The product of the breast and ovarian cancer predisposition gene, BRCAI, is a large protein with two main recognised motifs; the N-terrninal RING and C-terrninal BRCT. The N-tenninus has been reported to interact with at least eight proteins (MSH2, ATFI, ERalpha, BAPI, p300, NPMlB23, UbcH5A and BARDI). In order to test the relevance of these interacting partners to the BRCAI tumour suppressor activity, we intended to test patient derived missense variants upon each interaction. We found that of the eight potential partners, only BARDI and the ubiquitin conjugating enzyme UbcH5A interacted with the BRCAI N-terrninal region, by yeast two-hybrid assays. The BRCAI/UbcH5A interaction was also confirmed in mammalian cells by FRET analysis. To determine }Vhether these interactions, or their abrogation, might fonn part of the BRCAI-mediated tumorigenesis pathway we undertook an extensive mutagenesis study. Our results show that all known pathogenic missense mutations and the majority ofBRCAI N-terrninal unclassified variants, found in populations enriched for family history ofdisease, weakened or abolished the BRCAI/UbcH5A interaction. Loss of ubiquitin ligase activity measured biochemically, and in cells, correlated with BRCAI/UbcH5A disruption. Finally, in silico prediction, based on evolutionary conservation and protein structural analysis supported and complemented our mutagenesis results. Overall, our data provide functional evidence that the BRCAI/UbcH5A interaction is highly sensitive to missense substitution and suggest that the ubiquitin pathway is likely to play an important role in BRCAI-mediated tumorigenesis. Even though we could not detect a direct interaction between BRCAI and MSH2, our study on MSH2-deficient primary human cells implicated MSH2 in the homologous recombination repair pathway.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:486355 |
| Date | January 2008 |
| Creators | Pangon, Laurent Luc Gilles |
| Publisher | King's College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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