian, Q., Fernandes, A.F., Taylor, A., Wu, M., Pereira, P., Shang, F. 2008. Expression of k6w-ubiquitin in lens epithelial cells leads to up-regulation of a broad spectrum of molecular chaperones. Molecular Vision. 14:403-412.

Interpretive Summary: Many age-related diseases are associated with the growth of abnormal proteins. The ubiquitin-proteasome pathway (UPP) is the mechanism that removes damaged and irreparable proteins. Molecular chaperones also help in damaged-protein quality-control, helping to unfold misfolded proteins, but work against the UPP mechanism. To develop a further understanding of how both mechanisms work against each other, this study determines how molecular chaperones function when UPP is impaired in human lens epithelial cells (HLEC). To achieve this, the K6W-ubiquitin, a UPP inhibitor, was expressed through an adenoviral vector, while the protein levels for these chaperones were determined by Western blotting. Results showed that expression of K6W-ubiquitin obstructed the UPP-mediated proteolysis, which led to an accumulation of damaged protein in the HLEC. K6W-ubiquitin expression also resulted in increasing the expression of several molecular chaperones. mRNA for alphaB-crystallin, heat-shock proteins, caused Hsp70 and Hsp90 to increase 27-fold, 21-fold, and 2-fold as a result of K6W-ubiquitin expression, while the mRNA for Hsp60 also increased 1.6 fold. With K6W-ubiquitin expression, the expression pattern of these chaperones are similar to cell treated with proteasome-inhibitors or heat-shock. The results supported the hypothesis that UPP and molecular chaperones can back each other up during protein quality control and that up-regulation of these chaperones is associated with an increase of abnormal protein levels in compensating for UPP impairment in removing damaged proteins.

Technical 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 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 RT-PCR. Protein levels for these chaperones were determined by Western blotting. Results: Expression of K6W-ubiquitin impeded the UPP-mediated proteolysis and resulted in accumulation of damaged proteins in the cells. Expression of K6W-ubiquitin in HLEC also increased the expression of a broad spectrum of molecular chaperones. Among the heat-shock proteins, mRNA for aB-crystallin, Hsp70 and Hsp90 increased 27-fold, 21-fold and 2-fold, respectively, in response to expression of K6W-ubiquitin. Among the ER chaperones and ER-stress related factors, mRNA levels of protein disulfide isomerase ,Grp75, Grp78, Grp94, and CHOP increased from 1.7-fold to 3.7-fold. The mRNA for Hsp60 also increased 1.6 fold in response to expression of K6W-ubiquitin. The expression pattern of these chaperones in response to expression of K6W ubiquitin is similar to that when cells were treated with proteasome inhibitors or heat-shock. Conclusions: It appears that up-regulation 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 up-regulation of molecular chaperones in response to expression of dominant negative ubiquitin may compensate for the impairment of the UPP in degradation of abnormal proteins.