Identification of BRCA1 Ubiquitylation Targets

Institution: University of California, Irvine
Investigator(s): Peter Kaiser, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9IB-0124 Award: $75,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2003)
BRCA1 is a gene that is important in preventing the development of breast and ovarian cancers. The importance of BRCA1 in protection from these cancers is underlined by the fact that in 50-90% of hereditary breast and ovarian cancers BRCA1 is inactivated due to mutations. Given the importance of BRCA1 in breast cancer development many research groups are trying to understand how BRCA1 prevents breast cancer. However, despite intense research, our molecular understanding of BRCA1ís function in breast cancer prevention is very limited.

Recently, several research groups found that BRCA1 mediates the attachment of a small protein, called ubiquitin, to other proteins. Attachment of this small protein ubiquitin has long been known to regulate the function of the proteins it is attached to. Thus, identification of the proteins to which BRCA1 attaches ubiquitin is crucial to understand how BRCA1 protects from breast and ovarian cancers.

There is a large number of proteins to which ubiquitin gets attached with the help of various different cellular factors. BRCA1 is most likely only involved in attaching ubiquitin to a few proteins. The goal of this proposal is to identify these direct targets of BRCA1. To this end, we propose to compare the sets of proteins that have ubiquitin attached in normal cells and in cells from patients carrying a defective BRCA1 gene. Proteins that do not get ubiquitin attached in cells with defective BRCA1 are likely to be direct BRCA1 targets. Over the past years we have developed a technique that allows us to efficiently separate proteins that have ubiquitin attached from all other proteins and thus permit direct comparison of proteins with attached ubiquitin from different cells (e. g. normal cells and BRCA1 defective cells).

The identification of proteins to which BRCA1 attaches ubiquitin is currently one of the most important aspects in understanding of how BRCA1 prevents breast and ovarian cancers. Such a detailed understanding of the molecular role of BRCA1 is crucial to design new approaches to breast cancer prevention and therapy.


Final Report (2004)
The tumor suppressor gene BRCA1 is mutated in 50-90% of hereditary breast and ovarian cancers. The molecular basis of BRCA1ís tumor suppressor function is still unknown. The N-terminal part of BRCA1 contains a RING-finger domain. RING-finger domains have recently been implicated in ubiquitylation. These recent findings suggest that the molecular function of BRCA1 involves ubiquitylation of so far unknown target proteins. To understand the molecular pathways that are regulated by BRCA1 it is therefore crucial to identify the target proteins that are ubiquitylated by BRCA1 in vivo.

Our strategy is to identify ubiquitylated proteins from cells expressing functional BRCA1 (BRCA+) and cells that have no functional BRCA1 (BRCA-). Comparison of the two sets of identified proteins will reveal possible differences in ubiquitylation patterns and help to identify possible BRCA1 targets. To achieve this goal we developed an affinity tag that is fused to ubiquitin, which allows sequential purification of ubiquitylated proteins on two different affinity resins. During the last year we made several changes on this affinity tag so it is compatible with high expression levels in mammalian cells, which is crucial for the success of the approach. Because it became clear that the total number of ubiquitylated proteins is very high and it would probably be currently impossible to achieve comprehensive identification, we explored different strategies that focused on identification of the differences between BRCA+and BRCA- cells. After some technical difficulties we came to the conclusion that a novel approach called SILAC (stable isotope labeling by amino acids in cell culture) is ideal for our analysis. Proteins in BRCA+ will be labeled with a stable isotope, by replacing the arginine in the growth media by its stable 13C-labeled analog (13C-6-arginine). Proteins from BRCA-cells are unlabeled (grown in 12C-arginine). Total proteins from both samples are mixed and ubiquitylated proteins are purified. For each purified protein we can now identify two versions, one 13C-labeled (from BRCA+ cells) and one unlabeled version (from BRCA- cells). Data analysis can now be focused on proteins that shown higher abundance in the 13C-labeled form as compared to the unlabeled, form which is indicative for BRCA1 ubiquitylation targets.

In summary, we believe we have overcome most of the obstacles and have introduced a quantitative aspect into our approach, which will greatly improve our chances to reach our research goal of identification of BRCA1 ubiquitylation targets. This will be an important step towards understanding the role of BRCA1 in tumor prevention.


Symposium Abstract (2005)
In order to identify new target proteins of BRCA1, a tumor suppressor gene mutated in 50-90% of hereditary breast and ovarian cancers, we have developed a new tandem-affinity tag termed the HB-tag. Genetic data indicate that BRCA1 plays a major role in preventing breast and ovarian tumor formation. Despite intense research, the molecular basis of BRCA1ís tumor suppressor function is still unknown. Recent studies indicate that BRCA1 contains a specific structure called a RING-Finger domain, which is attracts and binds the protein ubiquitin. The attachment of ubiquitin to a protein is called ubiquitylation. Ubiquitylation either leads to the degradation of the target protein or may act as a regulatory switch in a distinct mechanism of a cell. BRCA1-mediated ubiquitylation occurs in response to cell replication stress, linking its ubiquitylation function to the DNA-damage response.

The HB-tag consists of a 6xHis and a biotin-binding domain sequence. In a two-step purification procedure, tagged proteins attach to 6xHis- or biotin-binding resins and can subsequently be concentrated to generate highly purified samples of ubiquitylated proteins. By applying a combination of extremely sensitive mass spectrometry methods we can then identify the complete set of proteins present in these samples. To determine the function of a certain protein involved in ubiquitylation we can analyze and compare samples derived from cell lines that are deficient of the protein of interest to samples that are derived from normal cell lines. The differences detected in the two protein profiles allow us to verify known ubiquitylation target proteins and to identify new target proteins.

We have recently applied this strategy to profiling of ubiquitylated proteins in budding yeast and mammalian cells, and are confident that a set of ubiquitylated proteins purified from BRCA1-deficient cell lines compared to wild type cell lines will reveal new candidate proteins regulated by BRCA1. This proteomic approach will determine all factors involved in the ubiquitylation process of BRCA1, which is crucial to study its molecular effects. Once the molecular function of BRCA1 ubiquitylation is clearly understood, a broad range of therapeutic approaches concerning the breast cancer problem can be pursued.


Symposium Abstract (2007)
BRCA1 is a gene that is important to prevent development of breast and ovarian cancers. The importance of BRCA1 in protection from these cancers is underlined by the fact that in 50-90% of hereditary breast and ovarian cancers BRCA1 is inactivated due to mutations. Despite intense research, our molecular understanding of BRCA1ís function in breast cancer prevention is very limited.

Recently, several research groups found that BRCA1 mediates the attachment of a small protein, called ubiquitin, to other proteins. Attachment of this small protein ubiquitin has long been known to regulate the function of the proteins it is attached to. Thus, identification of the proteins to which BRCA1 attaches ubiquitin is crucial to understand how BRCA1 protects from breast and ovarian cancers.

There are a large number of proteins to which ubiquitin gets attached with the help of various different cellular factors. BRCA1 is most likely only involved in attaching ubiquitin to a few proteins. The goal of our research is to develop strategies to identify these direct targets of BRCA1. To this end, we compare the sets of proteins that have ubiquitin attached in normal cells and in cells from patients carrying a defective BRCA1 gene. Proteins that do not get ubiquitin attached in cells with defective BRCA1 are likely to be direct BRCA1 targets. In addition, the function of BRCA1 to attach ubiquitin to other proteins has been shown to be stimulated after induction of DNA damage. We are therefore also detecting proteins that are modified with ubiquitin specifically in response to DNA damage. Lastly, we apply a new strategy to identify transient binding partners of BRCA1 as well as BARD1 with the aim to link BRCA1 with new cellular pathways and identify direct targets for BRCA1.