|Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70) - BioAssay Summary
Name: Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70) ..more
BioActive Compounds: 82
Depositor Specified Assays
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Richard Morimoto, Northwestern University
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 5 R21 NS056337-02
Grant Proposal PI: Richard Morimoto
External Assay ID: HSP70_AG_Lumi_1536_% Act
Name: Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)
The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival (1, 2). Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.
A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response (3, 4). Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells (5). Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins (6-8). High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.
1. Gupta, R.S., and Singh, B. 1994. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. Curr Biol 4:1104-1114.
2. Lindquist, S., and Craig, E.A. 1988. The heat-shock proteins. Annu Rev Genet 22:631-677.
3. Satyal, S.H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J.M., and Morimoto, R.I. 2000. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 97:5750-5755.
4. Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J., and Rubinsztein, D.C. 2000. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci U S A 97:2898-2903.
5. Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., and Zoghbi, H.Y. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148-154.
6. Krobitsch, S., and Lindquist, S. 2000. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A 97:1589-1594.
7. Kazemi-Esfarjani, P., and Benzer, S. 2000. Genetic suppression of polyglutamine toxicity in Drosophila. Science 287:1837-1840.
8. Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L., and Bonini, N.M. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat Genet 23:425-428.
Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, chaperone, agonist, activator, HTS, high throughput screen, primary screen, 1536, reporter gene, transcription, luciferase, luminescence, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
The purpose of this assay is to determine the ability of compounds from the MLSCN library to act as activators of Hsp70 expression. Induction of the heat shock response by test compound is measured in a Hela cell line stably expressing a luciferase reporter under control of the human Hsp70 promoter. As designed, a compound that acts as an activator of Hsp70 expression will activate the Hsp70 promoter, which will increase luciferase transcription, and thus increase well luminescence as detected with the appropriate substrate. Compounds were tested in singlicate at a final nominal concentration of 10 micromolar.
The hsp70.1pr-luc HeLa cell line was grown in tissue culture flasks in Dulbecco's Modified Eagle's Media supplemented with 10% v/v fetal bovine serum, 1% pen-strep-neomycin antibiotic mixture and 1% Geneticin at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH).
Prior to the start of the assay, cells were resuspended in growth media as above at a concentration of 750,000 cells/mL. Next, 5 ul of well-mixed cell suspension were dispensed into each well of 1536-well plates (3,750 cells per well). After incubation for 4 hours at 37 degrees C, 5% CO2 and 95% (RH), the assay was started by dispensing 50 nL of test compound in DMSO to sample wells, DMSO alone (1% final concentration) to negative control wells, or MG132 (final nominal EC100 concentration of 30 uM, set as 100% activation) to positive control wells. The plates were then incubated for 16 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Luminescence was measured on the ViewLux plate reader.
The percent activity was defined using the following mathematical formula:
% Activity = 100*((Test_Compound - Median_Low_Control) / (Median_High_Control - Median_Low_Control))
Test_Compound is defined as wells containing test compound
High_Control is defined as wells containing MG132
and Low_Control is define as wells containing DMSO
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
List of reagents:
Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092)
Fetal Bovine Serum (Hyclone, part SH 30088.03)
Geneticin (Invitrogen, part 10131-027)
Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)
1536-well plates (Greiner, part 789173)
T175 HYPERflasks (Corning, part 10010)
Reference agonist MG132 (American Peptide, part 81-5-15)
Due to the increasing size of the MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate luciferase activity directly, and compounds that quench or emit luminescence in the well. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)