Identification of BRCA1 Ubiquitylation Targets
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: Note: this grant is a competitive renewal of a grant funded by the CBCRP from 7/1/03 through 12/31/04.
 
 
 
 
 
 
 
 The tumor suppressor gene BRCA1 is mutated in 50-90% of hereditary breast and ovarian cancers, indicating that BRCA1 plays a major role in preventing tumor formation. Despite intense research, the molecular basis of BRCA1's tumor suppressor function is still unknown. BRCA1 functions as ubiquitin ligase. Attachment of ubiquitin, a small protein that is composed of 76 amino acids, “marks” proteins for proteolytic degradation by a complex cellular structure, called the proteasome. Attachment of ubiquitin can also directly regulate the function of a protein without inducing its degradation by the proteasome. Defects in the ubiquitin system affect a variety of cellular processes including cell proliferation and genomic stability and have been linked to many types of cancers. Many of the clinically important mutations in BRCA1 are predicted to interfere with the ubiquitin ligase function of BRCA1. Recent findings suggest that the molecular function of BRCA1 involves ubiquitination of, so far unknown, target proteins.
 
 
 
 
 
 
 
 To understand the molecular pathways that are regulated by BRCA1, it is crucial to identify the target proteins that are ubiquitinated. Our aims in this project are as follows: 
 
 1. Proteome-wide (i.e., screening all the protein components of a cell or tumor) identification of quantitative differences in ubiquitination profiles between BRCA1-positive and BRCA1-negative cells: We will optimize the purification procedure, stable isotope labeling, and data analysis of ubiquitinated proteins in cultured cells. We will then use the optimized strategy to detect quantitative differences in ubiquitination profiles caused by the loss of BRCA1.
 
 2. Validation and analyses of candidate BRCA1 ubiquitination targets: Candidate proteins that were identified in aim 1 will be analyzed individually for their ubiquitination status in BRCA1-positive and BRCA1-negative cells.
 
 For our studies we will express ubiquitin that is labeled with a new “tag” we have developed. This “tagged” ubiquitin allows efficient purification of all ubiquitinated proteins from a cell. Differences in ubiquitination patterns will be identified by a quantitative mass spectrometric approach. 
 
 
 
 
 
 
 
 The current screening procedure for BRCA1 mutations in families with increased breast cancer incidents relies on DNA sequencing. However, detection of a mutation in the BRCA1 gene does not necessarily indicate a gene defect. Since no functional test for BRCA1 exists, there is currently no way to identify mutations that affect BRCA1 function among the many mutations that are found in BRCA1 in the general population. Therefore, identification of BRCA1 ubiquitination targets could provide a useful way to identify the "meaningful" mutations in BRCA1, which in turn would lead to more reliable genetic counseling of members of families with high breast cancer incidents.