The breast and ovarian cancer tumor suppressor protein, BRCA1, supports a number of fundamental cellular processes. BRCA1 is required for normal proliferation during embryogenesis: in both mice and frogs, its absence results in early lethality. BRCA1 protein levels are up-regulated in proliferating breast epithelial cells, for example, during puberty, pregnancy, and lactation and loss of BRCA1 function in proliferating breast or ovarian epithelial cells has been shown to result in tumorigenesis. Inheritance of one mutant allele of BRCA1 increases a woman's risk of developing breast cancer from 1 in 8 to greater than 1 in 2. Although these observations, and many others, clearly point to one or more critical functions for BRCA1, we are still far from a biochemical understanding of what this key protein does. More than 50 proteins have been identified as BRCA1-binding proteins to date. Its multiple interactions imply that BRCA1 lies at the convergence of many cellular pathways. Our experimental plan recognizes the necessity of understanding the factors that govern the assembly of functional macromolecular complexes. The overall goals of the project are 1) elucidate the structural determinants of the recently described ubiquitin ligase activity of the heterodimeric BRCA1/BARD1 RING complex, 2) characterize functional and mechanistic aspects of the BRCA1/BARD1 ubiquitin ligase, including the identification of cellular targets of BRCA1-dependent ubiquitnation, 3) characterize the interaction between the N-terminal region of BRCA1 and two of its binding partners, BAP1, a ubiquitin hydrolase, and the estrogen receptor, ER-alpha, and 4) characterize protein complexes involving the C-terminal end of BRCA1, the BRCT. A combined biochemical and structural biological approach is proposed, involving techniques including multidimensional NMR, proteolytic mapping by MALDI-MS, yeast two-hybrid screens, Xenopus cell extract biochemistry, and others. In all aspects of the project, we will compare the properties and structures of wild-type domains with those harboring known cancer-predisposing mutations. The resulting description of the biochemical and structural properties of functional domains and complexes of BRCA1 will contribute significantly to the challenging task of discovering and understanding both the normal and pathological roles of BRCA1.
