Pharmacology, Pharmacy and Anaesthesiology Unit, School of Medicine and Pharmacology, University of Western Australia, Nedlands, Australia.
Mol Pharmacol. 2012 Nov;82(5):876-86. doi: 10.1124/mol.112.078956. Epub 2012 Aug 6.
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.
有毒的羰基化合物，如丙烯醛，参与许多退行性疾病。虽然亲核血管舒张药物肼屈嗪很容易在“试管”条件下捕获这些物质，但这些反应是否能充分解释其在羰基介导疾病的动物模型中的疗效尚不确定。我们之前已经表明，肼屈嗪在“加合物捕获”反应中攻击羰基加合物修饰的蛋白质，这种反应似乎优先于直接的“羰基隔离”反应，但这种反应如何提供细胞保护尚不清楚。本研究探讨了这样一种可能性，即通过增加丙烯醛加合物修饰蛋白的体积，加合物捕获可能改变伴侣蛋白向已知是丙烯醛靶标的受损细胞骨架蛋白的重新分布。使用 A549 肺腺癌细胞，在暴露于丙烯醛 3 小时后，从中间丝提取物中测量热休克蛋白 (Hsp) 40、Hsp70、Hsp90 和 Hsp110 等伴侣蛋白的水平。单独暴露于丙烯醛会适度增加所有四种伴侣蛋白的水平。同时暴露于肼屈嗪（10-100 μM）强烈抑制细胞 ATP 丢失，同时在中间丝中产生强烈的加合物捕获。最引人注目的是，肼屈嗪选择性地提高了与细胞骨架相关的 Hsp90 的水平，包括一种对 Hsp90 抑制剂 17-N-烯丙基-17-去甲氧基格尔德霉素敏感的高分子量物质。丙烯醛和肼屈嗪处理细胞的生化分级分离显示，肼屈嗪可能促进 Hsp90 从细胞质迁移到其他亚细胞隔室。短暂的热休克处理抑制 A549 细胞急性丙烯醛毒性的发现证实了 Hsp90 动员在细胞保护中的作用。总之，这些发现表明，通过增加羰基加合物修饰蛋白的空间位阻，加合物捕获药物触发关键分子伴侣 Hsp90 的细胞内动员。
