Abstract
Purpose:
We have already known that CHIP is a link between the chaperone and proteasome systems. The objective of this study was to investigate the effects of CHIP on the lens protein quality control system.
Methods:
A human lens epithelial cell line, HLE B3, was infected with lentivirus particles encoding CHIP short hairpin RNA (shRNA) (knock down, KD). The effects of CHIP were compared in the following groups: the CHIP-knock down (stably knocked-down), the rescue group (knocked-down cells transfected with plasmid expressing CHIP), the negative control (infected with lentivirus without target shRNA fragment, NC) and the blank (HLE B3 without infection, B3). Cells were subjected to a heat shock in incubator at 42-degree for 2h, then changes of levels of heat shock proteins(Hsp), such as Hsp27, Hsp70 and Hsp90, were determined with Western blotting analyses. Four mutant crystallins, R120G-αB, D140N-αB, T5P-γC, V76D-γD were transfected into cells with plasmid, and the percentage of aggresomes formation were observed with fluorescent microscopy.
Results:
When CHIP was knocked down, the expression of Hsp27 increased, however, Hsp70 and Hsp90 decreased. After heat shock, Hsp70 increased in B3 and NC, but decreased in KD, the same as Hsp90. The percentage of cells with mutant crystallin aggregates increased when CHIP was knocked down. Rescue of CHIP partially reversed the above-mentioned effects.
Conclusions:
Down regulation of CHIP affects both of the degradation and the refolding of abnormal proteins in lens epithelial cells. This suggested that CHIP plays an important role in lens protein quality control systems.
Keywords: 445 cataract •
450 chaperones •
662 proteolysis