Identifying substrate and E2 interactions of the BRCA1/BARD1 ubiquitin ligase
Christensen, Devin Eugene
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My thesis project addresses the need to understand the function of the Breast Cancer Susceptibility Protein, BRCA1, during the process of protein ubiquitination. A significant number of known cancer predisposing mutations are found within BRCA1's RING domain which, when associated with BARD1, functions as a ubiquitin ligase. Cancer associated RING mutations of BRCA1 eliminate ligase activity in vitro. Substrates of the BRCA1/BARD1 ubiquitin ligase are likely to be involved in important cellular processes related to the function of BRCA1 in tumor suppression, and provide a direct link between BRCA1's ligase function and the development of cancer.A ubiquitin ligase interacts with both a substrate and a ubiquitin conjugating enzyme (E2) to mediate the transfer of ubiquitin from the E2 to the substrate. To properly identify substrates, the E2s that interact and function with BRCA1 must also be determined. Using a "structure-based" yeast two-hybrid strategy, six new BRCA1-E2 interactions were identified for a total of ten E2s that bind to the BRCA1 RING. I found that for BRCA1 the E2 determines the type of ubiquitination product: depending on the E2 present during a ubiquitination assay different ubiquitination products are synthesized on a protein substrate including, mono-ubiquitin, Lys6-, Lys48-, or Lys63-linked poly-ubiquitin chains.I have identified two proteins that interact with BARD1, BABS and ZO-2. I also discovered that BABS is ubiquitinated by BRCA1/BARD1, in vitro , using a subset of the BRCA1-interacting E2s. Ubiquitin transfer is dictated by substrate-E2 interactions, as E2s that transfer ubiquitin to BABS share the common ability to interact with BABS. Though not tested as a substrate, ZO-2 may provide a link between BRCA1 and the tissue specific nature of cancer associated mutations. ZO-2 is a suspected tumor suppressor that has been linked to estrogen signaling and breast tumorigenesis. The identification of BRCA1/BARD1 substrates, and the fate conferred upon them by the ubiquitin tag, will lead to a better understanding of the association between loss-of-function mutations in BRCA1 and breast cancer.
- Biological chemistry