CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.
CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.
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CHIP 敲低降低了晶状体上皮细胞的热激反应和蛋白质质量控制能力。
DOI：
10.2174/1566524015666150831131636
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发表时间：
2015-07
影响因子：
2.5
通讯作者：
M. Wu
中科院分区：
文献类型：
--
作者：
Y. Qin;A. Taylor;F. Shang;M. Wu
蛋白质质量控制（PQC）系统，包括分子伴侣和泛素-蛋白酶体途径（UPP），在维持细胞内蛋白质稳态中起着重要作用。Hsc 70相互作用蛋白（CHIP）的羧基端连接分子伴侣和UPP，从而有助于修复或去除受损蛋白。CHIP的过表达以前曾被用于保护细胞免受环境胁迫。为了获得CHIP所赋予的优势的更多生理机制，我们诱导了CHIP敲低并监测细胞科普环境应激的能力。为了敲低CHIP，用编码CHIP短发夹RNA（shRNA）的慢病毒颗粒或阴性对照慢病毒颗粒转染人透镜上皮细胞系HLE B3。用嘌呤霉素选择稳定的CHIP敲低细胞（KD）和阴性对照细胞（NC）。热休克应激后，Hsp 90的表达没有观察到变化。与此相反，热休克蛋白70的水平显着增加NC细胞，但在KD细胞较少。热休克后Hsp 27水平也增加，但仅在NC细胞。当CHIP被敲低时，蛋白质泛素化减少。CHIP敲低降低了清除聚集蛋白的能力。当表达相同水平的易于聚集的RFP突变晶体蛋白融合蛋白RFP/V76 D-γD时，KD细胞中的聚集体比NC细胞中观察到的聚集体多约9倍。此外，KD细胞对氨基酸类似物刀豆氨酸的毒性比NC细胞更敏感。总之，这些数据表明CHIP是PQC所需的，并且CHIP敲低降低了透镜细胞中的细胞PQC能力。
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.