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| Author(s): |
Patricia Ribeiro de Moura
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2005-06-28 |
| Examining board members: |
Jörg Kobarg;
Debora Foguel;
Charlotte Marianna Harsi;
Beatriz Gomes Guimarães;
Gonçalo Amarante Guimarães Pereira
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| Advisor: | Jörg Kobarg |
| Abstract | |
Chronic infection of the hepatitis B virus (HBV) is one of the causes leading to liver cancer. The HBV genome encodes the 17 kDa onco-protein HBx, a multi-functional protein that contains 10 cysteine residues and is related to induce liver cancer in transgenic mice. The exact function of HBx is still unknown. However, it has been shown that HBx is a potent trans-activator, which activates transcription of many cellular and viral promoters indirecdy through protein-protein interactions, although it does not bind to double-stranded DNA direcdy. Besides, the HBx protein can affect viral replication and proliferation, and it interferes with cellular apoptosis and carcinogenesis. In this work, the recombinant HBx protein was expressed in E. coli as a GST or 6xHis fusion protein, and used in functional and structural assays. By Electrophoretic Mobility Shift Assay and UV cross-linking assays, it was observed that the HBx protein was able to bind to the "A", "V" and "AV" rich RNA oligonucleotides, and that the cysteine residues of the HBx protein were not required for its binding to the AV-rich RNA oligonucleotide (AV-38). The lack of cysteine residues in the HBx protein did not interfere with the pS3 promoter activation in the yeast one-hybrid system, or neither in the in vitro interaction through a co-precipitation assay of the HBx and human p53 protein. In HeLa cells, the HBx protein increased the cellular growth and caused a slight c-fos and c-myc mRNA stabilization. This mRNA stabilization could contribute for the transforming character of the onco-protein HBx. Both the circular dichroism and fluorescence spectroscopic assays had shown that the HBx protein was partially structured in aqueous solution, but the protein presented a propensity to gain secondary structure under specific experimental conditions. The HBx inherent conformational flexibility might explain its interaction with a wide array of cellular proteins and single-stranded nucleic acids, in a way that the HBx protein interferes with signaling cellular processes, modulates transcription, apoptosis and DNA repair, and contributes to the development of the liver cancer (AU) | |