Mechanism-based Discovery of Ubiquitin Ligase Targets

University Of Washington, Seattle WA

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Abstract

DESCRIPTION (provided by applicant): Defects in the ubiquitination pathway have an impact on many fundamental cellular processes. A growing number of proteins known to be involved in the development of cancer are also found to be involved in the ubiquitination process, either as targets themselves or acting as one of the protein components responsible for ubiquitination. The covalent attachment of ubiquitin to a target protein requires the sequential action of three protein activities E1, E2, and E3. Although remarkable progress has been made in recent years to identify proteins with E2 and E3 activity, little is known concerning the active cellular pairings of E2's and E3's and even less is known about the specific cellular protein target(s) for a given E3. Currently available protocols for "discovering" protein targets are to make an educated guess and test for function. While such a strategy may identify some targets, completely unexpected targets will not be found by this approach. In this pilot study, we propose two novel strategies to discover the cellular ubiquitination targets of the breast cancer susceptibility protein, BRCA1, which has recently been found to have ubiquitin ligase (E3) activity. Our approaches are based on mechanistic and structural insights gained from characterizations of BRCA1 and other ubiquitin pathway components. We have recently found that all known inherited missense mutations within the RING domain of BRCA1 abrogate its ubiquitin ligase activity, implying that the loss of this activity has severe consequences in the development of breast and ovarian cancer. Therefore, identification of the cellular targets of BRCAl-mediated ubiquitination will be a significant breakthrough towards our understanding of the role of BRCA1 in breast cancer biology. In addition, our mechanism-based strategy may be adapted to other ubiquitin ligase systems, providing an approach for discovering novel associations involved in ubiquitination processes.