The BRCA1/BARD1 ubiquitin ligase and its substrates

Abstract

Mutations in breast cancer type 1 susceptibility protein (BRCA1) and its heterodimeric binding partner BARD1 confer a high risk for the development of breast and ovarian cancers. The sole enzymatic function of the BRCA1/BARD1 complex is as a RING-type E3 ubiquitin (Ub) ligase, leading to the deposition of Ub signals onto a variety of substrate proteins. Distinct types of Ub signals deposited by BRCA1/BARD1 (i.e. degradative vs. non-degradative; mono-Ub vs. poly-Ub chains) on substrate proteins mediate aspects of its function in DNA double-stranded break repair, cell-cycle regulation, and transcriptional regulation. While cancer-predisposing mutations in both subunits lead to the inactivation of BRCA1/BARD1 ligase activity, controversy remains as to whether its Ub ligase activity directly inhibits tumorigenesis. Investigation of BRCA1/BARD1 substrates using rigorous, well-validated mutants and experimental systems will ultimately clarify the role of its ligase activity in cancer and possibly establish prognostic and diagnostic metrics for patients with mutations. In this review, we discuss the Ub ligase function of BRCA1/BARD1, highlighting experimental approaches, mechanistic considerations, and reagents that are useful in the study of substrate ubiquitylation. We also discuss the current understanding of two well-established BRCA1/BARD1 substrates (nucleosomal H2A and estrogen receptor α) and several recently discovered substrates (p50, NF2, Oct1, and LARP7). Lessons from the current body of work should provide a road map to researchers examining novel substrates and biological functions attributed to BRCA1/BARD1 Ub ligase activity.

Keywords: BARD1; BRCA1; ubiquitin ligases; ubiquitin signaling; ubiquitins.

Figure 1.. An overview of the features of the BRCA1/BARD1 Ubiquitin ligase.

A) The domain structure and function of BRCA1 and BARD1 domains. Folded domains of BRCA1 and BARD1 are depicted with ovals and their corresponding domain names: really interesting new gene (RING), BRCA1 C-terminal (BRCT), and ankyrin repeats (ARD). Substrate binding domains within the intrinsically disordered regions are represented with rectangles. Domain functions related to E3 ligase activity are listed above domains. Substrates are listed below the region of protein with which they interact. B) The solution structure of RING domains from BRCA1 and BARD1 (PDB 1JM7) are shown in magenta and pink respectively. The sidechains at mutation sites used to study the structure/function relationship are shown in spheres or sticks: BARD1 mutations are underlined, cancer-associated mutations are either labeled with an asterisks or in the case of zinc-coordinating mutation sites depicted in cyan sticks (BRCA1 C24R, C39S/R/Y/W, C44S/Y/F, C47S/Y/F, C61G, C64R/Y/W and BARD1 C53W, C71Y, and C83R). The colors of mutation site sidechains correspond with their functions in Panel C. C) Mutations are categorized in a Venn diagram according to the properties and functions they affect.

Figure 2:. BRCA1 and BARD1 bind the nucleosome substrate and E2 simultaneously.

The cryo-EM structure (PDB 7JZV) reveals that both BRCA1 and BARD1 RING domains (magenta and pink, respectively) contain critical residues (BRCA1 Arg 71 and BARD1 Trp 91, blue) that contact the nucleosome surface (gray). The E2, Ube2D3 (green), binds to a distinct interface on BRCA1 allowing the heterodimer to coordinate substrate and E2 simultaneously. The complex allows for transfer of ubiquitin from the E2 onto the dynamic C-terminal end of H2A (unresolved in the structure).