WWOX (WW domain-containing oxidoreductase) is a candidate tumor suppressor gene that has been shown to be involved in various cancers including breast, lung, prostate, gastric and hepatic. The Drosophila ortholog Wwox was identified and subjected to targeted ‘loss of function’ mutagenesis. The resulting mutants were found to be viable when homozygous with no obvious defects in the adult fly. As Wwox mutant flies were found to exhibit an increased sensitivity to ionising radiation (IR), a number of Wwox proteins specifically deleted or mutated at positions consisting of conserved functional protein motifs, or regions that are highly conserved among WWOX / Wwox homologs. The Wwox variants were tested for their ability to modify the IR sensitivity phenotype. In the course of this study, it was found that background mutations introduced during the generation of the mutant flies was responsible for the IR sensitivity phenotype. As a result, proteomic alterations resulting from changes in Wwox protein levels in Drosophila were investigated in order to ascertain the possible molecular functions of the Wwox protein. 2D-DIGE analysis was conducted on a number of different fly genotypes expressing differing levels of Wwox protein in both adult and embryonic flies. The proteomic changes resulting from lack of Wwox function as well as Wwox over-expression were detected with the proteins of interest identified by mass spectrometry (MS) using both MALDI-TOF/TOF-MS and LC-ESI-MS/MS. Label free quantitative MS analysis was also performed in order to determine the most abundant protein(s) in those spots found to contain multiple proteins. These proteomic studies identified changes in a wide variety of proteins with a significant number of metabolic proteins as well as proteins involved in oxidative stress response as a result of different levels of Wwox expression. Of particular interest, consistent changes in different isoforms of superoxide dismutase 1 (Sod1) were identified. Due to the known roles these proteins play in pro and anti-apoptotic pathways, it is possible that Sod1 and Wwox may work in concert to regulate the delicate balance of defence mechanisms in response to environmental stresses, particularly oxidative stress. The protein/gene targets identified in this work therefore offer some insights into normal Wwox function. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008
| Identifer | oai:union.ndltd.org:ADTP/264692 |
| Date | January 2008 |
| Creators | Colella, Alexander |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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