NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.NQO1-dependent pathway ͉ disruption of NQO1-p53 binding ͉ cancer hot-spot p53 mutant ͉ p53-induced apoptosis W ild-type p53 is a labile tumor suppressor protein whose cellular level is mainly regulated by its rate of proteasomal degradation (reviewed in ref. 1). WT p53 protein can induce growth arrest (1-4) or apoptosis (5-8) and can thus prevent accumulation of DNA-damaged cells that could lead to the development of cancer (reviewed in refs. 1, 9, and 10). This tumor-suppressing activity of WT p53 is abolished by mutations in the p53 gene that occur in Ͼ50% of human cancers (1,11,12), and the mutant proteins often accumulate in the cancer cells (13). Degradation of p53 is mediated by two alternative pathways, ubiquitin-independent (14, 15) or ubiquitin-dependent. Ubiquitin-dependent degradation of p53 is mediated by different ubiquitin E3 ligases, including Mdm2 (16,17), Pirh2 (18), and Cop1 (19), that bind and ubiquitinate p53, targeting it to degradation via the 26S proteasome. The ubiquitin-independent pathway is regulated by NAD(P)H:quinone oxidoreductase 1 (NQO1) (14,15,20,21) and is mediated via the 20S proteasome (22). We have shown that NQO1 stabilizes p53, so that NQO1 overexpression increases p53 levels (20, 21), whereas NQO1 knockdown by small interfering RNA (siRNA) decreases the level of p53 (14). NQO1 binds to p53 (23, 24) and dicoumarol, an inhibitor of NQO1 activity that competes with NAD(P)H for binding to NQO1, disrupts NQO1-p53 binding (23), and induces ubiquitin-independent proteasomal degradation of p53 (14,15,20,21,23). Various other coumarins and flavones that also compete with NAD(P)H for binding to NQO1 also induce ubiquitin-independent p53 degradation (23). However, unlike WT p53 and some p53 mutants, the most frequent hot-spot human cancer p53 mutants (11, 12) were resistant to dicoumarolinduced degradation by increased binding to NQO1 (23).Curcumin, a natural phenolic compound found in the spice ...

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent ''hot-spot'' p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.W ild-type p53 is a labile protein and its cellular level is mainly regulated by the rate of its proteasomal degradation (reviewed in ref. 1). p53 degradation is mediated by two alternative pathways that either depend on Mdm2 and ubiquitin (2, 3) or are independent of both (4). The Mdm2-and ubiquitinindependent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) (4-7). Our previous studies showed that NQO1 stabilizes p53 (5, 6) and that reducing the NQO1 level by small interfering RNA decreases the level of p53 (4). Dicoumarol is a competitive inhibitor of NQO1 activity that competes with NAD(P)H for binding to NQO1 (8) and induces ubiquitinindependent p53 degradation (4-7). We suggested that p53 stabilization requires the physical interaction of p53 with NQO1 (6), and recent studies have shown that NQO1 can bind to p53 (9). The tumor suppressor wild-type p53 is mutated in Ͼ50% of human cancers (1, 10, 11). We have now studied the control of ubiquitin-independent p53 degradation by using different NQO1 inhibitors, different p53 mutants, and binding of NQO1 to p53. Our results include the finding that the most frequent ''hot-spot'' p53 mutants in human cancer showed increased binding to NQO1 and resisted dicoumarol-induced degradation. These findings indicate that NQO1 plays a major role in stabilizing hot-spot p53 mutant proteins in human cancer cells. Materials and MethodsCells and Cell Culture. The cell lines used were: p53 null HCT116 human colon carcinoma cells (12), 293 human kidney cells, Huh 7 human hepatocellular carcinoma cells that carry the Y220P mutant 53 (1...

Eleven Israeli Holstein families including 5221 cows were analyzed by a daughter design for eight economic traits: milk, fat and protein production, fat and protein percentage, somatic cell score (SCS), herd-life, and female fertility. The cows were genotyped for 73 microsatellites with maximum spacing between markers of 53 cM. There were 86,304 informative genotypes. Preliminary analysis was by ANOVA of each trait, with the marker effect nested within sire. Significance was determined by controlling the false discovery rate at 0.4, after excluding markers with genome-wide significance for at least a single trait, and traits without any significant effects at this level. Thus, four markers on chromosomes 6 and 14 and female fertility were excluded. There remained 40 significant marker-trait combinations, and it is expected that 24 of these are true effects. To perform interval mapping for the families with significant contrasts, 21 additional markers were genotyped on chromosomes 2, 7, and 27. The bootstrap confidence intervals for gene effect did not include zero for protein percent on chromosome 2 and fat yield, protein yield, and SCS on chromosome 7. Quantitative trait locus heterozygosity was 33%, which is consistent with the hypothesis that only two alleles are segregating with unequal allele frequency.

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.

Background