Finding BRCA1 Ubiquitinated Substrates in Breast Cancer

Institution: The Burnham Institute for Medical Research
Investigator(s): Charles Spruck, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0130 Award: $191,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2009)

This project was originated by Dr. Sonia del Rincon and was completed by Dr. Charles Spruck.

Women that inherit a mutated BRCA1 gene have a lifetime risk of 36-85% for developing breast cancer. One of BRCA1’s tasks is “tagging” other proteins with a small protein, called ubiquitin. This task is called “ubiquitination”, a general cellular process that controls the stability, function, and intracellular localization of a wide variety of proteins. The most typical function of ubiquitin is labeling proteins for proteasomal degradation. Thus, BRCA1 is a critical ubiquitin ligase enzyme within cells. Interestingly, several inherited BRCA1 gene mutations prevent it from ubiquitinating proteins, suggesting the importance of this task in protecting breast epithelial cells from becoming cancerous.

Currently, little is known regarding how loss of BRCA1’s ubiquitin ligase function contributes to breast cancer development and this has primarily been due to an inability of researchers to identify the proteins tagged by BRCA1. We have developed a novel experimental method that allows us to identify proteins that become tagged with ubiquitin. Protein microarrays are glass slides that are spotted with more than 8000 human proteins as separate tiny “dots.” Some of these spotted proteins can become tagged by ubiquitin when the entire glass slide is overlaid with a biochemical reaction mix that includes a purified BRCA1 ubiquitin ligase. Once we have identified the specific proteins that are ubiquitinated, we will then use cell-based models for validation. Finally, we will use primary breast tumor samples derived from women that carry BRCA1 mutations to look at the biological outcomes associated when a specific protein is not ubiquitinated properly, thus giving insight into BRCA1-dependent processes that go awry in breast cancer.

If our studies are successful, we will be in a unique position to pinpoint what goes wrong when BRCA1 fails to function properly. This knowledge might ultimately be used to develop less radical breast cancer prevention strategies, other than bilateral mastectomies, for women harboring inherited BRCA1 mutations.




Final Report (2010)

Women that inherit a mutated copy of the BRCA1 gene have a lifetime risk of36-85% for developing breast cancer. One ofBRCA1 's cellular functions is to 'tag' proteins with a small protein called ubiquitin, in a function known as 'ubiquitination.' Proteins that do such a function are called ubiquitin ligases. Interestingly, several inherited mutations of BRCA1 have been shown to prevent its ubiquitinating activity, suggesting the importance of this function in protecting breast epithelial cells from becoming cancerous.

Currently, little is known regarding how loss of BRCA1 ubiquitin ligase function contributes to breast cancer development. This has been primarily attributed to the inability of researchers to identify the proteins that are targeted by BRCA1. To overcome this, we developed a novel experimental method that allows for the detection of proteins that become tagged with ubiquitin on a global scale. The technique uses protein microarrays as an experimental tool, which consists of glass slides spotted with more than 8000 different human proteins. Proteins targeted by BRCA 1are then identified by the attachment of a fluorescent signal. We established reaction conditions that allow for the targets of BRCA1 to be detected using both purified recombinant proteins as well as extracts prepared from BRCAI mutant and wild-type breast cancer cell lines. The recombinant proteins or cellular extracts are overlaid on the protein microarray device and substrates of BRCA1 identified by comparing the pattern of fluorescent spots between the BRCA1 mutant or wild-type specimen. This assay is extremely powerful and represents the first ever assay that allows for targets of ubiquitin ligases to be identified on a global scale.

This technique also has wide-spread applications to the studies of protein ubiquitylation in breast cancer development and progression. It can also be applied to global studies of other protein modifications in breast cancer. In the case of BRCA1, identification of its targets could shed light on the mechanism by which mutations increase breast cancer risk. This knowledge might ultimately be used to develop less radical breast cancer prevention strategies, other than bilateral mastectomies, for women harboring inherited BRCA1 mutations.



Development and validation of a method for profiling post-translational modification activities using protein microarrays.
Periodical:PLOS One
Index Medicus: PLOS One
Authors: Del Rincón SV, Rogers J, Widschwendter M, Sun D, Sieburg HB, Spruck C
Yr: 2010 Vol: 5 Nbr: 6 Abs: Pg:e11332