Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): chemiluminescence-based biochemical western blot assay for transcriptional activators of heat shock protein Hsp70
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): chemiluminescence-based biochemical western blot assay for transcriptional activators of heat shock protein Hsp70. ..more
BioActive Compound: 1
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_WESTERN BLOT ASSAY
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): chemiluminescence-based biochemical western blot assay for transcriptional activators of heat shock protein 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, Hsp 40, Hsp27, chaperone, agonist, activator, Western blot, immunoblot, protein, primary antibody, secondary antibody, HeLa cells, SDS-PAGE, gel electrophoresis, chemiluminescence, counterscreen, late stage, late stage AID, assay provider, powders, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
The purpose of this assay is to determine whether powder sample of a test compound identified as transcriptional activators of heat shock protein 70 (Hsp70) induces a change in the protein level of Hsp70. In this assay, HeLa cells are incubated with test compound, following by harvesting and western blot analysis of the reaction products using standard western blotting techniques. As designed, test compounds that induce a change in protein expression will result in a change in protein signal (compared to DMSO control) detected on the Western blot. Compound was tested at 10 uM.
HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with phenol red buffered with HEPES and supplemented with 10% v/v fetal bovine serum (FBS), 1% L-glutamine, and 100 U/ml penicillin/streptomycin. The cells were treated with test compound (10 uM), the positive controls celastrol (3 uM) and MG132 (10 uM), or left in vehicle (DMSO). The cells were harvested 8 hours after compound addition for analysis of chaperone expression by western blot analysis. Cells were lysed in a buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.9), 25% v/v glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol and 2 mg/ml of complete protease inhibitor cocktail for 30 minutes on ice. 15 ug of whole cell extracts were run on 7.5% SDS-PAGE gels and transferred to nitrocellulose. Primary antibody incubations were for 12 hours at 4 C in 10% BSA. The following primary antibodies were used: a mouse monoclonal Hsp70 antibody, a mouse monoclonal Hsp40 (alphaHdj-1 clone 25), and a mouse monoclonal Hsp27. All primary antibodies were used at a dilution of 1:10,000, except for the Hsp27 antibody, which was diluted 1:500. The anti-beta-tubulin antibody was diluted 1:5,000 and used to verify equal protein loading. The secondary antibody was an Alexa Fluor 680 goat anti mouse IgG diluted 1:5,000. Western analysis was performed with the Odyssey system. Protein expression levels were normalized to tubulin and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ on an Odyssey LI-COR imaging system.
PubChem Activity Outcome and Score:
Compounds that induced a minimum of 1.5-fold change in protein expression compared to the DMSO levels (set at 1.0) were active in this assay.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of reagents:
HeLa cells (supplied by Assay Provider)
Complete protease inhibitor cocktail (Roche, part 04 693 116 001)
Mouse monoclonal Hsp70 antibody (Affinity Bioreagents Inc., part 4g4)
Mouse monoclonal Hsp40 (supplied by Assay Provider)
Mouse monoclonal Hsp27 (Affinity Bioreagents, Inc., part MA3-0015)
anti-beta-tubulin antibody (Sigma, part 78328)
Alexa Fluor 680 goat anti-mouse IgG (Invitrogen, part A-21058)
This assay was performed by the assay provider with powder samples of purchased test compounds.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)