Abstract
Toxic carbonyls such as acrolein participate in many degenerative diseases. Although the nucleophilic vasodilatory drug hydralazine readily traps such species under “test-tube” conditions, whether these reactions adequately explain its efficacy in animal models of carbonyl-mediated disease is uncertain. We have previously shown that hydralazine attacks carbonyl-adducted proteins in an “adduct-trapping” reaction that appears to take precedence over direct “carbonyl-sequestering” reactions, but how this reaction conferred cytoprotection was unclear. This study explored the possibility that by increasing the bulkiness of acrolein-adducted proteins, adduct-trapping might alter the redistribution of chaperones to damaged cytoskeletal proteins that are known targets for acrolein. Using A549 lung adenocarcinoma cells, the levels of chaperones heat shock protein (Hsp) 40, Hsp70, Hsp90, and Hsp110 were measured in intermediate filament extracts prepared after a 3-h exposure to acrolein. Exposure to acrolein alone modestly increased the levels of all four chaperones. Coexposure to hydralazine (10–100 μM) strongly suppressed cell ATP loss while producing strong adduct-trapping in intermediate filaments. Most strikingly, hydralazine selectively boosted the levels of cytoskeletal-associated Hsp90, including a high-mass species that was sensitive to the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin. Biochemical fractionation of acrolein- and hydralazine-treated cells revealed that hydralazine likely promoted Hsp90 migration from cytosol into other subcellular compartments. A role for Hsp90 mobilization in cytoprotection was confirmed by the finding that brief heat shock treatment suppressed acute acrolein toxicity in A549 cells. Taken together, these findings suggest that by increasing the steric bulk of carbonyl-adducted proteins, adduct-trapping drugs trigger the intracellular mobilization of the key molecular chaperone Hsp90.
Footnotes
This work was supported by the National Health and Medical Research Council of Australia [Grants 403967, 572580].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- IF
- intermediate filament
- Hsp
- heat shock protein
- PMSF
- phenylmethanesulfonyl fluoride
- 17-AAG
- 17-N-allylamino-17-demethoxygeldanamycin
- BPBS
- Dulbecco's phosphate-buffered saline
- FBS
- fetal bovine serum
- PBS
- phosphate-buffered saline
- PAGE
- polyacrylamide gel electrophoresis.
- Received March 28, 2012.
- Accepted August 3, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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