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: DeltaF508-CFTR_ACT_FLUO

Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): yellow fluorescent protein (YFP) quenching assay to assess changes in trafficking of DeltaF508-CFTR.

Description:

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.


References:

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.

Keywords:

Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, chaperone, agonist, activator, cystic fibrosis, human bronchial epithelial cell line, epithelial cell line, cystic fibrosis transmembrane conductance regulator, CFTR, yellow fluorescent protein, YFP, halide, trafficking, proteostasis, proteostasis regulator, counterscreen, fluorescence, late stage, late stage AID, assay provider, powders, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.

Assay Overview:

The purpose of this assay is to determine whether a powder sample of a compound identified as a transcriptional activator of heat shock protein 70 (Hsp70) is active in the correction of defective trafficking in a cellular model of cystic fibrosis. This model corresponds to a human bronchial epithelial cell line (CFBE41o-) stably co-expressing the DeltaF508 alteration of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR) and a halide-sensing mutant of yellow fluorescent protein (YFPH148Q I152L). DeltaF508-CFTR is defective both in trafficking from the endoplasmic reticulum to the plasma membrane and in channel gating. As designed, correction of the defective trafficking of DeltaF508-CFTR with the proteostasis regulators would lead to an increased amount of active protein at the cell surface, allowing an increased flow of extracellular halides into the cell, which would result in a reduction, or quenching, of YFP fluorescence intensity.

Protocol Summary:

Human bronchial epithelial cells (CFBE41o-) stably expressing DeltaF508-CFTR as well as H148Q/I152L-YFP (CFBE41o- -YFP) were added to a 96-well black walled plate and grown to confluency in growth media (alpha-MEM containing 100 U/ml penicillin, 100 U/ml streptomycin, 10% v/v FBS, 2 mM L-glutamine, 2 ug/ml puromycin and 0.75 ug/ml G418). Cells were treated with the indicated concentration of compounds in complete growth media and incubated at 37 C, 5% CO2 for 24 hours. Cells were subsequently washed three times with 200 uL of PBS pH 7.4 (137 mM NaCl, 2.7 mM KCl, 0.7 mM CaCl2, 1.1 mM MgCl2, 1.5 mM KH2PO4, 8.1 mM Na2HPO4) and equilibrated in 40 uL of PBS pH 7.4 and maintained at 37 C throughout. Cells were stimulated with a final concentration of 10 uM forskolin (fsk) and 50 uM genistein (gen) for 15 minutes prior to addition of PBS + NaI (replacement of NaCl with 137 mM NaI). Fluorescence was monitored every second for a total of 30 seconds (3 seconds prior to addition of NaI and 27 seconds after addition of NaI). Data were normalized to the initial fluorescence to account for variations in the overall starting fluorescence. To ensure that the observed H148Q/I152L-YFP fluorescence decrease was the result of DeltaF508-CFTR activation and not the action of additional halide channels, the CFTR specific inhibitor (CFInh-172) was used. Each experimental condition was tested in duplicate.

The data were analyzed by subtracting the basal fluorescence value from an empty well from each time point. RFU were calculated by setting the fluorescence at time zero to 1 and expressing the fluorescence at each subsequent time point as a fraction of 1.

RFU = F_T=X / F_T=0

Where:

F_T=X = fluorescence at time X
F_T=0 = fluorescence at time 0

PubChem Activity Outcome and Score:

Compounds that resulted in a 15% quenching in fluorescence, or an RFU of 0.85, at any dose or time point, were considered active.

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:

Human bronchial epithelial cells (CFBE41o-) stably expressing DeltaF508-CFTR as well as H148Q/I152L-YFP (CFBE41o- -YFP) (provided by Assay Provider)
alpha-MEM (Invitrogen, 12714010S)
Penicillin Streptomycin (Invitrogen, 15640)
FBS (Invitrogen, 10082147)
L-glutamine (Invitrogen, 21051-024)
Puromycin (Invitrogen, A1113802)
G418 (Sigma A1720)
Forskolin (Invitrogen, B-7469)
Genistein (Sigma G6649)

This assay was performed by the assay provider with powder samples of purchased test compound.

Grant Number: 5 R21 NS056337-02