NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

Abstract

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells.Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.Modification of proteins by the covalent attachment of ubiquitin (Ub) 2 to the lysine (Lys) residue of a target protein is a key regulatory mechanism of many cellular processes including the DNA damage response (1, 2). In response to ionizing irradiation, ubiquitination occurs at DNA damage sites in a central DNA damage kinases ATM/ATR-dependent manner (3). Ubiquitin Lys63 (K63) polyubiquitin chain linkages play important roles in the recruitment of repair factors in the DNA damage response (3). K63-linked polyubiquitin chains are assembled through a conserved heterodimer of an E2 complex composed of a catalytically active Ubc13 subunit and its inactive sequence homolog Mms2 that lacks the enzymatic active site (4, 5). The formation of K63-polyubiquitins at DNA damage sites depends on two E3 ligases RNF8 and RNF168 (3,6). In addition, a HECT domain-containing E3 ligase HERC2 is also involved in facilitating the formation of K63-linked ubiquitin chains (7).Deubiquitinating enzymes (DUBs) catalyze the removal of Ub from Ub-conjugated substrate proteins and it has become increasingly obvious that Ub deconjugation plays important roles in regulating Ub-dependent pathways (8, 9). BRCC36 is a member of a small family of DUBs containing JAMM/MPNϩ domain proteins, which are Zn 2ϩ -binding metalloproteases (10 -12). It selectively cleaves K63-linked polyubiquitin (12-14), and is required for proper checkpoint regulation in response to DNA damage (13,(15)(16)(17). It plays an important role in recruiting BRCA1 to DNA damage sites, as BRCC36 deficiency leads to decreased BRCA1 accumulation at DNA damage sites (3).BRCC36 is a component of the BRCA1 A complex (13, 17, 18). The BRCA1 A complex contains at least five different components, Abraxas, NBA1/MERIT40, BRE, Rap80, and BRCC36. It associates with BRCA1 through an interaction of Abraxas with the BRCA1 C-termina...

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

“…Although the regulatory basis of its DUB activity is beginning to be understood (3)(4)(5)(6)34), it has remained unknown whether the BRCC36 ubiquitin hydrolyzing activity plays a role in mammalian DNA damage responses. In this study, we have systematically examined, by adopting both RNAi and genome editing approaches, whether BRCC36 and its DUB may be functionally important in driving cellular response to DSBs.…”

“…A more recent study also revealed that an additional phosphorylation event in the vicinity of pSPXF (serine 404) further stabilizes the Abraxas-BRCA1 interaction by formation of a stable dimer (16). Other components of the BRCA1-A complex, including Merit40 and BRE, are also key in maintaining the integrity of the macromolecular protein complex (6,9,17,18) and for BRCA1 accumulation at DSB sites. Together, these data underscore critically important roles of each of the subunits that make up the protein assembly.…”

“…BRCC36 DUB relies on its zinc-dependent JAB1/MPN/MOV4 metalloprotease domain (3) and its interaction with Abraxas/KIAA0157 (3)(4)(5). Although BRCC36 forms distinct nuclear and cytoplasmic complexes (5,6) and plays pleiotropic roles during cell proliferation, the K63-Ub DUB is arguably best known for its strong links with the breast and ovarian susceptible gene product BRCA1 in DNA damage responses (DDRs). Indeed, BRCC36 resides within the BRCA1-A macromolecular protein complex, which comprises the ubiquitin-binding protein RAP80, BRCC45/BRE, Abraxas/CCDC98, Merit40/NBA1, and BRCA1.…”

Protein Ubiquitination in IR-Induced DNA Damage Response

“…BRCC36 DUB activity and localization are regulated via its interactions within the context of each protein complex. In the BRCA1-A complex, ABRA1 and BRE are essential for BRCC36 DUB activity and localization at DNA damage site (100,122,127,128). Although the exact function of BRCC36 deubiqutination at DNA damage site is not known, it has been suggested that BRCC36 catalyzes the deubiquitination of chromatin-associated proteins that lead to the chromatin remodeling and amplification of damage signal.…”

“…Aside from RAP80 and BRCA1/BARD1, many components of the BRCA1-A complex possess structural domains implicating them in ubiquitination pathway. BRCC36 has a JAM domain characteristic of lysine 63-specific deubiquitinating enzymes, and Abraxas and BRE contain predicted Mpr-1/Pad1 N-terminal (MPN -) and ubiquitin E2 variant (UEV) domains respectively (122,(125)(126)(127)(128). Possible targets of BRCC36 include RAP80 and di-ubiquitinated H2A/H2AX.…”