BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1 C61G , does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.Germline mutations of BRCA1 predispose women to breast and ovarian cancers (1). BRCA1 contains several domains that interact with a variety of molecules and is potentially responsible for multiple functions in DNA damage repair, transcription, and cell-cycle regulation (2-4). BARD1 was identified in a yeast two-hybrid screen as a protein that interacts with BRCA1 (5). Both BRCA1 and BARD1 proteins contain a RING finger (5) and exist as homodimers or preferentially form stable heterodimers (6). The heterodimeric interaction is mediated by the flanking regions of the RING finger motif of the two molecules (6). Although a transcriptional function in the C terminus of BRCA1 has been recently reported (3), the biochemical function of the heterodimeric RING finger constituted from the N termini of BRCA1 and BARD1 is not known.Previously, we and others identified a highly conserved small RING finger protein, ROC1 (also called Rbx1 and Hrt1), as an essential subunit of the SCF Ub 1 ligase (7-10). The Ub ligase (E3) catalyzes the formation of polyubiquitin chains onto substrate proteins via isopeptide bonds utilizing the Ubs that have been sequentially activated by enzymes E1 and E2. Polyubiquitinated substrates are then rapidly degraded by the 26 S proteasome (11). The SCF and the APC are the two major Ub ligase complexes that regulate ubiquitin-mediated proteolysis during G 1 /S and anaphase (12), and contain the small RING finger proteins ROC1 and APC11, respectively (7-10). Point mutations in the RING finger domain of ROC1 completely disrupted the Ub ligase activity, suggesting an essential role of the domain for its activity (7). APC11 also contains Ub ligase activity in vitro (7). More recently, several large RING finger proteins, such as MDM2, c-Cbl, IAP, and AO7, with otherwise diverse structures and functions were linked to ubiquitination (13-16), suggesting a potentially broad and general function for RING fingers in activating Ub ligase activity. One...

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, and BRCA1-BARD1 co-expression in cells causes NPM stabilization rather than degradation. This is consistent with the notion that this ligase catalyzes untraditional polyubiquitin chains. Given the many overlapped functions between NPM and BRCA1, we propose that NPM is a strong candidate as a substrate of the BRCA1-BARD1 ubiquitin ligase.

Based on the enzymatic saccharification of the various pulps in the previous 0.8 l ultrasonic stirred tank reactor, the ultrasound-enhanced saccharification of waste papers such as newspaper, carton paper, office paper etc. was carried out in the same reactor as well as larger scale stirred tank reactors of size 3.2 and 6.4 l. The saccharification of each waste paper was less enhanced in the larger reactor at a given ultrasonic intensity. This could be attributed to the decrease in the ultrasonic intensity per reaction volume, i.e., the specific ultrasonic intensity. Most waste papers were more efficiently hydrolyzed with increasing specific ultrasonic intensities, although newspaper was less efficiently done for a too high specific intensity. Such an adverse effect might be due to the fact that some impurities in the newspaper such as lignin were activated by an intensive ultrasonic irradiation to form a rigid and closed network, which inhibited the access and adsorption of cellulase on to the substrate surface. The previous kinetic model was found to be applicable to analyze and simulate the saccharification of each waste paper in the different ultrasonic reactors. The ultimate conversion of a substrate based on the total sugar concentration estimated for an infinite reaction time could be correlated as a function of the ratio of initial substrate to enzyme concentrations at a fixed specific ultrasonic intensity. Either the apparent rate constant or the ultimate conversion increased and tended to approach a constant with an increase in the specific ultrasonic intensity except for the case of newspaper, while neither the apparent Michaelis constant, product inhibition constant nor glucose formation equilibrium constant was influenced by the specific ultrasonic intensity.

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