A Proteomics Approach to Study Biological Activity of HE-145 and Protein-Protein Interaction of Rhodostomin
| Autor: | Jin-Yuan Ho, 何晉元 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 In recent years, proteomics has become a widely used research method in life sciences. There are two major techniques in this rapid approach to study proteins: two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). In this thesis, I will report my results in applying the proteomics approach to study the protein-protein interaction of rhodostomin (from snake venom) and biological activity of a natural product, HE-145. Rhodostomin was known to interact with the membrane proteins of platelet. Here, I applied proteomics approach and chemical cross-linking to study its protein-protein interaction and spatial arrangement of resulting protein complex. In the in vitro binding experiment with cell lysate of platelet, I have successfully identified integrin alphaIIb/beta3, actin, myosin, and gelsolin from rhodostomin-associated complex. Furthermore, when applying cross linkers with intact platelet, I found two additional proteins, thrombospondin and endothelial cell multimerin, that were previously reported to interact with integrin alphaIIb/beta3. With all these protein identifications, I intend to establish a protocol to illustrate the function of rhodostomin-associated complex. The second part of my thesis is to study the anti-HBV activity of HE-145. Hepatitis B virus (HBV) has a great propagation rate in Asia. From the epidemiological studies, chronic infection with HBV has high correlation with hepatocellular carcinoma (HCC) and related liver disease. HE-145 (helioxanthin) is a natural product and was identified from Taiwania cryptomeroides Hayata by Prof. Kuo in Department of Chemistry at NTU. During the screening experiments of antiviral activity by Prof. Yen in Institute of Biochemistry at NYMU, HE-145 was shown to inhibit HBsAg secretion and promote cell proliferation. In this thesis, I reported the utilization of 2DE to analyze the proteome change of human hepatoma cell HepA2 before/after the treatment of HE-145. The differentially expressed proteins were then characterized by mass spectrometry. There are 25 down-regulated proteins, 50 up-regulated proteins and 7 proteins changed their modification status. Among these proteins, we observed that the down-stream proteins of INF-gamma were up-regulated by HE-145 treatment. In addition, we also found that PPAR-alpha is down-regulated due to the treatment of helioxanthin. With all these information, these will provide us a lead to study the regulatory mechanism. By combining the linkage of these proteins and comprehensive literature search, we intend to establish a regulatory mechanism illustrate how HE-145 inhibits HBsAg expression. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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