Identification of BRCA1 Ubiquitylation Targets

Institution: University of California, Irvine
Investigator(s): Peter Kaiser, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11NB-0177 Award: $199,600
Award Type: IDEA Competitive Renewal
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2005)
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.

Final Report (2007)
Despite intense research, little is known about the molecular basis for how BRCA1 prevents formation of breast cancer. A number of findings strongly suggest that the tumor suppressor function of BRCA1 is executed by its ubiquitin-ligase activity. Ubiquitin ligases help to attach the small protein ubiquitin to other proteins. The important in vivo ubiquitination substrates of BRACT remain to be identified.

We used CBCRP support to develop and apply an unbiased proteome-wide approach to detect changes in ubiquitination profiles between cells grown under different conditions or between normal cells and cells lacking specific functions, such as cell lines deficient for BRCA1 function. The goal is to apply this strategy to identify BRCA1 ubiquitination targets in vivo. During the funding period we have developed a “tandem-affinity” purification strategy and applied it to proteome-wide purification of ubiquitinated proteins. To allow sensitive detection of differences in ubiquitination profiles we have combining this purification strategy with a quantitative mass spectrometric approach (SILAC). Analyses software was developed to streamline detection of changes in the compared profiles. Because BRCA1-dependent ubiquitination has recently been shown to be activated in response to DNA damage, we opted to generate ubiquitination profiles after gamma-irradiation of cells. These experiments allowed us to quantitatively describe changes in ubiquitination in response to DNA damage. To define changes in ubiquitination events that specifically depend on BRCA1 we sought to compare the response of cells deficient in BRCA1 ubiquitin ligase function and normal cell lines. These experiments were delayed because the stable cell lines generated experienced a dramatic loss of expression of the tagged ubiquitin during their adaptation to the SILAC-compatible growth media. These problems were overcome by establishing new stable cell lines, and ubiquitination profiles in response to DNA damage are being analyzed. In summary, we have established an effective procedure that allows quantitative comparison of ubiquitination profiles. This allowed us to described ubiquitination substrates in mammalian cells on a proteome-wide scale.

We identified many precise ubiquitination sites and established ubiquitin-chain linkage architecture in mammalian cells. Furthermore, quantitative changes in ubiquitination profiles in response to DNA-damage induced by gamma-irradiation were established, and DNA damage induced changes in ubiquitin-chain architecture were measured. These are very promising results and we are continuing with this approach to identify direct substrates of BRCA1. A high-end mass spectrometer with superior performance is now available for these experiments through an NIH-funded instrumentation grant. The instrument was funded in part to continue these experiments. We are preparing to re-analyze samples generated over the past year, which will help to describe BRCA1-dependent ubiquitination events.

A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivocross-linking.
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Tagwerker C, Flick K, Cui M, Guerrero C, Dou Y, Auer B, Baldi P, Huang L, Kaiser P.
Yr: 2006 Vol: 5 Nbr: 4 Abs: Pg:737-48

An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network
Periodical:Molecular and Cellular Proteomics
Index Medicus: Mol Cell Proteomics
Authors: Guerrero C, Tagwerker C, Kaiser P, Huang L
Yr: 2005 Vol: 5 Nbr: 2 Abs: Pg:366-78

Global approaches to understanding ubiquitination.
Periodical:Genome Biology
Index Medicus: Genome Biology
Authors: Kaiser P, Huang L
Yr: 2005 Vol: 6 Nbr: 10 Abs: Pg:233

Expanding horizons at Big Sky. Symposium on Ubiquitin and Signaling
Periodical:Embo Journal
Index Medicus: EMBO J
Authors: Kaiser P, Fon EA
Yr: 2007 Vol: 8 Nbr: 9 Abs: Pg:817-22

Mass spectrometric characterization of the affinity-purified human 26S proteasome complex.
Index Medicus: Biochemistry
Authors: Wang X, Chen CF, Baker PR, Chen PL, Kaiser P, Huang L.
Yr: 2007 Vol: 46 Nbr: 11 Abs: Pg:3553-65