摘要
蛋白质的质量控制(PQC)系统包括分子伴侣和泛素-蛋白酶体通路(UPP),它在维持细胞内蛋白质的平衡起着重要的作用。热休克相互作用蛋白羧基末端(CHIP)与UPP相连接,从而有助于修复或去除损坏的蛋白质。CHIP的过度表达常被用来保护细胞免受环境压力。为了获得更多CHIP所赋予的生理机制优势,我们对CHIP进行诱导抑制并监测细胞应对环境压力的能力。为了抑制CHIP,用编码短发夹RNA(shRNA)的慢病毒颗粒感染人晶状体上皮细胞系HLE B3或者设置慢病毒颗粒阴性对照组。利用嘌呤霉素对稳定的CHIP抑制细胞(KD)细胞和阴性对照组组细胞(NC)进行筛选。热应激处理后,HSP90的表达并没有发生改变。相比之下,NC细胞的HSP70水平显著提高但低于KD细胞。热应激后,只有NC细胞中HSP27水平有所增加。当CHIP所受到抑制后,蛋白质泛素化降低,清理聚合蛋白的能力降低。当同一水平的具有聚集倾向的RFP突变体蛋白的融合蛋白RFP/V76D-γD得到表达时,KD细胞存在9倍于NC细胞的聚集体,此外,KD细胞对氨基酸模拟刀豆氨酸的毒性更敏感。这些数据都表明CHIP抑制对蛋白质的质量控制(PQC十分重要)并且CHIP抑制能减弱晶状体细胞PQC能力。
关键词: CHIP,晶状体蛋白,热休克蛋白,晶状体上皮细胞,压力,泛素-蛋白酶通路
Current Molecular Medicine
Title:CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells
Volume: 15 Issue: 7
Author(s): W. Zhang, Z. Liu, X. Bao, Y. Qin, A. Taylor, F. Shang and M. Wu
Affiliation:
关键词: CHIP,晶状体蛋白,热休克蛋白,晶状体上皮细胞,压力,泛素-蛋白酶通路
摘要: 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.
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Cite this article as:
W. Zhang, Z. Liu, X. Bao, Y. Qin, A. Taylor, F. Shang and M. Wu , CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells, Current Molecular Medicine 2015; 15 (7) . https://dx.doi.org/10.2174/1566524015666150831131636
DOI https://dx.doi.org/10.2174/1566524015666150831131636 |
Print ISSN 1566-5240 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5666 |
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