Background: Metabolic enzymes have been hypothesized to assemble into complex to respond to cellular metabolism changes. Results: De novo purine biosynthesis increases in purinosome-containing cells. Conclusion: Purine metabolism is adjusted by purinosome assembly. Significance: This study indicates that purinosome is a functional multienzyme complex.

Mutations in the WNT/beta-catenin pathway are responsible for initiating the majority of colorectal cancers (CRCs). We have previously shown that hyperactivation of this signaling by histone deacetylase inhibitors (HDACis) such as butyrate, a fermentation product of dietary fiber, promotes CRC cell apoptosis. The extent of association between beta-catenin and the transcriptional coactivator CREB-binding protein (CBP) influences WNT/catenin signaling and, therefore, colonic cell physiology. CBP functions as a histone acetylase (HAT); therefore, we hypothesized that the modulation of WNT/catenin activity by CBP modifies the ability of the HDACi butyrate to hyperinduce WNT signaling and apoptosis in CRC cells. Our findings indicate that CBP affects the hyperinduction of WNT activity by butyrate. ICG-001, which specifically blocks association between CBP and beta-catenin, abrogates the butyrate-triggered increase in the number of CRC cells with high levels of WNT/catenin signaling. Combination treatment of CRC cells with ICG-001 and butyrate results in cell type-specific effects on apoptosis. Further, both butyrate and ICG-001 repress CRC cell proliferation, with additive effects in suppressing cell growth. Our study strongly suggests that ICG-001-like agents would be effective against butyrate/HDACi-resistant CRC cells. Therefore, ICG-001-like agents may represent an important therapeutic option for CRCs that exhibit low-fold hyperactivation of WNT activity and apoptosis in the presence of HDACis. The findings generated from this study may lead to approaches that utilize modulation of CBP activity to facilitate CRC therapeutic or chemopreventive strategies.

Inflammation is a hallmark of inflammatory bowel disease (IBD) that involves macrophages. Given the inverse link between selenium (Se) status and IBD-induced inflammation, our objective was to demonstrate that selenoproteins in macrophages were essential to suppress pro-inflammatory mediators, in part, by the modulation of arachidonic acid metabolism. Acute colitis was induced using 4% DSS in wild type mice maintained on Se-deficient (<0.01 ppm Se), Se-adequate (0.1 ppm; sodium selenite), and two supraphysiological levels in the form of Se-supplemented (0.4 ppm; sodium selenite) and high Se (1.0 ppm; sodium selenite) diets. Transfer RNASec (tRNA[sec]) knockout mice (Trspfl/flLysMCre) were used to examine the role of selenoproteins in macrophages on disease progression and severity using histopathological evaluation, expression of pro-inflammatory and anti-inflammatory genes, and modulation of prostaglandin (PG) metabolites in urine and plasma. While Se-deficient and Se-adequate mice showed increased colitis and exhibited poor survival, Se supplementation at 0.4 and 1.0 ppm increased survival of mice and decreased colitis-associated inflammation with an up-regulation of expression of pro-inflammatory and anti-inflammatory genes. Metabolomic profiling of urine suggested increased oxidation of PGE2 at supraphysiological levels of Se that also correlated well with Se-dependent upregulation of 15-hydroxy-PG dehydrogenase (15-PGDH) in macrophages. Pharmacological inhibition of 15-PGDH, lack of selenoprotein expression in macrophages, and depletion of infiltrating macrophages indicated that macrophage-specific selenoproteins and upregulation of 15-PGDH expression were key for Se-dependent anti-inflammatory and pro-resolving effects. Selenoproteins in macrophages protect mice from DSS-colitis by enhancing 15-PGDH-dependent oxidation of PGE2 to alleviate inflammation, suggesting a therapeutic role for Se in IBD.

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.Expression of the Bacillus subtilis tryptophan biosynthetic (trp) genes is negatively regulated in response to tryptophan by TRAP, 1 the trp RNA-binding attenuation protein, which is the product of the mtrB gene (reviewed in 1). TRAP-mediated regulation of the trp genes includes a transcription attenuation and two translation control mechanisms. The untranslated trp operon leader transcript contains inverted repeats that allow folding of the transcript to form three RNA secondary structures. Two of these structures, the antiterminator and terminator, overlap by four nucleotides and therefore are mutually exclusive ( Fig. 1) (2-5). The TRAP binding target in the trp leader transcript consists of 11 (G/U)AG repeats, six of which are present within the antiterminator (6). When activated by tryptophan, TRAP binds to the 11 triplet repeats, which prevents formation of the antiterminator structure. This allows formation of the overlapping terminator, which promotes transcription termination upstream of the trp structural genes. In the absence of TRAP binding, the antiterminator structure forms, which results in transcription of the entire operon (7,8). An additional RNA secondary structure forms at the extreme 5Ј-end of the B. subtilis trp leader transcript. Disruption of the 5Ј-stem loop results in a substantial reduction in trp operon regulation in vivo and reduced affinity of TRAP for trp leader RNA in vitro (9). TRAP-5Ј-stem loop RNA interaction reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus it appears that the 5Ј-structure increases the rate of TRAP-trp leader RNA association, promoting the likelihood that tryptop...

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsible for mediating the cellular response to the toxic compound 2,3,7,8,-tetrachlorodibenzo-p-dioxin. An essential role for the AhR in cellular biology has been established previously, but no high-affinity endogenous ligand has yet been identified. We have confirmed the presence of a putative endogenous ligand(s) in CV-1 cells through transient transfection with various cytochrome P450 isoforms. Expression of cytochromes P450 1A1, 1A2, or 1B1 reduced AhR-mediated luciferase reporter activity, whereas cytochrome P450 2E1 exhibited no significant effect. Studies with 2,4,3Ј,5Ј-tetramethoxystilbene, a potent and specific inhibitor of cytochrome P450 1B1, was able to partially block cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR-regulated cytochromes P450 from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands, including electrophoretic mobility shift assay analyses, which demonstrated that these ligands directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr-null mouse tissues were also analyzed for the presence of AhR activators. Studies revealed that Ahr-null mouse lung tissue had a 4-fold increase in AhR-mediated reporter activity in cells. Quantitative polymerase chain reaction analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.