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CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells

[ Vol. 15 , Issue. 7 ]

Author(s):

W. Zhang, Z. Liu, X. Bao, Y. Qin, A. Taylor, F. Shang and M. Wu   Pages 652 - 662 ( 11 )

Abstract:


Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells.

Keywords:

CHIP, crystallin, heat shock proteins, lens epithelial cell, stress, ubiquitin proteasome pathway.

Affiliation:

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, China.



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