The breast and ovarian cancer tumor suppressor protein, BRCA1, and its obligate protein partner, BARD1 are critical to proper functioning of fundamental cellular processes that support genomic stability. To date, a single biochemical activity has been identified for BRCA1/BARD1, namely, they function together as a ubiquitin E3 ligase. Although it is believed that the ability of BRCA1/BARD1 to modify specific cellular proteins with ubiquitin is fundamental to its role as a tumor suppressor, the details and ramifications of this relationship remain to be elucidated. During the past grant period, we discovered that BRCA1/BARD1 can function with ten human ubiquitin-conjugating enzymes (E2s) and that the ultimate product generated by BRCA1/BARD1 on substrates depends on the E2 present. We also identified several new substrates, including the estrogen receptor. In the next grant period we will expand our focus to include the essential subunit, BARD1, more explicitly and will participate in a collaborative effort to develop new molecular insights and tools for another RING E3 ligase that is critical in cancers, Mdm2/MdmX. The overall goals for the next grant period are 1) elucidate structural and functional determinants of mono-ubiquitin transfer and poly-ubiquitin chain formation by BRCA1/BARD1 and its interacting E2s, 2) investigate the molecular and structural determinants of BRCA1/BARD1-substrate interactions, 3) characterize BARD1 and its interactions, and 4) identify the human E2s that interact with Mdm2/MdmX, the ligase responsible for p53 regulation in vivo. A broad experimental approach will be undertaken, including biochemical, structural, molecular biological, and cellular techniques. Results from these studies will provide new insights into BRCA1/BARD1 function and will contribute to the general understanding of protein ubiquitination.

Public Health Relevance

The breast and ovarian cancer tumor suppressor protein, BRCA1, plays a role in the maintenance of genomic stability and its loss or dysfunction leads to widespread chromosomal abnormalities. Inheritance of a mutant form of BRCA1 increases a woman's lifetime risk of developing breast cancer from 1 in 8 to greater than 1 in 2. Some of the most common inherited mutations abrogate BRCA1's function as a ubiquitin ligase, implying that this function is central to the health of a cell. A full description of the molecular interactions that are critical to BRCA1 function will provide new insight into the early events associated with loss of BRCA1 that lead to tumorogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088055-13
Application #
8309249
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Wehrle, Janna P
Project Start
1998-12-21
Project End
2013-07-31
Budget Start
2012-07-01
Budget End
2013-07-31
Support Year
13
Fiscal Year
2012
Total Cost
$509,677
Indirect Cost
$179,075
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Dove, Katja K; Stieglitz, Benjamin; Duncan, Emily D et al. (2016) Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms. EMBO Rep 17:1221-35
Dove, Katja K; Klevit, Rachel E; Rittinger, Katrin (2015) pUBLically unzipping Parkin: how phosphorylation exposes a ligase bit by bit. EMBO J 34:2486-8
Wu, Wenwen; Nishikawa, Hiroyuki; Fukuda, Takayo et al. (2015) Interaction of BARD1 and HP1 Is Required for BRCA1 Retention at Sites of DNA Damage. Cancer Res 75:1311-21
Vittal, Vinayak; Stewart, Mikaela D; Brzovic, Peter S et al. (2015) Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases. J Biol Chem 290:21244-51
Vittal, Vinayak; Shi, Lei; Wenzel, Dawn M et al. (2015) Intrinsic disorder drives N-terminal ubiquitination by Ube2w. Nat Chem Biol 11:83-9
Metzger, Meredith B; Pruneda, Jonathan N; Klevit, Rachel E et al. (2014) RING-type E3 ligases: master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination. Biochim Biophys Acta 1843:47-60
Pruneda, Jonathan N; Smith, F Donelson; Daurie, Angela et al. (2014) E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis. EMBO J 33:437-49
Warfield, Linda; Tuttle, Lisa M; Pacheco, Derek et al. (2014) A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface. Proc Natl Acad Sci U S A 111:E3506-13
Soss, Sarah E; Klevit, Rachel E; Chazin, Walter J (2013) Activation of UbcH5c~Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B. Biochemistry 52:2991-9
Starita, Lea M; Pruneda, Jonathan N; Lo, Russell S et al. (2013) Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis. Proc Natl Acad Sci U S A 110:E1263-72

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