Design of a Flexible Cell-Based Assay for the Evaluation of Heat Shock Protein 70 Expression Modulators
Design of a Flexible Cell-Based Assay for the Evaluation of Heat Shock Protein 70 Expression Modulators
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DOI：
10.1089/adt.2010.0327
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发表时间：
2011-06-01
影响因子：
1.8
通讯作者：
Chiosis, Gabriela
中科院分区：
文献类型：
--
作者：
Ahn, James H.;Luo, Wenjie;Chiosis, Gabriela
热休克蛋白70 （Hsp70）是一种伴侣蛋白，有助于防止细胞应激，这种功能可能被用来对抗人类疾病。特别是，上调Hsp70可以抑制错误折叠蛋白的神经毒性，提示神经退行性疾病可能的治疗策略。另外，在高水平的Hsp70抑制内在和外在凋亡途径的癌细胞中，Hsp70水平的降低可能会诱导细胞凋亡。为了评估和鉴定诱导或抑制内源性Hsp70的小分子，我们设计并优化了一种依赖于Hsp70全细胞免疫检测的微滴度测定方法。该试验利用了最少数量的神经元或癌细胞，但具有足够的灵敏度和可重复性，可以进行定量测定。我们使用一组Hsp70调节剂进一步验证了该实验。总之，我们已经开发出一种快速、稳健、经济高效的检测方法。因此，它可以在大多数研究实验室实施。该试验将大大提高新的Hsp70诱导剂和表达抑制剂的鉴定和评估速度。
Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.