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F. Shang, Q. Bian, A. Taylor, M. Wu, J. J. Liang; Enhancement of Ubiquitin-Proteasome Pathway (UPP) Reduces Aggregation of Mutant Crystallins and Provides Cytoprotection. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6358.
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Accumulation and precipitation of abnormal proteins are associated with many age-related diseases, including cataract. To maintain cell function and protein homeostasis, organisms evolved protein quality control mechanisms, which include the UPP to selectively degrade damaged proteins, and molecular chaperones to refold misfolded proteins. The objective of this study is to determine the interaction between the UPP and molecular chaperones and to test the effects of enhancement of the UPP on aggregation of mutant crystallins and cytoprotection against environmental stress.
To enhance the UPP function we overexpressed a rate-limiting ubiquitin ligase (CHIP) or CHIP together with its functional partner, Ubc5.CHIP or CHIP together with Ubc5 were expressed in confluent human lens epithelial cells (HLEC) via an adenoviral vector. Levels of CHIP, Ubc5, ubiquitin conjugates and molecular chaperones were determined by Western blot. mRNA levels of molecular chaperones were determined by Real-Time RT-PCR. RFP-tagged Wt and R120G mutant αB-crystallins were co-expressed with CHIP or CHIP and Ubc5 in HeLa cells. Aggregates of αB-crystallins in HeLa cells were detected using confocal microscope. Cell viability was determined by MTS assay.
Over-expression of CHIP in HLEC increased the levels of ubiquitin conjugates and many cytoplasmic chaperones, but not ER chaperones. Among the cytoplasmic chaperones, mRNA level of αB-crystallin, Hsp70 and Hsc70 increased 3-fold, 6-fold and 2-fold respectively. Over-expression of CHIP in HLEC significantly reduced the cytotoxcity caused by L-canavaline, an amino acid analog which results in abnormal proteins. Over-expression of CHIP together with CHIP confers stronger protective effects than expression of CHIP alone. Furthermore, expression of CHIP and Ubc5 in HeLa cells reduced the number and size of perinuclear aggregates of R120G mutant αB-crystallin.
These data show that the UPP plays important role in coping with abnormal proteins. Enhancement of functions of the UPP via over-expression of CHIP or CHIP and Ubc5 may be an effective therapeutic approach to prevent protein aggregation and protein aggregation-associated diseases, such as cataract.
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