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. 2008 Oct;7(10):4384-95.
doi: 10.1021/pr800376w. Epub 2008 Aug 23.

Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin

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Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin

Shujia Dai et al. J Proteome Res. 2008 Oct.

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, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.

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Figures

Figure 1
Figure 1
Thiolutin selectively inhibits endothelial cell proliferation. (A) HUVEC (5000 cells/well) were plated on 96-well culture plates and incubated for 72 h in EGM + 2% FCS with the indicated treatment agents, Thiolutin, ADT (5-[p-methoxyphenyl]-3H-1,2-dithiol-3-thione), and D3T (1,2-dithiole 3-thione). Cell proliferation was assayed via the colorimetric change obtained after incubation with MTS reagent using a microplate reader at 490 nm. (B) Proliferation of breast carcinoma (MCF-7), prostate carcinoma (PC3), ovarian carcinoma (CaOV3), and small cell lung carcinoma (OH-1) cells was assessed in the presence of the indicated concentrations of thiolutin. Results are expressed as percentage of control and represent the mean ± SD of at least three separate experiments.
Figure 2
Figure 2
Thiolutin selectively stimulates Hsp27 phosphorylation in endothelial cells. (A) HUVEC were treated with 1 µM of thiolutin, ADT, and D3T for the time indicated. Western blots were performed with anti-Hsp27 phosphorylation specific antibodies as described in Materials and Methods. The membranes were then reprobed with antiactin antibody for sample loading control. (B) HUVEC were treated with thiolutin at 0.5 or 0.1 µM for the time indicated. Right panel: HUVEC were treated with the indicated concentrations of thiolutin for 60 min. Samples were collected for Western blotting using anti-pS78 Hsp27, antiactin, and anti-Hsp27 antibodies. (C) Indicated tumor cell lines were treated with 0.5 or 10 µM thiolutin for 30 min. Cell lysates were analyzed by Western blotting probed with anti-Hsp27, anti-pS78 Hsp27, and antiactin antibodies.
Figure 3
Figure 3
p38 is required for Hsp27 phosphorylation but not activated by thiolutin. (A) Selective activation of MAP kinases by thiolutin. HUVEC were grown in full growth media, switched to EGM + 2% FBS for 24 h, and then treated with the indicated concentrations of thiolutin in EGM + 0.1% BSA. Lysates were analyzed by Western blots probed with antiphospho-ERK, phos-pho-JNK, or phospho-p38 antibodies. (B) HUVEC were treated with (+) or without (−) 10 µM of JNK inhibitor (SP600125) for 20 min followed with (+) or without (−) 1 µM thiolutin for 1 h. Samples were collected for Western blotting using anti-pS78 Hsp27 and antiactin antibodies. (C) HUVEC were treated with the indicated concentrations of the p38 inhibitor (SB203680) for 20 min and then treated with (+) or without (−) thiolutin at 0.5 µM for 1 h. Samples were collected for Western blotting using anti-pS78 Hsp27 and antiactin antibodies.
Figure 4
Figure 4
(A) Workflow for the IP of Hsp27 from HUVEC. Each cell lysate (either treated with thiolutin at 1 µM for 60 min or without treatment) was IP by two different antibodies of HSP27, one against the backbone of the C-terminal end of Hsp27 and the other against phosphorylation at pS78 region. A total of four IPs were obtained for subsequent LC–MS analysis. (B) Workflow for LC–MS analysis. Each IP was first digested by Lys-C, and then one-third of the sample was further digested by trypsin and the other one thirds by Glu-C. Each digest (Lys-C plus trypsin or Lys-C plus Glu-C or Lys-C only) was subsequently analyzed by online LC–MS analysis.
Figure 5
Figure 5
Summary of Hsp27 Peptides Identified by LC–MS Analysis. (A) The Hsp 27 peptides were identified from a Lys-C plus trypsin digested sample with the sequence coverage of 89.3% (amino acids in bold). (B) The Hsp 27 peptides were identified from the Lys-C plus Glu-C digested sample with the sequence coverage of 69.3% (amino acids in bold). The total sequence coverage was 96.1% when combining the identified peptides from the both digests. Fragments that contain the phosphorylation sites (S* in the figure) are underlined.
Figure 6
Figure 6
Identification of acetylated species of the Lys-C fragment 2–112 of Hsp27 by LC–MS analysis. The base ion chromatogram of the Lys-C digest (A), the FTICR measurement of the precursor ion at 37.14 min with the ion of the highest charge state (14+ and m/z 907.9143) shown in the insert (B), the CID-MS2 product ions of the precursor ion (m/z 907.9143) using the FTICR measurement with the highest product ion (y66, 7+) shown in the insert (C).
Figure 7
Figure 7
Identification of the Glu-C fragment 65–87 of Hsp27 with pS82 by LC–MS analysis. The base ion chromatogram of the Lys-C plus Glu-C digest (A), the FTICR measurement of the precursor ion at 43.43 min (B), the CID-MS2 product ions of the precursor ion (m/z 795.7603) at 43.43 min (C), and the CID-MS3 product ions of the neutral loss ion (m/z 966.7) generated from the previous CID-MS2 fragmentation (D).
Figure 8
Figure 8
Extracted ion chromatograms (XIC) of phosphorylated isoforms of the Glu-C fragment 65–87 of Hsp27. (A) Base peak XIC of the Glu-C fragment 65–87 of Hsp27 with monophosphorylation. The phosphorylated Hsp27, IP from the antibody against the C-terminal end, was indicated in the insert of (A). The extracted ion chromatogram (m/z 966.7) was isolated from the CID-MS3 fragmentation, which was generated from the fragmentation of the ion (m/z 1015.6), and the 1015.6 ion was from the CID-MS2 of the precursor ion (m/z 795.7). (B) Base peak XIC of the Glu-C fragment 65–87 of Hsp27 with monophosphorylation. The phosphorylated Hsp27, IP from the antibody against the pS78 region, was indicated in the insert of (B). The extracted ion chromatogram (m/z 966.7) was isolated from the same procedure as in (A).

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