Abstract
Purpose: :
Accumulation and precipitation of abnormal proteins is associated with many age-related diseases. To maintain cell function and homeostasis, organisms evolved protein quality control mechanisms, including the ubiquitin-proteasome pathway (UPP) which selectively degrades damaged proteins, and molecular chaperones which refolds misfolded proteins. The objective of this study is to determine the effects of over-expression of C-terminus of Hsp70 interacting protein (CHIP), a co-chaperone with ubiquitin ligase activity, on the expression of molecular chaperones, UPP function and cytoprotection in human lens epithelial cells (HLEC).
Methods: :
CHIP 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. To mimic physiological and pathological stress, HLECs were exposed to H2O2 or L-canavanine. Cell viability was determined by MTS assay. Proteasome activity was monitored by using fluorogenic peptides as substrates. Levels of ubiquitin conjugates were determined by Western blotting.
Results: :
Over-expression of CHIP in HLEC increased the expression of heat-shock proteins, but not ER chaperones. Among the heat-shock proteins, mRNA levels of B-crystallin, Hsp70 and Hsc70 increased 3-fold, 6-fold and 2-fold respectively. Over-expression of CHIP significantly increased the protein level of Hsp70. Exposure of HLEC s to 40 µM H2O2 for 3 hours resulted in an ~50% reduction in chymotrypsin-like activity of the proteasome. Over-expression of CHIP partially protected the proteasome from this oxidative inactivation. Over-expression of CHIP increased the ubiquitin conjugating activity and cell viability under conditions of physiological and pathological stress.
Conclusions: :
These data show that over-expression of CHIP leads to the up-regulation of heat shock proteins and protects the proteasome from oxidative inactivation. The data also provide further insight into the functional relationship between molecular chaperones and UPP in protein quality control. CHIP might be a therapeutic target to prevent protein aggregation and its associated diseases, such as cataract.
Keywords: chaperones • proteolysis • cell survival