title: CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.
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.
authors: W
authors: W
authors: Zhang
authors: Z
authors: Z
authors: Liu
authors: X
authors: X
authors: Bao
authors: Y
authors: Y
authors: Qin
authors: A
authors: A
authors: Taylor
authors: F
authors: F
authors: Shang
authors: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, China. wumingx@mail.sysu.edu.cn.
authors: M
authors: M
authors: Wu
mesh_terms: D002172:Canavanine / Q000494:pharmacology; D002460:Cell Line; D003459:Crystallins / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D018869:Heat-Shock Response; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D059748:Proteolysis; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D003459:Crystallins; D002172:Canavanine; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases