| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 104 === Andrographis paniculata is an herbaceous plant and widely used in Europe and Asia to treat infection and other diseases. The major active constituent of Andrographis paniculatais is andrographolide (ANDRO) which exhibits multiple pharmacological activities, including the antimicrobial, anti-inflammatory, antioxidant, cardiovascular, antidiabetic and anticancer activities. Previous studies indicated that ANDRO affects NF-kB function through direct labeling with cysteine residue of p50. Our laboratory also discovered that ANDRO induced Hsp90 cleavage. Since Hsp90 is a molecular chaperone protein that maintains the structures and functions of its client proteins, including several oncoproteins in cancer cells, it is crucial to investigate where ANDRO labels Hsp90 and which mechanism such labeling affects Hsp90. In this study, the recombinant Hsp90-WT (wild type), Hsp90-M (middle domain) and Hsp90 mutants carrying different cysteine mutation were first prepared. Subsequently, fluorescent ANDRO derivative (ANDRO-NBD) and mass spectroscopic analysis were used to detect and measure the labeling of Hsp90 by ANDRO. Firstly, fluorescent intensities of Hsp90-WT upon ANDRO-NBD treatment were increased in a dose-dependent manner from 0.1µM to 10 µM, indicating that wild type Hsp90 is a target of ANDRO-NBD. In addition, the fluorescent signals were abolished by iodoacetamide (IAA) treatment, confirming that ANDRO-NBD labels Hsp90 through cysteine residues. Similarly, ANDRO-NBD labels middle domain of Hsp90 in the same way and tendency. Upon treatment of ANDRO-NBD, all Hsp90 mutants carrying cysteine mutation gave similar fluorescent signals, indicating that no cysteine exhibits specific preference for the labeling of ANDRO-NBD. Replacement of IAA with IAA derivatives containing stable isotopes (IAAh6 or IAAd6) showed that IAAh6 or IAAd6 is able to label cysteines of Hsp90 and prevent its labeling with ANDRO-NBD. The tendency of relative reactivity with Hsp90-WT is IAA > IAAd6> IAAh6, whereas the tendency of relative reactivity with Hsp90-M is IAA ≈ IAAd6> IAAh6. Finally, a technique using stable isotope labeling was proposed to analyze the Hsp90 proteins respectively labeled with IAAh6, IAAd6, ANDRO and ADNRO-NBD by LC-MS/MS spectroscopic analysis. Currently, results of MS analysis were still unable to reveal the direct labeling status of ANDRO-NBD on Hsp90 and extensive investigation is suggested.
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