Investigation of the andrographolide-induced downregulation of Hsp90 client protein, Bcr-Abl
| Autor: | Jie-Ling Lee, 李婕綾 |
|---|---|
| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 Andrographolide (ANDRO) is a natural product of diterpenoid lactone, which is isolated from the leaves of Andrographis paniculata. Professor Shu-Ling Fu (NYMU) previously reported that a cleaved fragment of Hsp90 was identified in ANDRO-treated cells by a proteomics approach. In addition, Fu’s lab also reported that ANDRO induced Hsp90 cleavage and decreased levels of Bcr-Abl in K562 cell, a cell line derived from patients with chronic myelogenous leukemia. A previous proteomics research previously reported that Hsp90 was a targeting protein of ANDRO. We speculated that ANDRO promoted the Bcr-Abl degradation and hence suppressed cancer cell proliferation via forming chemical bonds with Hsp90. In order to identify the target proteins of ANDRO, we have collaborated with Professor Lee-Chiang Lo (NTU) to design a fluorescence-based derivative of ANDRO (ANDRO-NBD), which possesses an acetoxy group at C-14 and a nitrobenzoxadiazole (NBD) fluorophore at C-19 hydroxyl group. The fluorescent signal of ANDRO-NBD was detected at the position of p50 on SDS-PAGE gel, and confirmed that ANDRO-NBD covalently labels with a known protein target of ANDRO. In addition, the heating-mediated decrease of fluorescent signal of ANDRO-NBD on target proteins was due to the loss of protein conformation. Furthermore, we confirmed that Hsp90 is a target protein of ANDRO-NBD, and ANDRO-NBD exhibits a stronger activity of protein targeting than ANDRO. Previous studies indicated that ANDRO affects NF-κB function through direct labeling with cysteine residue of p50. I also confirmed that ANDRO-NBD labels cysteine as ANDRO in target proteins. The ANDRO-labeled peptide fragment containing Cys572 of Hsp90 was identified by LC-MS/MS. In addition, the ANDRO-NBD-labeled peptide fragments containing Cys572 and Cys529 were also identified, and I further found that ANDRO-NBD induces crosslinking of two Hsp90 peptides, TKFENLC572K and DYC481TR. Our results suggested that ANDRO/ANDRO-NBD inhibited Hsp90 activity by binding to a cysteine residue. Furthermore, ANDRO-NBD can induce crosslinking of different hsp90 cysteines via 2nd labeling. Moreover, we found that ANDRO-induced Bcr-Abl downregulation was slightly recovered in the cell constitutively expressing Hsp90 C572S in K562 cells. Therefore, this finding cannot jump to a conclusion. In summary, this thesis identified the targeting site(s) of Hsp90 inhibition by ANDRO and ANDRO-NBD. In addition, ANDRO-NBD also induces crosslinking of different cysteines, which may result in conformational change of Hsp90 and the disruption of interaction between Hsp90 and its client proteins. These findings provide a possible mechanism explaining why ANDRO-NBD exhibits better anti-cancer effect than ANDRO. In the future, development of ANDRO analogs, with higher labeling to Cys572 of Hsp90 protein, may exhibit higher inhibitory effect on Hsp90 and merit further development. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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