Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin.
Abstract
Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser1...Continue Reading
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