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Ubiquitin (Ub)2 is a protein of 76 residues that is highly conserved from yeast to humans. Conjugation of Ub needs a cascade of reactions that involve E1, E2, and E3 enzymes, which ultimately lead to the formation of an isopeptide bond between C-terminal Gly of Ub and a Lys residue on the substrate. Ubiquitin contains seven lysine residues (at positions 6, 11, 27, 29, 33, 48, and 63), and polyubiquitin chain assembly can occur at any of these lysine residues. Lys48-linked polyubiquitination of proteins is quite common and normally targets substrates for proteolysis by 26 S proteasome, whereas Lys63-linked polyubiquitination is not typically associated with protein degradation. Instead, Lys63-linked ubiquitination modification is often a signaling event and has been shown to participate in diverse cellular functions, including endocytosis, autophagy, NF-κB activation, and DNA damage repair.
Lys-63-specific deubiquitinase BRCC36 is an enzyme that is encoded by the BRCC3 gene in humans. BRCC36 is a member of the JAMM/MPN+ family of zinc metalloproteases that specifically cleaves Lys 63-linked polyubiquitin chains in vitro. And BRCC36 is a component of the BRCA1-A complex, which consists of RAP80, CCDC98/ABRAXAS, BRCC45/BRE, MERIT40/NBA1, BRCC36, and BRCA1. This complex participates in the regulation of BRCA1 localization in response to DNA damage. In stable BRCC36 knockdown cell lines, chromatin-associated Lys63-linked Ub chains in these cells are accumulation, indicating that BRCC36 is normally involved in the regulation of Lys63-linked ubiquitin chain formation in the nucleus. In the BRCA1-A complex, BRCC36 specifically removes 'Lys-63'-linked ubiquitin on histones H2A and H2AX (the phosphorylated form of the H2AX protein), antagonizing the RNF8-dependent ubiquitination at double-strand breaks (DSBs).
BRCC36 also mediates the specific Lys63-specific deubiquitination associated with the COP9 signalosome complex (CSN), via the interaction of the BRISC complex with the CSN complex. BRCC36’s Lys63-specific deubiquitination activity is insensitive to both N-ethyl-maleimide and ubiquitin aldehyde, indicating that it lacks an active site cysteine residue. Using a biochemical approach, Eric, et al. found that the activity was intrinsic to 19S (PA700) portion of the 26S proteasome and the Brcc36 isopeptidase complex (BRISC), but that the CSN-associated activity was due entirely to an interaction with Brcc36. None of the complexes cleave K6, K11, K29, K48 or α-linked polyubiquitin, but they do cleave K63 linkages within mixed-linkage chains. Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here, we identify serine hydroxymethyl transferase (SHMT) as a previously unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub-mediated internalization and lysosomal degradation. BRISC-deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a mechanism of DUB regulation and suggest a therapeutic use of BRISC inhibitors for treating pathophysiological processes driven by elevated IFN responses.
There’re also a fraction of BRCC36 existing in the cytoplasm, which is activated by a CCDC98-like protein KIAA0157. The KIAA0157-BRCC36 complex mainly exists in cytosol and may regulate cytoplasmic function of BRCC36, whereas CCDC98 determines the nuclear localization of BRCC36, and they form a nuclear complex with three additional components, RAP80, BRCC45, and MERIT40, which is important for nuclear function of BRCC36, especially in response to DNA damage. Although the binding of KIAA0157 to BRCC36 is sufficient to activate BRCC36, the association of CCDC98 with BRCC36 is not. These observations clearly indicate that these two scaffold proteins differentially regulate BRCC36 catalytic activities.
Fig 1. A model of BRCC36 in two distinct protein complexes. (Feng L, et al. 2010).