Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess Hsp70 gene expression changes
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess Hsp70 gene expression changes. ..more
BioActive Compound: 1
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_FLUO_QPCR
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess Hsp70 gene expression changes.
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, QPCR, RTPCR, qRT-PCR, RNA, polymerase chain reaction, PCR, HeLa, celastrol, primers, cDNA, mRNA, counterscreen, fluorescence, 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 samples of a compound identified as transcriptional activators of heat shock protein 70 (Hsp70) modulates the gene expression of Hsp70 involved in the heat shock response and protein homeostasis. In this assay, HeLa cells are incubated with test compound and harvested. RNA is purified and subjected to quantitative reverse transcription (qRT-PCR) of Hsp70 and GAPDH (control). Gene expression is normalized to GAPDH and compared to levels in cells treated with DMSO only. As designed, test compounds that induce an increase in Hsp70 gene expression will result in an increase in amplified RNA product.
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, the positive controls celastrol (3 uM), CdCl2 (50 uM) and MG132 (10 uM), or left in vehicle (DMSO). The cells were harvested 4 hours after compound addition for analysis of chaperone expression by quantitative reverse transcription (qRT)-PCR. RNA was purified using the RNeasy Mini kit according to the manufacturer's instructions. After the reverse transcription reaction, PCR was performed using PCR primers specific for human Hsp70 and GAPDH. PCR products were amplified with Taq polymerase by using standard cycling conditions. Gene expression was normalized to GAPDH and compared to levels in cells treated with DMSO (mRNA levels set as 1.0) using ImageJ.
PubChem Activity Outcome and Score:
Compounds that induced a minimum of 1.5-fold induction in gene 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 (provided by Assay Provider)
RNeasy Mini kit (Qiagen, part 74106)
Taq polymerase (Promega, part M3001)
PCR primers were ordered as appropriate. The primer pair sequences for the human Hsp70 and GAPDH gene targets are indicated below:
Hsp70 forward: 5'-AGAGCCGAGCCGACAGAG-3'
Hsp70 reverse: 5'-CACCTTGCCGTGTTGGAA-3'
GAPDH forward: 5'-GTCGGAGTCAACGGATT-3'
GAPDH reverse: 5'-AAGCTTCCCGTTCTCAG-3'
This assay was performed by the assay provider with powder samples of purchased test compounds.
** Test Concentration.
Data Table (Concise)