Structural and Functional Characterization of BRCA1

Summary

Principal Investigator: Rachel Klevit
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 1998-12-21 - 2009-04-30
more information: NIH RePORT

Top Publications

  1. pmc Estrogen receptor alpha is a putative substrate for the BRCA1 ubiquitin ligase
    Catherine M Eakin
    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
    Proc Natl Acad Sci U S A 104:5794-9. 2007
  2. ncbi Structural Biology: Parkin's Serpentine Shape Revealed in the Year of the Snake
    Katja K Dove
    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
    Curr Biol 23:R691-3. 2013
  3. pmc Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination
    Devin E Christensen
    Department of Biochemistry, University of Utah, Salt Lake City, UT 98195, USA
    FEBS J 276:5381-9. 2009
  4. pmc Structural and functional characterization of the monomeric U-box domain from E4B
    Kyle A Nordquist
    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA
    Biochemistry 49:347-55. 2010
  5. ncbi A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination
    Peter S Brzovic
    Department of Biochemistry, University of Washington, Seattle, 98195, USA
    Mol Cell 21:873-80. 2006
  6. ncbi Ubiquitin transfer from the E2 perspective: why is UbcH5 so promiscuous?
    Peter S Brzovic
    Department of Biochemistry, University of Washington, Seattle, Washington 98195 7742, USA
    Cell Cycle 5:2867-73. 2006
  7. ncbi E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages
    Devin E Christensen
    Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington 98195 7350, USA
    Nat Struct Mol Biol 14:941-8. 2007

Detail Information

Publications7

  1. pmc Estrogen receptor alpha is a putative substrate for the BRCA1 ubiquitin ligase
    Catherine M Eakin
    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
    Proc Natl Acad Sci U S A 104:5794-9. 2007
    ..The identification of ERalpha as a putative BRCA1/BARD1 ubiquitination substrate reveals a potential link between the loss of BRCA1/BARD1 ligase activity and tissue-specific carcinoma...
  2. ncbi Structural Biology: Parkin's Serpentine Shape Revealed in the Year of the Snake
    Katja K Dove
    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA
    Curr Biol 23:R691-3. 2013
    ..Parkin is an E3 ubiquitin ligase, mutations in which are responsible for autosomal recessive juvenile parkinsonism. Recently, several structures of Parkin have been solved, revealing its serpentine shape and modes of auto-inhibition. ..
  3. pmc Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination
    Devin E Christensen
    Department of Biochemistry, University of Utah, Salt Lake City, UT 98195, USA
    FEBS J 276:5381-9. 2009
    ..Defining the set of E2s that interact with other RING and U-box E3s will open the door for predictive models and lead to a better understand of substrate ubiquitination...
  4. pmc Structural and functional characterization of the monomeric U-box domain from E4B
    Kyle A Nordquist
    Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232, USA
    Biochemistry 49:347-55. 2010
    ..The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization...
  5. ncbi A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination
    Peter S Brzovic
    Department of Biochemistry, University of Washington, Seattle, 98195, USA
    Mol Cell 21:873-80. 2006
    ..Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1...
  6. ncbi Ubiquitin transfer from the E2 perspective: why is UbcH5 so promiscuous?
    Peter S Brzovic
    Department of Biochemistry, University of Washington, Seattle, Washington 98195 7742, USA
    Cell Cycle 5:2867-73. 2006
    ..This unique property allows activated UbcH5 approximately Ub complexes to self-assemble and has a profound influence on poly-ubiquitin chain synthesis...
  7. ncbi E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages
    Devin E Christensen
    Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington 98195 7350, USA
    Nat Struct Mol Biol 14:941-8. 2007
    ..Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING...