BAP1 is a novel regulator of HIF-1α.

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

Altered Fluorescence of Buccal Cells as a Candidate Biomarker for Areca Nut Chewing.

Background: Betel nut is used by an estimated 600 million people globally and is the 4th most widely used psychoactive substance in the world. Its use has been shown to cause oral and esophageal cancers. Therefore, cessation programs are needed in which an effective biomarker can be employed. Objectives: Buccal cells are highly exposed to the betel nut during its use and are also easy to collect. However, it is unknown if there are significant changes to these cells upon exposure or how long any changes may last as the turnover of buccal cells is relatively fast. We sought to determine if optical changes could be detected on buccal cells after exposure to betel nut and if detected, how long these changes were sustained. Methods: Flow cytometry was employed to determine whether fluorescence intensities differ between buccal cells exposed to betel nut and naïve cells. We further characterized the optical signature of buccal cells exposed to betel nut and other polyphenol-rich substances using lambda scans performed on a laser scanning confocal microscope. Results: We demonstrate that the fluorescence of betel nut exposed cells is greater than that of cells exposed to other optically active compounds such as polyphenol-rich foods. We also demonstrate that the fluorescence spectra of betel nut quid exposed cells are distinct from that of cells exposed to other polyphenol-rich substances. Conclusions: We conclude that detecting the altered fluorescence of buccal cells following exposure to betel nut quid may serve as a candidate biomarker for betel nut quid use.

Ursodeoxycholic acid accelerates bile acid enterohepatic circulation.

BACKGROUND AND PURPOSE: Ursodeoxycholic acid (UDCA) is the first-line treatment for primary biliary cholangitis, but its effects on the enterohepatic circulation of bile acid (BA) have been under-investigated. Therefore, we studied the influence of UDCA on BA enterohepatic circulation in vivo and the mechanisms by which UDCA affects the BA kinetics. EXPERIMENTAL APPROACH: Mice were treated with UDCA and other BAs to observe changes in BA pool and BA transporters involved in enterohepatic circulation. Isotope dilution techniques and biochemical analyses were applied to study BA kinetics after oral administration of UDCA, and the mechanism involved. KEY RESULTS: Oral administration of UDCA in mice reduced the overall BA pool and produced a unique BA profile with high-abundance conjugated UDCA species, including tauroursodeoxycholic acid (TUDCA) and GUDCA. We found increased expression of several main BA transporters in the ileum and liver. BA kinetic experiment showed that feeding UDCA shortened cycling time of BA and accelerated BA enterohepatic circulation. Additionally, we found evidence that the effect of UDCA administration on accelerating BA enterohepatic circulation was due to the inhibition of farnesoid X receptor (FXR) signalling in the ileum and FGF15/19 in the liver. CONCLUSION AND IMPLICATIONS: Oral administration of UDCA produced a unique BA profile with high-abundance TUDCA and GUDCA and significantly accelerated BA enterohepatic circulation through the inhibition of intestinal FXR signalling and reduced level of FGF15/19, which in turn, induced the expression of BA transporters in the liver. These findings highlight a critical role for UDCA in maintaining the homeostasis of BA enterohepatic circulation in vivo.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Biotin synthase exhibits burst kinetics and multiple turnovers in the absence of inhibition by products and product-related biomolecules.

Biotin synthase (BS) is a member of the "SAM radical" superfamily of enzymes, which catalyze reactions in which the reversible or irreversible oxidation of various substrates is coupled to the reduction of the S-adenosyl-l-methionine (AdoMet) sulfonium to generate methionine and 5'-deoxyadenosine (dAH). Prior studies have demonstrated that these products are modest inhibitors of BS and other members of this enzyme family. In addition, the in vivo catalytic activity of Escherichia coli BS requires expression of 5'-methylthioadenosine/S-adenosyl-l-homocysteine nucleosidase, which hydrolyzes 5'-methylthioadenosine (MTA), S-adenosyl-l-homocysteine (AdoHcy), and dAH. In the present work, we confirm that dAH is a modest inhibitor of BS (K(i) = 20 µM) and show that cooperative binding of dAH with excess methionine results in a 3-fold enhancement of this inhibition. However, with regard to the other substrates of MTA/AdoHcy nucleosidase, we demonstrate that AdoHcy is a potent inhibitor of BS (K(i) ≤ 650 nM) while MTA is not an inhibitor. Inhibition by both dAH and AdoHcy likely accounts for the in vivo requirement for MTA/AdoHcy nucleosidase and may help to explain some of the experimental disparities between various laboratories studying BS. In addition, we examine possible inhibition by other AdoMet-related biomolecules present as common contaminants in commercial AdoMet preparations and/or generated during an assay, as well as by sinefungin, a natural product that is a known inhibitor of several AdoMet-dependent enzymes. Finally, we examine the catalytic activity of BS with highly purified AdoMet in the presence of MTAN to relieve product inhibition and present evidence suggesting that the enzyme is half-site active and capable of undergoing multiple turnovers in vitro.

Protein residues that control the reaction trajectory in S-adenosylmethionine radical enzymes: mutagenesis of asparagine 153 and aspartate 155 in Escherichia coli biotin synthase.

Biotin synthase catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This reaction is initiated by the reductive cleavage of the sulfonium center of S-adenosyl-L-methionine (AdoMet), generating methionine and a transient 5'-deoxyadenosyl radical that functions as an oxidant by abstracting hydrogen atoms from DTB. Biotin synthase contains a highly conserved sequence motif, YNHNLD, in which Asn153 and Asp155 form hydrogen bonds with the ribose hydroxyl groups of AdoMet. In the present work, we constructed four individual site-directed mutations to change each of these two residues in order to probe their role in the active site. We used molecular weight filtration assays to show that for most of the mutant enzymes binding of the substrates was only slightly affected. In vitro assays demonstrate that several of the mutant enzymes were able to reductively cleave AdoMet, but none were able to produce a significant amount of biotin. Several of the mutants, especially Asn153Ser, were able to produce high levels of the stable intermediate 9-mercaptodethiobiotin. Some of the mutants, such as Asp155Asn and Asn153Ala, produced instead an alternate product tentatively identified by mass spectrometry as 5'-mercapto-5'-deoxyadenosine, generated by direct attack of the 5'-deoxyadenosyl radical on the [4Fe-4S](2+) cluster. Collectively, these results suggest that the protein residues that form hydrogen bonds to AdoMet and DTB are important for retaining intermediates during the catalytic cycle and for targeting the reactivity of the 5'-deoxyadenosyl radical.

9-Mercaptodethiobiotin is formed as a competent catalytic intermediate by Escherichia coli biotin synthase.

Biotin synthase (BS) catalyzes the oxidative addition of a sulfur atom to dethiobiotin (DTB) to generate the biotin thiophane ring. This enzyme is an S-adenosylmethionine (AdoMet) radical enzyme that catalyzes the reductive cleavage of AdoMet, generating methionine and a transient 5'-deoxyadenosyl radical. In our working mechanism, the 5'-deoxyadenosyl radical oxidizes DTB by abstracting a hydrogen from C6 or C9, generating a dethiobiotinyl carbon radical that is quenched by a sulfide from a [2Fe-2S] (2+) cluster. A similar reaction sequence directed at the other position generates the second C-S bond in the thiophane ring. Since the BS active site holds only one AdoMet and one DTB, it follows that dissociation of methionine and 5'-deoxyadenosine and binding of a second equivalent of AdoMet must be intermediate steps in the formation of biotin. During these dissociation-association steps, a discrete DTB-derived intermediate must remain bound to the enzyme. In this work, we confirm that the conversion of DTB to biotin is accompanied by the reductive cleavage of 2 equiv of AdoMet. A discrepancy between DTB consumption and biotin formation suggests the presence of an intermediate, and we use liquid chromatography and mass spectrometry to demonstrate that this intermediate is indeed 9-mercaptodethiobiotin, generated at approximately 10% of the total enzyme concentration. The amount of intermediate observed is increased when the reaction is run with substoichiometric levels of AdoMet or with the defective enzyme containing the Asn153Ser mutation. The retention of 9-mercaptodethiobiotin as a tightly bound intermediate is consistent with a mechanism involving the stepwise radical-mediated oxidative abstraction of sulfide from an iron-sulfur cluster.

Increased cell proliferation and granule cell number in the dentate gyrus of protein repair-deficient mice.

Recent studies have demonstrated that mice lacking protein L-isoaspartate (D-aspartate) O-methyltransferase (Pcmt1-/- mice) have alterations in the insulin-like growth factor-I (IGF-I) and insulin receptor pathways within the hippocampal formation as well as other brain regions. However, the cellular localization of these changes and whether the alterations might be associated with an increase in cell number within proliferative regions, such as the dentate gyrus, were unknown. In this study, stereological methods were used to demonstrate that these mice have an increased number of granule cells in the granule cell layer and hilus of the dentate gyrus. The higher number of granule cells was accompanied by a greater number of cells undergoing mitosis in the dentate gyrus, suggesting that an increase in neuronal cell proliferation occurs in this neurogenic zone of adult Pcmt1-/- mice. In support of this, increased doublecortin labeling of immature neurons was detected in the subgranular zone of the dentate gyrus. In addition, double immunofluorescence studies demonstrated that phosphorylated IGF-I/insulin receptors in the subgranular zone were localized on immature neurons, suggesting that the increased activation of one or both of these receptors in Pcmt1-/- mice could contribute to the growth and survival of these cells. We propose that deficits in the repair of isoaspartyl protein damage leads to alterations in metabolic and growth-receptor pathways, and that this model may be particularly relevant for studies of neurogenesis that is stimulated by cellular damage.

Diet-dependent survival of protein repair-deficient mice.

Protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein-repair enzyme, and mice lacking this enzyme accumulate damaged proteins in multiple tissues, die at an early age from progressive epilepsy and have an increased S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy) ratio in brain tissue. It has been proposed that the alteration of AdoMet and AdoHcy levels might contribute to the seizure phenotype, particularly as AdoHcy has anticonvulsant properties. To investigate whether altered AdoMet and AdoHcy levels might contribute to the seizures and thus the survivability of the repair-deficient mice, a folate-deficient amino acid-based diet was administered to the mice in place of a standard chow diet. We found that the low-folate diet significantly decreases the AdoMet/AdoHcy ratio in brain tissue and results in an almost threefold extension of mean life span in the protein repair-deficient mice. These results indicate that the increased AdoMet/AdoHcy ratio may contribute to the lowered seizure threshold in young PCMT1-deficient mice. However, mean survival was also extended almost twofold for mice on a control folate-replete amino acid-based diet compared to mice on the standard chow diet. Survival after 40 days was similar in the mice on the low- and high-folate amino acid-based diets, suggesting that the survival of older PCMT1-deficient mice is not affected by the higher brain AdoMet/AdoHcy ratio. Additionally, the surviving older repair-deficient mice have a significant increase in body weight when compared to age-matched normal mice, independent of the type of diet. This weight increase was not accompanied by an increase in consumption levels, indicating that the repair-deficient mice may also have an altered metabolic state.

Activation of the PI3K/Akt signal transduction pathway and increased levels of insulin receptor in protein repair-deficient mice.

Protein L-isoaspartate (D-aspartate) O-methyltransferase is an enzyme that catalyses the repair of isoaspartyl damage in proteins. Mice lacking this enzyme (Pcmt1-/- mice) have a progressive increase in brain size compared with wild-type mice (Pcmt1+/+ mice), a phenotype that can be associated with alterations in the PI3K/Akt signal transduction pathway. Here we show that components of this pathway, including Akt, GSK3beta and PDK-1, are more highly phosphorylated in the brains of Pcmt1-/- mice, particularly in cells of the hippocampus, in comparison with Pcmt1+/+ mice. Examination of upstream elements of this pathway in the hippocampus revealed that Pcmt1-/- mice have increased activation of insulin-like growth factor-I (IGF-I) receptor and/or insulin receptor. Western blot analysis revealed an approximate 200% increase in insulin receptor protein levels and an approximate 50% increase in IGF-I receptor protein levels in the hippocampus of Pcmt1-/- mice. Higher levels of the insulin receptor protein were also found in other regions of the adult brain and in whole tissue extracts of brain, liver, heart and testes of both juvenile and adult Pcmt1-/- mice. There were no significant differences in plasma insulin levels for adult Pcmt1-/- mice during glucose tolerance tests. However, they did show higher peak levels of blood glucose, suggesting a mild impairment in glucose tolerance. We propose that Pcmt1-/- mice have altered regulation of the insulin pathway, possibly as a compensatory response to altered glucose uptake or metabolism or as an adaptive response to a general accumulation of isoaspartyl protein damage in the brain and other tissues.

Altered levels of S-adenosylmethionine and S-adenosylhomocysteine in the brains of L-isoaspartyl (D-Aspartyl) O-methyltransferase-deficient mice.

L-Isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein repair enzyme that initiates the conversion of abnormal D-aspartyl and L-isoaspartyl residues to the normal L-aspartyl form. In the course of this reaction, PCMT1 converts the methyl donor S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Due to the high level of activity of this enzyme, particularly in the brain, it seemed of interest to investigate whether the lack of PCMT1 activity might alter the concentrations of these small molecules. AdoMet and AdoHcy were measured in mice lacking PCMT1 (Pcmt1-/-), as well as in their heterozygous (Pcmt1+/-) and wild type (Pcmt1+/+) littermates. Higher levels of AdoMet and lower levels of AdoHcy were found in the brains of Pcmt1-/- mice, and to a lesser extent in Pcmt1+/- mice, when compared with Pcmt1+/+ mice. In addition, these levels appear to be most significantly altered in the hippocampus of the Pcmt1-/- mice. The changes in the AdoMet/AdoHcy ratio could not be attributed to increases in the activities of methionine adenosyltransferase II or S-adenosylhomocysteine hydrolase in the brain tissue of these mice. Because changes in the AdoMet/AdoHcy ratio could potentially alter the overall excitatory state of the brain, this effect may play a role in the progressive epilepsy seen in the Pcmt1-/- mice.