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Published on: July 17, 2020
Design of a flexible cell-based assay for the evaluation of heat shock protein 70 expression modulators
James H Ahn1, Wenjie Luo, Joungnam Kim
1Department of Medicine and Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York.
Abstract:
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.
Insights
A new microtiter assay enables rapid identification of small molecules that modulate Heat Shock Protein 70 (Hsp70). This tool aids research into neurodegenerative diseases and cancer by evaluating Hsp70
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- Heat shock protein 70 (Hsp70) is a crucial chaperone protein involved in cellular stress response.
- Hsp70's role in disease includes suppressing neurotoxicity in neurodegenerative conditions and inhibiting apoptosis in cancer.
- Targeting Hsp70 offers potential therapeutic strategies for both neurodegenerative diseases and cancer.
Purpose of the Study:
- To develop and optimize a single, high-throughput assay for identifying small molecules that modulate endogenous Heat Shock Protein 70 (Hsp70) levels.
- To create a cost-effective and robust method for evaluating Hsp70 inducers and inhibitors.
Main Methods:
- A microtiter assay format was designed and optimized using whole-cell immunodetection of Hsp70.
- The assay utilizes a minimal number of neuronal or cancer cells for quantitative determinations.
- Assay performance was validated using a panel of known Hsp70 modulators.
Main Results:
- The developed assay is sensitive, reproducible, and capable of quantitative measurements.
- The assay format is efficient, requiring minimal cell numbers.
- Validation confirmed the assay's reliability in identifying Hsp70 modulators.
Conclusions:
- A fast, robust, and cost-efficient assay for Hsp70 modulation has been successfully developed.
- This assay can be readily implemented in most research laboratories.
- The assay is expected to significantly accelerate the discovery and evaluation of novel Hsp70-targeting therapeutics.
